Engineered pyruvate oxidase variants

Abstract

The present invention provides engineered pyruvate oxidase polypeptides useful for the conversion of pyruvate, phosphate, and oxygen to acetyl phosphate, carbon dioxide, and hydrogen peroxide, as well as compositions and methods of utilizing these engineered polypeptides.

Description

ENGINEERED PYRUVATE OXIDASE VARIANTSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 661,199, filed June 18, 2024, and U.S. Provisional Application No. 63 / 706, 183, filed October 1 1 , 2024, the contents of which are incorporated by reference herein.TECHNICAL FIELD

[0002] The present disclosure provides engineered pyruvate oxidase polypeptides useful for generating acetyl phosphate from pyruvate, as well as compositions and methods of utilizing these engineered polypeptides.REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

[0003] The official copy of the Sequence Listing is submitted concurrently with the specification as an XML file, with a file name of “CX10-272WO2_ST26.xml”, a creation date of June 17, 2025, and a size of 2,161,937 bytes. The Sequence Listing filed is part of the specification and is incorporated in its entirety by reference herein.BACKGROUND

[0004] Pyruvate oxidase (POx, EC 1.2.3.3) is a tetrameric peripheral membrane enzyme known to catalyze the conversion of pyruvate, phosphate, and oxygen to form acetyl phosphate, hydrogen peroxide, and carbon dioxide. Pyruvate oxidase requires thiamine pyrophosphate (TPP), which covalently binds to pyruvate, and flavin adenine dinucleotide (FAD), which oxidizes the covalent intermediate to eliminate CO2 and create the reactive intermediate.

[0005] Pyruvate oxidases have been used for decades as metabolic sensors and to measure various disease states or enzymatic reactions.

[0006] Pyruvate oxidases also find use in phosphate donor recycling systems and as auxiliary enzymes in enzyme cascades. One recent publication describes use of a pyruvate oxidase to both drive the equilibrium of a kinase reaction by consuming inorganic phosphate and to generate acetyl phosphate to support ATP recycling (WO 2022 / 133289). Similarly, several wild-type pyruvate oxidases are used together with acetate kinase and catalase to recycle phosphate and concomitantly recycle ATP in a uridine synthesis reaction (WO2022 / 173507). A related report describes use of several wild-type pyruvate oxidases with acetate kinase and catalase in the synthesis of antiviral nucleosides (WO2022 / 133205).

[0007] Previously, mutants of a pyruvate oxidase from Lactobacillus plantarum were created that have better stability in salts and in the alkaline pH range (DE3833601).

[0008] Pyruvate oxidases with improved characteristics under industrial conditions, including increased activity, increased thermostability, increase activity at high temperatures, and increased solubility or protein expression are necessary to enable commercially useful reactions, including phosphate and nucleoside triphosphate recycling reactions.SUMMARY

[0009] The present disclosure provides engineered pyruvate oxidase polypeptides useful for the conversion of pyruvate, phosphate, and oxygen to acetyl phosphate, carbon dioxide, and hydrogen peroxide, as well as compositions and methods of utilizing these engineered polypeptides. The engineered pyruvate oxidases of the present disclosure are variants of the wild-type pyruvate oxidase gene from Jeotgalibaca sp. PTS2502 (SEQ ID NO: 6).

[0010] In some embodiments, the present disclosure provides an engineered pyruvate oxidase polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence selected from SEQ ID NO: 6, 16, 56, 184, 264, 290, 400, or 696, comprising at least one substitution or one substitution set at one or more amino acid residue positions, wherein the positions are numbered with reference to SEQ ID NO: 6, 16, 56, 184, 264, 290, 400, or 696, and wherein the engineered pyruvate oxidase polypeptide has increased activity, increased thermostability, increased soluble protein expression, and / or increased activity at high temperatures, as compared to a wild-type or reference pyruvate oxidase. These engineered pyruvate oxidase polypeptides with one or more amino acid substitutions or substitution sets are described below, in the detailed description of the invention.

[0011] In some additional embodiments, the engineered polypeptide comprises an amino acid sequence with at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to any even-numbered sequence selected from SEQ ID NOs: 14-636 and 692-876. In some embodiments, the engineered pyruvate oxidase comprises an amino acid sequence having at least 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-604 of an even- numbered SEQ ID NO. of SEQ ID NOs: 6, 14-636, and 692-876, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 6, 14-636, and 692-876, or a functional fragment thereof, wherein the amino acid sequence comprises one or more amino acid residue differences relative to a reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, 16, 56, 184, 264, 290, 400, or 696, or relative to a reference sequence corresponding to SEQ ID NO: 6, 16, 56, 184, 264, 290, 400, or 696.

[0012] In some embodiments, the engineered pyruvate oxidase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 14- 636 and 692-876, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 14-636 and 692-876, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, or relative to the reference sequence corresponding to SEQ ID NO: 6.

[0013] In some embodiments, the engineered pyruvate oxidase comprises an amino acid sequence comprising at least amino acid residue difference at amino acid position 14, 15, 21, 31, 32, 45, 54, 57, 61, 62, 74, 108, 117, 127, 134, 135, 139, 150, 153, 161, 163, 167, 168, 177, 183, 187, 192, 193, 197, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 214, 215, 217, 218, 220, 226, 228, 233,235, 237, 239, 242, 244, 250, 259, 302, 307, 308, 314, 317, 325, 326, 331, 336, 342, 345, 351, 357,367, 374, 377, 380, 385, 389, 393, 404, 409, 417, 432, 458, 469, 471, 480, 482, 496, 504, 506, 510,511, 512, 515, 521, 523, 524, 527, 528, 530, 536, 555, 561, 563, 565, 567, 572, 575, 576, 579, 580,585, 587, 591, 594, or 604, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, or relative to the reference sequence corresponding to SEQ ID NO: 6.

[0014] In some embodiments, the engineered pyruvate oxidase comprises an amino acid sequence comprising at least amino acid residue difference(s) at (a) amino acid position 168, 220, 242, 308, 377, 432, or 512, or combinations thereof; (b) amino acid position 61, 168, 202, 233, 242, 244, 308, 336, 480, 510, 512, or 528, or combinations thereof; or (c) at amino acid position(s) 54, 54 / 57, 54 / 57 / 168, 54 / 57 / 168 / 482 / 510 / 512, 54 / 57 / 336, 54 / 57 / 482, 54 / 57 / 510, 54 / 57 / 510 / 512, 54 / 168, 54 / 168 / 336 / 512, 54 / 168 / 482 / 510, 54 / 168 / 510, 54 / 168 / 510 / 512, 54 / 242 / 482 / 512, 54 / 336 / 510, 54 / 336 / 512, 54 / 482, 54 / 482 / 512 / 528, 54 / 510, 54 / 512 / 528, 57, 57 / 168 / 482 / 510 / 512, 57 / 336 / 512, 57 / 482, 57 / 510, 57 / 512, 57 / 512 / 528, 61 / 202 / 308 / 469 / 536, 61 / 244, 61 / 308 / 469, 61 / 308 / 471 / 536, 61 / 471 / 536, 168 / 242 / 336 / 510 / 512, 168 / 242 / 482 / 512 / 528, 168 / 512, 202 / 244 / 308, 202 / 244 / 308 / 471, 202 / 244 / 536, 202 / 308 / 432 / 536, 202 / 308 / 469 / 471, 202 / 308 / 469 / 536, 202 / 308 / 536, 202 / 471 / 536, 202 / 536, 242 / 512 / 528, 244 / 308 / 469, 308 / 469 / 471, 336 / 482, 336 / 482 / 510 / 512, 469, 482, 482 / 510, 482 / 510 / 512 / 528, or 536, or combinations thereof; wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, or relative to the reference sequence corresponding to SEQ ID NO: 6.

[0015] In some embodiments, the engineered pyruvate oxidase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696, or to the reference sequence corresponding to SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the referencesequence corresponding to amino acid residues 12-604 of SEQ ID NO: 16, 56, 184, 264, 290, 400, or696, or relative to the reference sequence corresponding to SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696.

[0016] In some embodiments, the engineered pyruvate oxidase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 14- 636 and 692-876, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 14-636 and 692-876, or a functional fragment thereof, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696, or relative to the reference sequence corresponding to SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696.

[0017] In some embodiments, the engineered pyruvate oxidase comprises an amino acid sequence comprising at least amino acid residue difference at amino acid position 14, 15, 21, 31, 32, 45, 54, 57, 61, 62, 74, 108, 117, 127, 134, 135, 139, 150, 153, 161, 163, 167, 168, 177, 183, 187, 192, 193, 197, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 214, 215, 217, 218, 220, 226, 228, 233,235, 237, 239, 242, 244, 250, 259, 302, 307, 308, 314, 317, 325, 326, 331, 336, 342, 345, 351, 357,367, 374, 377, 380, 385, 389, 393, 404, 409, 417, 432, 458, 469, 471, 480, 482, 496, 504, 506, 510,511, 512, 515, 521, 523, 524, 527, 528, 530, 536, 555, 561, 563, 565, 567, 572, 575, 576, 579, 580,585, 587, 591, 594, or 604, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696, or relative to the reference sequence corresponding to SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696.

[0018] In some embodiments, the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference at amino acid position 14, 199, 201, 202, 215, 218, 496, 575, or 580, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 56, or relative to the reference sequence corresponding to SEQ ID NO: 56.

[0019] In some embodiments, the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference at amino acid position 21, 183, 193, 201, 203, 204, 205, 206, 207, 209, 214, 235, 307, 325, 345, 374, 393, 510, 527, 530, 561, 563, 565, 580, 594, or 604, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0020] In some embodiments, the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference(s) at amino acid position(s) 177, 177 / 209, 177 / 209 / 336, 177 / 209 / 563, 177 / 336, 177 / 336 / 471, 177 / 336 / 563, 177 / 563, 206 / 308, 206 / 308 / 469, 206 / 308 / 536 / 591, 206 / 469, 308, 308 / 469, 308 / 469 / 536, 336 / 471, 336 / 563, 469, 469 / 536, or 563, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 184, or relative to the reference sequence corresponding to SEQ ID NO: 184.

[0021] In some embodiments, the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference at (a) amino acid position 135, 153, 161, or 504, or combinations thereof; (b) amino acid position 32, 127, 134, 135, 153, 163, 192, 197, 200, 317, 331, 385, 504, or 567, or combinations thereof; or (c) amino acid position 32, 74, 108, 117, 127, 134, 135, 139, 150, 153, 163, 177, 192, 197, 200, 226, 237, 250, 259, 302, 314, 317, 331, 385, 404, 409, 458, 504, 506, 511, 515, 523, or 567, or combinations thereof; wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 264, or relative to the reference sequence corresponding to SEQ ID NO: 264.

[0022] In some embodiments, the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference(s) at amino acid position(s) 32 / 134 / 135 / 523 / 567, 32 / 134 / 139 / 163 / 250 / 331 / 523, 32 / 134 / 163 / 409, 32 / 134 / 331 / 567, 32 / 139 / 163 / 250, 32 / 139 / 163 / 409, 32 / 139 / 163 / 567, 32 / 139 / 250 / 567, 32 / 139 / 409, 32 / 163, 32 / 163 / 250, 32 / 163 / 250 / 567, 32 / 163 / 331, 32 / 163 / 331 / 409 / 523 / 567, 32 / 163 / 331 / 409 / 567, 32 / 163 / 409 / 567, 32 / 163 / 523 / 567, 32 / 163 / 567, 32 / 250, 32 / 250 / 409, 32 / 250 / 523 / 567, 32 / 250 / 567, 32 / 331 / 567, 32 / 409, 32 / 409 / 567, 32 / 567, 127, 127 / 153, 127 / 153 / 192 / 197, 127 / 153 / 192 / 197 / 200 / 317, 127 / 153 / 192 / 200 / 317 / 385, 127 / 153 / 192 / 504, 127 / 153 / 201 / 385, 127 / 153 / 317 / 385, 127 / 153 / 317 / 567, 127 / 192 / 197 / 200 / 385 / 504, 127 / 192 / 197 / 200 / 504, 127 / 192 / 197 / 317 / 504 / 567, 127 / 192 / 197 / 385 / 504, 127 / 192 / 197 / 504, 127 / 192 / 317 / 385 / 504, 127 / 192 / 385 / 504 / 567, 127 / 197 / 200 / 317, 127 / 200, 127 / 200 / 317 / 567, 127 / 317, 127 / 385, 127 / 385 / 567, 134 / 135 / 163 / 567, 134 / 163 / 331 / 409 / 567, 134 / 250, 134 / 409 / 567, 139, 139 / 163, 139 / 163 / 250 / 331 / 523, 139 / 163 / 331 / 523, 139 / 163 / 567, 139 / 523 / 567, 153, 153 / 192 / 197 / 200 / 385 / 567, 153 / 192 / 197 / 200 / 504, 153 / 192 / 197 / 317 / 504, 153 / 192 / 197 / 317 / 567, 153 / 192 / 200 / 504, 153 / 192 / 317 / 385, 153 / 197 / 504 / 567, 153 / 200, 153 / 317, 153 / 317 / 385, 153 / 317 / 504, 153 / 317 / 504 / 567, 153 / 385, 153 / 504, 153 / 504 / 567, 163, 163 / 250, 163 / 250 / 567, 163 / 331, 163 / 331 / 409, 163 / 331 / 409 / 567, 163 / 331 / 567, 163 / 409, 163 / 523 / 567, 163 / 567, 192 / 197 / 200, 192 / 197 / 200 / 317 / 567, 192 / 197 / 200 / 385, 192 / 197 / 200 / 385 / 567, 192 / 197 / 317, 192 / 197 / 385 / 504 / 567, 192 / 197 / 504, 192 / 200, 192 / 200 / 385, 192 / 317, 192 / 504, 197 / 200 / 567, 197 / 317, 197 / 317 / 385 / 504 / 567, 200 / 317 / 385, 200 / 385 / 567, 250, 250 / 331, 250 / 331 / 567, 250 / 523, 250 / 567, 317, 317 / 385 / 567, 317 / 567, 331 / 409, 331 / 567, 385, 385 / 567, 409 / 523, 523, 523 / 567, or 567, or combinations thereof, wherein the amino acid positionsare relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 290, or relative to the reference sequence corresponding to SEQ ID NO: 290.

[0023] In some embodiments, the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference(s) at (a) amino acid position(s) 32, 32 / 135, 134, 134 / 200 / 331, 134 / 331, or 331, or combinations thereof; (b) amino acid position(s) 62 / 117, 117 / 139 / 331 / 523, 117 / 200 / 523, 117 / 523, 134, or 200 / 523, or combinations thereof; (c) amino acid position 31, 45, 167, 208, 210, 217, 228, 351, 357, 367, 380, or 579, or combinations thereof; wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 400, or relative to the reference sequence corresponding to SEQ ID NO: 400.

[0024] In some embodiments, the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference(s) at (a) amino acid position 15, 203, 206, 239, 326, 417, 524, 527, 555, 572, 587, or 594, or combinations thereof; (b) amino acid position 15, 168, 183, 187, 203, 206, 214, 389, 417, 521, 527, 572, 575, 580, 585, 587, or 594, or combinations thereof; (c) amino acid position(s) 15, 203 / 206, 203 / 206 / 521, 203 / 521, 206, 206 / 342 / 521, 206 / 342 / 576, 206 / 521, 206 / 585 / 587, 342 / 576, 342 / 576 / 580, 342 / 585 / 587, or 576, or combinations thereof; or (d) amino acid position(s) 15, 15 / 183, 15 / 389, 203 / 206, 203 / 206 / 342 / 576, 203 / 206 / 521, 203 / 206 / 576, 206, 206 / 342, 206 / 521, 342 / 576, 576, or 576 / 580, or combinations thereof; wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 696, or relative to the reference sequence corresponding to SEQ ID NO: 696.

[0025] In some embodiments, the engineered pyruvate oxidase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 6, 14-636, and 692-876, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 6, 14-636, and 692-876.

[0026] In some embodiments, the engineered pyruvate oxidase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696, or to a reference sequence corresponding to SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696.

[0027] In some embodiments, the engineered pyruvate oxidase comprises an amino acid sequence comprising amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 14-636 and 692-876, or an amino acid sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOs: 14-636 and 692-876.

[0028] In some embodiments, the engineered pyruvate oxidase comprises an amino acid sequence comprising amino acid residues 12-604 of SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696, or an amino acid sequence comprising SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696.

[0029] The present disclosure also provides an engineered polynucleotide encoding at least one engineered polypeptide described herein and in the above paragraphs.

[0030] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34-1812 of SEQ ID NO: 5, 15, 55, 183, 263, 289, 399, or 695, or to a reference polynucleotide sequence of SEQ ID NO: 5, 15, 55, 183, 263, 289, 399, or 695, wherein the recombinant polynucleotide encodes a polypeptide having pyruvate oxidase activity.

[0031] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34-1812 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 13-635 and 691-875, or to a reference polynucleotide sequence of SEQ ID NOs: 13-635 and 691 -875, wherein the recombinant polynucleotide encodes a polypeptide having pyruvate oxidase activity.

[0032] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising nucleotide residues 34-1812 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 13-635 and 691-875, or comprising an odd-numbered SEQ ID NO. of SEQ ID NOs: 13-635 and 691-875.

[0033] In another aspect, the present disclosure further provides vectors, e.g., an expression vector, comprising at least one recombinant polynucleotide described above and herein. In some embodiments, the vectors further comprise at least one control sequence.

[0034] In a further aspect, the present disclosure provides host cells comprising the vectors provided herein. In some embodiments, the host cell produces at least one engineered polypeptide provided herein.

[0035] The present disclosure further provides methods of producing an engineered pyruvate oxidase polypeptide, comprising the steps of culturing the host cell provided herein under conditions such that the engineered polynucleotide is expressed. In some embodiments, the methods further comprise recovering and / or purifying the expressed polypeptide.

[0036] In another aspect, the present disclosure provides a method of producing acetyl phosphate, comprising contacting an engineered pyruvate oxidase describe herein with pyruvate under suitable reaction conditions for the production of acetyl phosphate.

[0037] In some embodiments, the method further comprises providing a second enzyme in a paired reaction, wherein the second enzyme uses acetylphosphate as a substrate or degrades hydrogen peroxide.DESCRIPTION OF THE INVENTION

[0038] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein and the laboratory procedures of cell culture, molecular genetics, microbiology, organic chemistry, analytical chemistry and nucleic acid chemistry described below are those well-known and commonly employed in the art. Such techniques are well- known and described in numerous texts and reference works well known to those of skill in the art. Standard techniques, or modifications thereof, are used for chemical syntheses and chemical analyses. All patents, patent applications, articles and publications mentioned herein, both supra and infra, are hereby expressly incorporated herein by reference.

[0039] Although any suitable methods and materials similar or equivalent to those described herein find use in the practice of the present invention, some methods and materials are described herein. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art. Accordingly, the terms defined immediately below are more fully described by reference to the invention as a whole.

[0040] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present invention. The section headings used herein are for organizational purposes only and not to be construed as limiting the subject matter described. Numeric ranges are inclusive of the numbers defining the range. Thus, every numerical range disclosed herein is intended to encompass every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein. It is also intended that every maximum (or minimum) numerical limitation disclosed herein includes every lower (or higher) numerical limitation, as if such lower (or higher) numerical limitations were expressly written herein.

[0041] As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “a polypeptide” includes more than one polypeptide. Similarly, “comprise,” “comprises,” “comprising” “include,” “includes,” and “including” are interchangeable and not intended to be limiting.

[0042] It is to be understood that where descriptions of various embodiments use the term “comprising,” those skilled in the art would understand that in some specific instances, anembodiment can be alternatively described using language “consisting essentially of’ or “consisting of.” It is to be further understood that where descriptions of various embodiments use the term “optional” or “optionally” the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not. It is to be understood that both the foregoing general description, and the following detailed description are exemplary and explanatory only and are not restrictive of this disclosure. The section headings used herein are for organizational purposes only and not to be construed as limiting the subject matter described.Abbreviations

[0043] The abbreviations used for the genetically encoded amino acids are conventional and are as follows:

[0044] When the three-letter abbreviations are used, unless specifically preceded by an “L” or a “D” or clear from the context in which the abbreviation is used, the amino acid may be in either the L- or D-configuration about cc-carbon (C«). For example, whereas “Ala” designates alanine withoutspecifying the configuration about the acarbon, “D-Ala” and “L-Ala” designate D-alanine and L- alanine, respectively.

[0045] When the one-letter abbreviations are used, upper case letters designate amino acids in the L- configuration about the a-carbon and lower case letters designate amino acids in the D-configuration about the a-carbon. For example, “A” designates L-alanine and “a” designates D-alanine. When polypeptide sequences are presented as a string of one-letter or three-letter abbreviations (or mixtures thereof), the sequences are presented in the amino (N) to carboxy (C) direction in accordance with common convention.

[0046] The abbreviations used for the genetically encoding nucleosides are conventional and are as follows: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). These abbreviations are also used interchangeably for nucleosides and nucleotides (nucleosides with one or more phosphate groups). Unless specifically delineated, the abbreviated nucleosides or nucleotides may be either ribonucleosides (or ribonucleotides) or 2’ -deoxyribonucleosides (or 2’- deoxyribonucleotides). The nucleosides or nucleotides may also be modified at the 3’ position. The nucleosides or nucleotides may be specified as being either ribonucleosides (or ribonucleotides) or 2’ - deoxyribonucleosides (or 2 ’-deoxyribonucleotides) on an individual basis or on an aggregate basis. When nucleic acid sequences are presented as a string of one-letter abbreviations, the sequences are presented in the 5’ to 3’ direction in accordance with common convention, and the phosphates are not indicated.Definitions

[0047] In reference to the present invention, the technical and scientific terms used in the descriptions herein will have the meanings commonly understood by one of ordinary skill in the art, unless specifically defined otherwise. Accordingly, the following terms are intended to have the following meanings.

[0048] “EC” number refers to the Enzyme Nomenclature of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). The IUBMB biochemical classification is a numerical classification system for enzymes based on the chemical reactions they catalyze.

[0049] “ATCC” refers to the American Type Culture Collection whose biorepository collection includes genes and strains.

[0050] ‘ ‘NCBI” refers to National Center for Biological Information and the sequence databases provided therein.

[0051] ‘ ‘Protein,” “polypeptide,” and “peptide” arc used interchangeably herein to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation, phosphorylation, lipidation, myristoylation, ubiquitination, etc.). Included within this definition are D- and L-amino acids, and mixtures of D- and L-amino acids, as well as polymers comprising D- and L-amino acids, and mixtures of D- and L- amino acids.

[0052] ‘ ‘Amino acids” are referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single letter codes.

[0053] As used herein, “polynucleotide,” “oligonucleotide,” and “nucleic acid’ ’ are used interchangeably herein and refer to two or more nucleosides or nucleotides that are covalently linked together. The polynucleotide may be wholly comprised of ribonucleotides (i.e., RNA), wholly comprised of 2’ deoxyribonucleotides (i.e., DNA), wholly comprised of other synthetic nucleotides or comprised of mixtures of synthetic, ribo- and / or 2’ deoxyribonucleotides. The polynucleotides may also include modified nucleotides with substitutions, including 2’ substitutions (e.g., 2’-flouro, 2’-O- methyl, 2’-O-methoxyethyl, locked or constrained ethyl modifications, and others known to those skilled in the art). Nucleosides will be linked together via standard phosphodiester linkages or via one or more non-standard linkages, including but not limited to phosphorothioate linkages. The polynucleotide may be single-stranded or double-stranded or may include both single-stranded regions and double-stranded regions. Moreover, while a polynucleotide will typically be composed of the naturally occurring encoding nucleobases (i.e., adenine, guanine, uracil, thymine and cytosine), it may include one or more modified and / or synthetic nucleobases, such as, for example, inosine, xanthine, hypoxanthine, etc. In some embodiments, such modified or synthetic nucleobases are nucleobases encoding amino-acid sequences. Nucleobases that are modified or synthetic may comprise any known or hypothetical or future discovered modification or structure that would be recognized by one of skill in the art as a modified or synthetic nucleobase. Similarly, the terms “polynucleotide,” “oligonucleotide,” and “nucleic acid’ ’ are intended to comprise any modified or synthetic structure that is now known or discovered in the future that would be recognized by one of skill in the art as being or having the function of a “polynucleotide,” “oligonucleotide,” or “nucleic acid.” An example of a modified or synthetic structure having the function of a “polynucleotide,” “oligonucleotide,” or “nucleic acid’ ’ is PNA or peptide nucleic acid.

[0054] As used herein, “NTP,” “NDP,” “NMP,” “NQP,” “nucleoside,” “nucleotide” or similar terms are intended to generically refer to ribo, deoxyribo, and / or 2’ modified (modifications other than H or OH) versions of these molecules, unless specifically defined in a specific instance. These molecules may also have additional modifications at other positions, and these terms are intended to encompass these variations, absent any specific definition to the contrary.

[0055] “Coding sequence” refers to that portion of a nucleic acid (e.g., a gene) that encodes an amino acid sequence of a protein.

[0056] “Pyruvate oxidase” or “POx” (EC 1.2.3.3) refers to an enzyme that catalyzes the reaction between pyruvate, phosphate, and oxygen to generate acetyl phosphate, carbon dioxide, and hydrogen peroxide. In some embodiments, the pyruvate oxidase is derived from Jeotgalibaca sp. PTS2502 and is a tetrameric peripheral membrane enzyme utilizing cofactors TPP, FAD, and a divalent metal. In some embodiments, the engineered pyruvate oxidases of the present disclosure include an N-terminal his tag.

[0057] ‘ ‘Acetate kinase (“AcK”) refers to enzymes that are capable of catalyzing the phosphorylation of nucleoside diphosphates or analogues thereof, to nucleoside triphosphates or the corresponding analogues, using acetyl phosphate or another phosphoryl group donor. Acetate kinases as used herein includes naturally occurring, wild-type enzymes or engineered enzymes. In some embodiments, acetate kinases are naturally occurring, wild-type basic metabolic enzymes found primarily in prokaryotes that catalyze the phosphorylation of acetate to acetyl-CoA in the presence of ATP. In some embodiments, acetate kinases are derived from the naturally occurring, wild-type enzymes.

[0058] ‘ ‘Catalase” refers to an enzyme that converts hydrogen peroxide (H2O2) to H2O and O2. Catalase can be used to remove residual hydrogen peroxide in applications where hydrogen peroxide is present or is a product in a process. In some embodiments, catalases include enzymes classified in EC 1.11.1.6.

[0059] “Naturally-occurring” or “wild-type” refers to the form found in nature. For example, a naturally occurring or wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a source in nature and which has not been intentionally modified by human manipulation.

[0060] As used herein, “recombinant,” “engineered,” and “non-naturally occurring” when used with reference to a cell, nucleic acid, or polypeptide, refer to a material, or a material corresponding to the natural or native form of the material, that has been modified in a manner that would not otherwise exist in nature. In some embodiments, the cell, nucleic acid or polypeptide is identical a naturally occurring cell, nucleic acid or polypeptide, but is produced or derived from synthetic materials and / or by manipulation using recombinant techniques. Non-limiting examples include, among others, recombinant cells expressing genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise expressed at a different level.

[0061] “Percentage of sequence identity” and “percentage homology” are used interchangeably herein to refer to comparisons among polynucleotides or polypeptides and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions ordeletions (i.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentage may be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Alternatively, the percentage may be calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Those of skill in the art appreciate that there are many established algorithms available to align two sequences. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 1981, 2:482), by the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 1970, 48:443), by the search for similarity method of Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 1988, 85:2444), by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection, as known in the art. Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity include, but are not limited to the BLAST and BLAST 2.0 algorithms, which are described by Altschul et al. (See, Altschul et al., J. Mol. Biol., 1990, 215:403- 410; and Altschul et al., Nucl. Acids Res., 1997, 25(17):3389-3402, respectively). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as, the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and acomparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (See, Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 19889, 89: 10915). Exemplary determination of sequence alignment and % sequence identity can employ the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison WI), using default parameters provided.

[0062] “Reference sequence” refers to a defined sequence used as a basis for a sequence comparison. A reference sequence may be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. Generally, a reference sequence is at least 20 nucleotide or amino acid residues in length, at least 25 residues in length, at least 50 residues in length, or the full length of the nucleic acid or polypeptide. Since two polynucleotides or polypeptides may each (1) comprise a sequence (z.e., a portion of the complete sequence) that is similar between the two sequences, and (2) may further comprise a sequence that is divergent between the two sequences, sequence comparisons between two (or more) polynucleotides or polypeptide are typically performed by comparing sequences of the two polynucleotides or polypeptides over a “comparison window” to identify and compare local regions of sequence similarity. In some embodiments, a “reference sequence” can be based on a primary amino acid sequence, where the reference sequence is a sequence that can have one or more changes in the primary sequence. For instance, a “reference sequence based on SEQ ID NO: 6 having at the residue corresponding to X14 an alanine” or X14A refers to a reference sequence in which the corresponding residue at X14 in SEQ ID NO: 6, which is an aspartic acid, has been changed to alanine.

[0063] “Comparison window” refers to a conceptual segment of contiguous nucleotide positions or amino acids residues wherein a sequence may be compared to a reference sequence. In some embodiments, the comparison window is at least 15 to 20 contiguous nucleotides or amino acids and wherein the portion of the sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. In some embodiments, the comparison window can be longer than 15-20 contiguous residues, and includes, optionally 30, 40, 50, 100, or longer windows.

[0064] As used herein, “substantial identity” refers to a polynucleotide or polypeptide sequence that has at least 80 percent sequence identity, at least 85 percent identity, at least between 89 to 95 percent sequence identity, or more usually, at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 residue positions, frequently over a window of at least 30-50 residues, wherein the percentage of sequence identity is calculated by comparing the reference sequence to a sequence that includes deletions or additions which total 20 percent or less of the reference sequence over the window of comparison. In some specific embodiments applied to polypeptides, the term “substantial identity” means that two polypeptide sequences, when optimallyaligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80 percent sequence identity, preferably at least 89 percent sequence identity, at least 95 percent sequence identity or more (e.g., 99 percent sequence identity). In some embodiments, residue positions that are not identical in sequences being compared differ by conservative amino acid substitutions.

[0065] “Corresponding to,” “reference to,” and “relative to” when used in the context of the numbering of a given amino acid or polynucleotide sequence refer to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue number or residue position of a given polymer is designated with respect to the reference sequence rather than by the actual numerical position of the residue within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as that of an engineered pyruvate oxidate, can be aligned to a reference sequence by introducing gaps to optimize residue matches between the two sequences. In these cases, although the gaps are present, the numbering of the residue in the given amino acid or polynucleotide sequence is made with respect to the reference sequence to which it has been aligned.

[0066] “Amino acid difference” or “residue difference” refers to a change in the amino acid residue at a position of a polypeptide sequence relative to the amino acid residue at a corresponding position in a reference sequence. The positions of amino acid differences generally are referred to herein as “Xn,” where n refers to the corresponding position in the reference sequence upon which the residue difference is based. For example, a “residue difference at position X21 as compared to SEQ ID NO: 6” refers to a change of the amino acid residue at the polypeptide position corresponding to position 21 of SEQ ID NO: 6. Thus, if the reference polypeptide of SEQ ID NO: 6 has a threonine at position 21, then a “residue difference at position X21 as compared to SEQ ID NO: 6” an amino acid substitution of any residue other than threonine at the position of the polypeptide corresponding to position 21 of SEQ ID NO: 6. In most instances herein, the specific amino acid residue difference at a position is indicated as “XnY” where “Xn” specified the corresponding position as described above, and “Y” is the single letter identifier of the amino acid found in the engineered polypeptide (i.e., the different residue than in the reference polypeptide). In some embodiments, more than one amino acid can appear in a specified residue position (i.e., the alternative amino acids can be listed in the form XnY / Z, where Y and Z represent alternate amino acid residues). In some instances (e.g., in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, and 23.2.) the present invention also provides specific amino acid differences denoted by the conventional notation “AnB”, where A is the single letter identifier of the residue in the reference sequence, “n” is the number of the residue position in the reference sequence, and B is the single letter identifier of the residue substitution in the sequence of the engineered polypeptide. Furthermore, in some instances, a polypeptide of the present invention can include one or more amino acid residue differences relativeto a reference sequence, which is indicated by a list of the specified positions where changes are made relative to the reference sequence. In some additional embodiments, the present invention provides engineered polypeptide sequences comprising both conservative and non-conservative amino acid substitutions.

[0067] As used herein, “conservative amino acid substitution” refers to a substitution of a residue with a different residue having a similar side chain, and thus typically involves substitution of the amino acid in the polypeptide with amino acids within the same or similar defined class of amino acids. By way of example and not limitation, an amino acid with an aliphatic side chain is substituted with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid with an hydroxyl side chain is substituted with another amino acid with a hydroxyl side chain (e.g., serine and threonine); an amino acid having aromatic side chains is substituted with another amino acid having an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain is substituted with another amino acid with a basis side chain (e.g., lysine and arginine); an amino acid with an acidic side chain is substituted with another amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid); and / or a hydrophobic or hydrophilic amino acid is replaced with another hydrophobic or hydrophilic amino acid, respectively. Exemplary conservative substitutions are provided in Table 1 below.

[0068] “Non-conservative substitution” refers to substitution of an amino acid in the polypeptide with an amino acid with significantly differing side chain properties. Non-conservative substitutions may use amino acids between, rather than within, the defined groups and affects (a) the structure of the peptide backbone in the area of the substitution (e.g., proline for glycine), (b) the charge or hydrophobicity, or (c) the bulk of the side chain. By way of example and not limitation, an exemplary non-conservative substitution can be an acidic amino acid substituted with a basic or aliphatic amino acid; an aromatic amino acid substituted with a small amino acid; and a hydrophilic amino acid substituted with a hydrophobic amino acid.

[0069] “Deletion” refers to modification to the polypeptide by removal of one or more amino acids from the reference polypeptide. Deletions can comprise removal of 1 or more amino acids, 2 or moreamino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids, or up to 20% of the total number of amino acids making up the reference enzyme while retaining enzymatic activity and / or retaining the improved properties of an engineered pyruvate oxidase enzyme. Deletions can be directed to the internal portions and / or terminal portions of the polypeptide. In various embodiments, the deletion can comprise a continuous segment or can be discontinuous.

[0070] ‘ ‘Insertion” refers to modification to the polypeptide by addition of one or more amino acids from the reference polypeptide. In some embodiments, the improved engineered pyruvate oxidase enzymes comprise insertions of one or more amino acids to the naturally occurring polypeptide as well as insertions of one or more amino acids to other improved pyruvate oxidase polypeptides. Insertions can be in the internal portions of the polypeptide, or to the carboxy or amino terminus. Insertions as used herein include fusion proteins as is known in the art. The insertion can be a contiguous segment of amino acids or separated by one or more of the amino acids in the naturally occurring polypeptide.

[0071] “Fragment” as used herein refers to a polypeptide that has an amino-terminal and / or carboxyterminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the sequence. Fragments can be at least 20 amino acids long, at least 50 amino acids long, at least 100 amino acids long, at least 200 amino acids long, or longer, and up to 70%, 80%, 90%, 95%, 98%, and 99% of the full-length pyruvate oxidase polypeptide, for example the polypeptide of SEQ ID NO: 6 or a pyruvate oxidase provided in the even-numbered sequences of SEQ ID NOs: 14- 636 and 692-876.

[0072] ‘ ‘Functional fragment” and “biologically active fragment” are used interchangeably herein to refer to a polypeptide that has an amino-terminal and / or carboxy-terminal deletion(s) and / or internal deletions, but where the remaining amino acid sequence is identical to the corresponding positions in the sequence to which it is being compared (e.g., a full-length engineered pyruvate oxidase of the present invention) and that retains substantially all of the activity of the full-length polypeptide.

[0073] ‘ ‘Isolated polypeptide” refers to a polypeptide which is substantially separated from other contaminants that naturally accompany it, e.g., protein, lipids, and polynucleotides. The term embraces polypeptides which have been removed or purified from their naturally -occurring environment or expression system (e.g., host cell or in vitro synthesis). The engineered pyruvate oxidase enzymes may be present within a cell, present in the cellular medium, or prepared in various forms, such as lysates or isolated preparations. As such, in some embodiments, the engineered pyruvate oxidase enzyme can be an isolated polypeptide.

[0074] “Substantially pure polypeptide” refers to a composition in which the polypeptide species is the predominant species present (i.e., on a molar or weight basis it is more abundant than any otherindividual macromolecular species in the composition) and is generally a substantially purified composition when the object species comprises at least about 50 percent of the macromolecular species present by mole or % weight. Generally, a substantially pure pyruvate oxidase composition will comprise about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more of all macromolecular species by mole or % weight present in the composition. In some embodiments, the object species is purified to essential homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered macromolecular species. In some embodiments, the isolated engineered pyruvate oxidase polypeptide is a substantially pure polypeptide composition.

[0075] “Improved enzyme property” refers to at least one improved property of an enzyme. In some embodiments, the present invention provides engineered pyruvate oxidase polypeptides that exhibit an improvement in any enzyme property as compared to a reference pyruvate oxidase polypeptide and / or a wild-type pyruvate oxidase polypeptide, and / or another engineered pyruvate oxidase polypeptide. For the engineered pyruvate oxidase polypeptides described herein, the comparison is generally made to the wild-type enzyme from which the pyruvate oxidase is derived, although in some embodiments, the reference enzyme can be another improved engineered pyruvate oxidase. Thus, the level of “improvement” can be determined and compared between various pyruvate oxidase polypeptides, including wild-type, as well as engineered pyruvate oxidases. Improved properties include, but are not limited, to such properties as enzymatic activity (which can be expressed in terms of percent conversion of the substrate), thermo stability, solvent stability, pH activity profile, cofactor requirements, refractoriness to inhibitors (e.g., substrate or product inhibition), activity at elevated temperatures, increased soluble expression, decreased by-product formation, increased specific activity on substrates, and / or increased activity (including enantioselectivity).

[0076] ‘ ‘Increased enzymatic activity” refers to an improved property of the pyruvate oxidase polypeptides, which can be represented by an increase in specific activity (e.g., product produced / time / weight protein) or an increase in percent conversion of the substrate to the product (e.g., percent conversion of starting amount of substrate to product in a specified time period using a specified amount of pyruvate oxidase) as compared to the reference pyruvate oxidase enzyme. Exemplary methods to determine enzyme activity are provided in the Examples. Any property relating to enzyme activity may be affected, including the classical enzyme properties of Km,or kcat, changes of which can lead to increased enzymatic activity. Improvements in enzyme activity can be from about 1.2 times the enzymatic activity of the corresponding wild-type enzyme, to as much as 2 times, 5 times, 10 times, 20 times, 25 times, 50 times or more enzymatic activity than the naturally occurring or another engineered pyruvate oxidase from which the pyruvate oxidase polypeptides werederived. Pyruvate oxidase activity can be measured by any one of standard assays, such as by monitoring changes in properties of substrates, cofactors, or products. In some embodiments, enzyme activity may be measured indirectly using a coupled reaction, wherein the amount of substrate produced or product consumed is measured in one or more paired reactions. In some embodiments, the amount of products generated can be measured by Liquid Chromatography-Mass Spectrometry (LC-MS), HPLC, capillary electrophoresis (CE) or other methods, as known in the art. Comparisons of enzyme activities are made using a defined preparation of enzyme, a defined assay under a set condition, and one or more defined substrates, as further described in detail herein. Generally, when lysates are compared, the numbers of cells and the amount of protein assayed are determined as well as use of identical expression systems and identical host cells to minimize variations in amount of enzyme produced by the host cells and present in the lysates.

[0077] ‘ ‘Conversion” refers to the enzymatic conversion of the substrate(s) to the corresponding product(s). “Percent conversion” refers to the percent of the substrate that is converted to the product within a period of time under specified conditions. Thus, the “enzymatic activity” or “activity” of a pyruvate oxidase polypeptide can be expressed as “percent conversion” of the substrate to the product.

[0078] ‘ ‘Thermostable” refers to a pyruvate oxidase polypeptide that maintains similar activity (more than 60% to 80% for example) after exposure to elevated temperatures (e.g., 40-80 °C) for a period of time (e.g., 0.5-24 hrs) compared to the wild-type enzyme or another reference enzyme exposed to the same elevated temperature.

[0079] ‘ ‘Solvent stable” refers to a pyruvate oxidase polypeptide that maintains similar activity (more than e.g., 60% to 80%) after exposure to varying concentrations (e.g., 5-99%) of solvent (ethanol, isopropyl alcohol, dimethylsulfoxide (DMSO), tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene, butyl acetate, methyl tert-butyl ether, etc.) for a period of time (e.g., 0.5-24 hrs) compared to the wild-type enzyme exposed to the same concentration of the same solvent.

[0080] “Thermo- and solvent stable” refers to a pyruvate oxidase polypeptide that is both thermostable and solvent stable.

[0081] “Stringent hybridization conditions” is used herein to refer to conditions under which nucleic acid hybrids are stable. As known to those of skill in the art, the stability of hybrids is reflected in the melting temperature (Tm) of the hybrids. In general, the stability of a hybrid is a function of ion strength, temperature, G / C content, and the presence of chaotropic agents. The Tmvalues for polynucleotides can be calculated using known methods for predicting melting temperatures (See e.g., Baldino et al., Meth. Enzymol., 1989, 168:761-777; Bolton et al., Proc. Natl. Acad. Sci. USA, 1962, 48:1390; Bresslauer et aL, Proc. Natl. Acad. Sci. USA, 1986, 83:8893-8897; Freier et al., Proc. Natl. Acad. Sci. USA, 1986, 83:9373-9377; Kierzek et al., Biochem., 1986, 25:7840-7846; Rychlik et al., 1990, Nucl. Acids Res., 18:6409-6412 (erratum, Nucl. Acids Res., 1991, 19:698); Suggs et al., 1981,in Developmental Biology Using Purified Genes, Brown et al. (eds.), pp. 683-693, Academic Press, Cambridge, MA (1981); and Wetmur, Crit. Rev. Biochem. Mol. BioL, 1991, 26:227-259). In some embodiments, the polynucleotide encodes the polypeptide disclosed herein and hybridizes under defined conditions, such as moderately stringent or highly stringent conditions, to the complement of a sequence encoding an engineered pyruvate oxidase enzyme of the present invention.

[0082] “Hybridization stringency” relates to hybridization conditions, such as washing conditions, in the hybridization of nucleic acids. Generally, hybridization reactions are performed under conditions of lower stringency, followed by washes of varying but higher stringency. The term “moderately stringent hybridization” refers to conditions that permit target-DNA to bind a complementary nucleic acid that has about 60% identity, preferably about 75% identity, about 85% identity to the target DNA, with greater than about 90% identity to target-polynucleotide. Exemplary moderately stringent conditions are conditions equivalent to hybridization in 50% formamide, 5x Denhart's solution, 5xSSPE, 0.2% SDS at 42 °C, followed by washing in 0.2xSSPE, 0.2% SDS, at 42 °C. “High stringency hybridization” refers generally to conditions that are about 10 °C or less from the thermal melting temperature Tmas determined under the solution condition for a defined polynucleotide sequence. In some embodiments, a high stringency condition refers to conditions that permit hybridization of only those nucleic acid sequences that form stable hybrids in 0.018M NaCl at 65 °C (i.e., if a hybrid is not stable in 0.018M NaCl at 65 °C, it will not be stable under high stringency conditions, as contemplated herein). High stringency conditions can be provided, for example, by hybridization in conditions equivalent to 50% formamide, 5x Denhart's solution, 5xSSPE, 0.2% SDS at 42 °C, followed by washing in O.lxSSPE, and 0.1% SDS at 65 °C. Another high stringency condition is hybridizing in conditions equivalent to hybridizing in 5X SSC containing 0.1% (w:v) SDS at 65 °C and washing in 0.1 x SSC containing 0.1% SDS at 65 °C. Other high stringency hybridization conditions, as well as moderately stringent conditions, are described in the references cited above.

[0083] “Heterologous” polynucleotide refers to any polynucleotide that is introduced into a host cell by laboratory techniques and includes polynucleotides that are removed from a host cell, subjected to laboratory manipulation, and then reintroduced into a host cell.

[0084] “Codon optimized” refers to changes in the codons of the polynucleotide encoding a protein to those preferentially used in a particular organism such that the encoded protein is efficiently expressed in the organism of interest. Although the genetic code is degenerate in that most amino acids are represented by several codons, called “synonyms” or “synonymous” codons, it is well known that codon usage by particular organisms is nonrandom and biased towards particular codon triplets. This codon usage bias may be higher in reference to a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and the aggregate protein coding regions of an organism's genome. In some embodiments, the polynucleotides encodingthe pyruvate oxidase enzymes may be codon optimized for optimal production from the host organism selected for expression.

[0085] As used herein, “preferred, optimal, high codon usage bias codons” refers interchangeably to codons that are used at higher frequency in the protein coding regions than other codons that code for the same amino acid. The preferred codons may be determined in relation to codon usage in a single gene, a set of genes of common function or origin, highly expressed genes, the codon frequency in the aggregate protein coding regions of the whole organism, codon frequency in the aggregate protein coding regions of related organisms, or combinations thereof. Codons whose frequency increases with the level of gene expression are typically optimal codons for expression. A variety of methods are known for determining the codon frequency (e.g., codon usage, relative synonymous codon usage) and codon preference in specific organisms, including multivariate analysis, for example, using cluster analysis or correspondence analysis, and the effective number of codons used in a gene (See e.g., GCG CodonPreference, Genetics Computer Group Wisconsin Package; CodonW, Peden, University of Nottingham; McInerney, Bioinform., 1998, 14:372-73; Stenico et al., NucL Acids Res., 1994, 222437-46; Wright, Gene, 1990, 87:23-29). Codon usage tables are available for many different organisms (See e.g., Wad et aL, Nucl. Acids Res., 1992, 20:2111-2118; Nakamura et al., Nucl. Acids Res., 2000, 28:292; Henaut and Danchin, In Escherichia coli and Salmonella, Neidhardt, et al. (eds.), ASM Press, Washington D.C., p. 2047-2066 (1996)). The data source for obtaining codon usage may rely on any available nucleotide sequence capable of coding for a protein. These data sets include nucleic acid sequences actually known to encode expressed proteins (e.g., complete protein coding sequences-CDS), expressed sequence tags (ESTS), or predicted coding regions of genomic sequences (See e.g., Mount, Bioinformatics: Sequence and Genome Analysis, Chapter 8, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); Uberbacher, Meth. Enzymol., 1996, 266:259- 281 ; and Tiwari et al., Comput. Appl. Biosci., 1997, 13:263-270).

[0086] “Control sequence” is defined herein to include all components, which are necessary or advantageous for the expression of a polynucleotide and / or polypeptide of the present invention. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding a polypeptide.

[0087] “Operably linked” is defined herein as a configuration in which a control sequence is appropriately placed i.e., in a functional relationship) at a position relative to a polynucleotide ofinterest such that the control sequence directs or regulates the expression of the polynucleotide and / or polypeptide of interest.

[0088] “Promoter sequence” refers to a nucleic acid sequence that is recognized by a host cell for expression of a polynucleotide of interest, such as a coding sequence. The promoter sequence contains transcriptional control sequences, which mediate the expression of a polynucleotide of interest. The promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

[0089] “Suitable reaction conditions” refer to those conditions in the biocatalytic reaction solution (e.g., ranges of enzyme loading, substrate loading, cofactor loading, temperature, pH, buffers, cosolvents, etc.) under which a pyruvate oxidase polypeptide of the present invention is capable of converting one or more substrate compounds to a product compound. Exemplary “suitable reaction conditions” are provided in the present invention and illustrated by the Examples.

[0090] “Composition” refers to a mixture or combination of one or more substances, wherein each substance or component of the composition retains its individual properties. As used herein, a biocatalytic composition refers to a combination of one or more substances useful for biocatalysis.

[0091] “Loading”, such as in “compound loading” or “enzyme loading” or “cofactor loading” refers to the concentration or amount of a component in a reaction mixture at the start of the reaction.

[0092] “Substrate” in the context of a biocatalyst mediated process refers to the compound or molecule acted on by the biocatalyst. For example, a pyruvate oxidase biocatalyst used in the synthesis processes disclosed herein acts on pyruvate, oxygen, and phosphate.

[0093] “Product” in the context of a biocatalyst mediated process refers to the compound or molecule resulting from the action of the biocatalyst. For example, an exemplary product for a pyruvate oxidase biocatalyst used in a process disclosed herein is acetyl phosphate, carbon dioxide, and hydrogen peroxide.

[0094] “Alkyl” refers to saturated hydrocarbon groups of from 1 to 18 carbon atoms inclusively, either straight chained or branched, more preferably from 1 to 8 carbon atoms inclusively, and most preferably 1 to 6 carbon atoms inclusively. An alkyl with a specified number of carbon atoms is denoted in parenthesis (e.g., (Ci-C6)alkyl refers to an alkyl of 1 to 6 carbon atoms).

[0095] “Alkenyl” refers to hydrocarbon groups of from 2 to 12 carbon atoms inclusively, either straight or branched containing at least one double bond but optionally containing more than one double bond.

[0096] “Alkynyl” refers to hydrocarbon groups of from 2 to 12 carbon atoms inclusively, either straight or branched containing at least one triple bond but optionally containing more than one triple bond, and additionally optionally containing one or more double bonded moieties.

[0097] “Heteroalkyl, “heteroalkenyl,” and heteroalkynyl,” refer respectively, to alkyl, alkenyl and alkynyl as defined herein in which one or more of the carbon atoms are each independently replaced with the same or different heteroatoms or heteroatomic groups. Heteroatoms and / or heteroatomic groups which can replace the carbon atoms include, but arc not limited to-O-, -S-, -S-O-, -NRY-, -PH-, -S(O)-, -S(O)2-, -S(O) NR'-, -StOjiNR', and the like, including combinations thereof, where each RYis independently selected from hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl.

[0098] “Amino” refers to the group -NH2. Substituted amino refers to the group -NHR’1, NR^R11, and NR^R^R11, where each R11is independently selected from substituted or unsubstituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy, aryl, heteroaryl, heteroarylalkyl, acyl, alkoxycarbonyl, sulfanyl, sulfinyl, sulfonyl, and the like. Typical amino groups include, but are limited to, dimethylamino, diethylamino, trimethylammonium, triethylammonium, methylysulfonylamino, furanyl-oxy- sulfamino, and the like.

[0099] “Aminoalkyl” refers to an alkyl group in which one or more of the hydrogen atoms are replaced with one or more amino groups, including substituted amino groups.

[0100] “Aminocarbonyl” refers to -C(O)NH2. Substituted aminocarbonyl refers to -C(O)NRr|R11, where the amino group NR^R11is as defined herein.

[0101] “Oxy” refers to a divalent group -O-, which may have various substituents to form different oxy groups, including ethers and esters.

[0102] “Alkoxy” or “alkyloxy” are used interchangeably herein to refer to the group -OR1’, wherein R’ is an alkyl group, including optionally substituted alkyl groups.

[0103] “Carboxy” refers to -COOH.

[0104] “Carbonyl” refers to -C(O)-, which may have a variety of substituents to form different carbonyl groups including acids, acid halides, aldehydes, amides, esters, and ketones.

[0105] “Carboxyalkyl” refers to an alkyl in which one or more of the hydrogen atoms are replaced with one or more carboxy groups.

[0106] “Aminocarbonylalkyl” refers to an alkyl substituted with an aminocarbonyl group, as defined herein.

[0107] “Halogen” or “halo” refers to fluoro, chloro, bromo and iodo.

[0108] “Haloalkyl” refers to an alkyl group in which one or more of the hydrogen atoms are replaced with a halogen. Thus, the term “haloalkyl” is meant to include monohaloalkyls, dihaloalkyls, trihaloalkyls, etc. up to perhaloalkyls. For example, the expression “(Ci - C2) haloalkyl” includes 1- fluoromethyl, difluoromethyl, trifluoromethyl, 1 -fluoroethyl, 1,1 -difluoroethyl, 1,2-difluoroethyl, 1,1,1 trifluoroethyl, perfluoroethyl, etc.

[0109] “Hydroxy” refers to -OH.

[0110] “Hydroxyalkyl” refers to an alkyl group in which in which one or more of the hydrogen atoms are replaced with one or more hydroxy groups.

[0111] “Thiol” or “sulfanyl” refers to -SH. Substituted thiol or sulfanyl refers to -S-R'1, where R'1is an alkyl, aryl or other suitable substituent.

[0112] “Sulfonyl” refers to -SO2-. Substituted sulfonyl refers to -SO2-R11, where R11is an alkyl, aryl or other suitable substituent.

[0113] “Alkylsulfonyl" refers to -SCh-RS where R is an alkyl, which can be optionally substituted. Typical alkylsulfonyl groups include, but are not limited to, methylsulfonyl, ethylsulfonyl, n- propylsulfonyl, and the like.

[0114] “Phosphate” as used herein refers to a functional group comprised of an orthophosphate ion (phosphorous atom covalently linked to four oxygen atoms). The orthophosphate ion is commonly found with one or more hydrogen atoms or organic groups. A phosphate group or chain may be modified, as further described herein.

[0115] “Phosphorylated” as used herein refers to the addition or presence of one of more phosphoryl groups (phosphorous atom covalently linked to the three oxygen atoms).

[0116] “thiophosphate” refers to an instance where a non-bridging oxygen in a phosphate group of a phosphodiester bond, NMP, NDP, NTP or NQP is replaced with a sulfur.

[0117] “dithiophosphate” refers to an instance where two non-bridging oxygens in a phosphate group of a phosphodiester bond, NMP, NDP, NTP or NQP are replaced with two sulfurs.

[0118] “Optionally substituted” as used herein with respect to the foregoing chemical groups means that positions of the chemical group occupied by hydrogen can be substituted with another atom (unless otherwise specified) exemplified by, but not limited to carbon, oxygen, nitrogen, or sulfur, or a chemical group, exemplified by, but not limited to, hydroxy, oxo, nitro, methoxy, ethoxy, alkoxy, substituted alkoxy, trifluoromethoxy, haloalkoxy, fluoro, chloro, bromo, iodo, halo, methyl, ethyl, propyl, butyl, alkyl, alkenyl, alkynyl, substituted alkyl, trifluoromethyl, haloalkyl, hydroxyalkyl, alkoxyalkyl, thio, alkylthio, acyl, carboxy, alkoxycarbonyl, carboxamido, substituted carboxamido, alkylsulfonyl, alkylsulfinyl, alkylsulfonylamino, sulfonamido, substituted sulfonamido, cyano, amino,substituted amino, alkylamino, dialkylamino, aminoalkyl, acylamino, amidino, amidoximo, hydroxamoyl, phenyl, aryl, substituted aryl, aryloxy, arylalkyl, arylalkenyl, arylalkynyl, pyridyl, imidazolyl, heteroaryl, substituted heteroaryl, heteroaryloxy, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, substituted cycloalkyl, cycloalkyloxy, pyrrolidinyl, piperidinyl, morpholino, heterocycle, (heterocycle)oxy, and (heterocycle)alkyl; where preferred heteroatoms are oxygen, nitrogen, and sulfur. Additionally, where open valences exist on these substitute chemical groups they can be further substituted with alkyl, cycloalkyl, aryl, heteroaryl, and / or heterocycle groups, that where these open valences exist on carbon they can be further substituted by halogen and by oxygen-, nitrogen-, or sulfur-bonded substituents, and where multiple such open valences exist, these groups can be joined to form a ring, either by direct formation of a bond or by formation of bonds to a new heteroatom, preferably oxygen, nitrogen, or sulfur. It is further contemplated that the above substitutions can be made provided that replacing the hydrogen with the substituent does not introduce unacceptable instability to the molecules of the present invention and is otherwise chemically reasonable. One of ordinary skill in the art would understand that with respect to any chemical group described as optionally substituted, only sterically practical and / or synthetically feasible chemical groups are meant to be included. “Optionally substituted” as used herein refers to all subsequent modifiers in a term or series of chemical groups. For example, in the term "optionally substituted arylalkyl,” the “alkyl” portion and the “aryl” portion of the molecule may or may not be substituted, and for the series “optionally substituted alkyl, cycloalkyl, aryl and heteroaryl,” the alkyl, cycloalkyl, aryl, and heteroaryl groups, independently of the others, may or may not be substituted.

[0119] “Reaction” as used herein refers to a process in which one or more substances or compounds or substrates is converted into one or more different substances, compounds, or processes.Pyruvate Oxidases for Phosphate Donor Recycling

[0120] Pyruvate oxidases with improved properties can be important for commercial uses under industrial process conditions, including use in phosphate donor recycling systems and as auxiliary enzymes in enzyme cascades.

[0121] Biocatalysis, particularly for industrial applications, often utilizes cascades of enzymes with paired reactions. One widespread strategy is to provide auxiliary enzymes to regenerate substrates, which often has the additional advantage of removing an inhibitory side product and / or of shifting the reaction equilibrium to promote conversion of additional substrate to product.

[0122] As a specific example, ATP or NTP recycling is often used to regenerate NTPs for reactions that utilize an NTP as a phosphate donor. Various NTP regenerating systems are known in the art including pyruvate kinase / phosphoenol pyruvate, acetate kinase / acetyl phosphate, and creatine kinase / creatine phosphate (see, e.g., Lian et al., Appl Biochem Biotechnol., 2014, 174:2351-2367). Inacetate kinase based NTP recycling, acetate kinase catalyzes the reversible conversion of an NDP to an NTP and acetate using acetyl phosphate as a secondary phosphate donor. In this reaction, the secondary phosphate donor, acetyl phosphate must be added to the reaction.

[0123] However, use of pyruvate oxidase as a phosphate donor recycling enzyme to generate acetyl phosphate from pyruvate has the advantage of generating an unstable, moisture sensitive, and expensive substrate (acetyl phosphate) from stable, readily available, and inexpensive reagents (pyruvate, atmospheric oxygen, and potassium phosphate buffer).

[0124] By way of example and not limitation, Scheme 1, below, depicts an enzyme cascade that uses pyruvate oxidase to generate acetyl phosphate that is used as a phosphate donor by acetate kinase. The pyruvate oxidase supports all three steps of the primary enzyme cascade. In the first two steps of the cascade, a nucleoside is sequentially phosphorylated by a 5’O-kinase to form a nucleoside monophosphate and then by a nucleoside monophosphate kinase to form a nucleoside diphosphate. The last step of the enzyme cascade is the conversion of the nucleoside diphosphate to a nucleoside triphosphate by the acetate kinase. Each of these steps requires consumption of acetyl phosphate by acetate kinase, either directly or as part of NTP recycling. As depicted in Scheme 1, the acetate kinase enzyme is further coupled with a pyruvate oxidase enzyme to generate acetyl phosphate from pyruvate.Scheme 1

[0125] While Scheme 1 depicts a use of pyruvate oxidase in a biocatalytic cascade to generate nucleoside triphosphates, the improved pyruvate oxidases of the present disclosure are not limited tothis use. A person of skill in the art will immediately envisage a range of potential uses, both as a phosphate donor recycling enzyme for NTPs, as well as other uses.

[0126] In some embodiments, phosphate is present in a phosphate buffer, such as potassium phosphate buffer. In some other embodiments, inorganic phosphate is generated by a paired reaction in an enzyme cascade.

[0127] As the skilled artisan will recognize, the yield of enzyme cascades may be impacted by one or more rate limiting steps, as well as the presence of various substrates, products, by-products, or cofactors present to facilitate a system of several reactions. Similarly, optimization of yield can be accomplished by coordination of temperature, pressure, pH, and other industrial process conditions. Therefore, improved pyruvate oxidases with improved properties in multi-enzyme cascades and / or under industrial process conditions can be useful in a variety of applications.

[0128] Accordingly, the present disclosure provides pyruvate oxidases with improved properties, including, among others, increased activity, increased thermostability, increased activity at high temperatures, and increased solubility or protein expression.

[0129] In some embodiments, the present invention provides an engineered pyruvate oxidase polypeptide comprising an amino acid sequence having at least 70% sequence identity to an amino acid reference sequence of SEQ ID NO: 6 and further comprising one or more amino acid residue differences as compared to the reference amino acid sequence, wherein the engineered pyruvate oxidase polypeptide has increased activity, increased thermostability, increase activity at high temperatures, and increased solubility or protein expression, as compared to a wild-type or reference pyruvate oxidase.

[0130] In particular, the engineered pyruvate oxidase polypeptides of the present disclosure have been engineered for improved properties in multi-enzyme cascades and / or under industrial process conditions.Engineered Pyruvate Oxidase Polypeptides

[0131] The present disclosure provides engineered pyruvate oxidase polypeptides useful for the conversion of pyruvate, phosphate, and oxygen to acetyl phosphate, carbon dioxide, and hydrogen peroxide, as well as compositions and methods of utilizing these engineered polypeptides. Where the description relates to polypeptides, it is to be understood that it can describe the polynucleotides encoding the polypeptides.

[0132] Suitable reaction conditions under which the above-described improved properties of the engineered polypeptides carry out the desired reaction can be determined with respect to concentrations or amounts of polypeptide, substrate, cofactors, buffer, solvent, pH, conditionsincluding temperature and reaction time, and / or conditions with the polypeptide immobilized on a solid support, as further described below and in the Examples.

[0133] In some embodiments, the exemplary engineered pyruvate oxidases comprise an amino acid sequence that has one or more residue differences as compared to a reference sequence of SEQ ID NO: 6, 16, 56, 184, 264, 290, 400, or 696 at the residue positions indicated in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, and 23.2.

[0134] The structure and function information for the exemplary engineered polypeptides of the present invention are based on the conversion of pyruvate, phosphate, and oxygen to acetyl phosphate, carbon dioxide, and hydrogen peroxide, the results of which are shown below in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, and 23.2, as further described in the Examples. The odd numbered sequence identifiers (i.e., SEQ ID NOs) in these Tables refer to the nucleotide sequence encoding the amino acid sequence provided by the even numbered SEQ ID NOs in these Tables. Exemplary sequences are provided in the electronic sequence listing file accompanying this invention, which is hereby incorporated by reference herein. The amino acid residue differences are based on comparison to a reference sequence of SEQ ID NO: 6, 16, 56, 184, 264, 290, 400, or 696.

[0135] Various pyruvate oxidases have been identified in many species. In some embodiments, the pyruvate oxidase is a pyruvate oxidase of Bifidobacterium mongoliense (A0A087C4V4) (SEQ ID NO: 2), Pisciglobus halotolerans (A0A1I3CCM7) (SEQ ID NO: 4), Jeotgalibaca sp PTS2502 (A0A1U7E9W7) (SEQ ID NO: 6), Vagococcus fluvialis (A0A8I2AXT4) (SEQ ID NO: 8), Candidatus Gracilibacteria bacterium (A0A2M7FGE0) (SEQ ID NO: 10), or Bavariicoccus seileri (A0A3D4S346) (SEQ ID NO: 12).

[0136] In some embodiments of the present disclosure, one or more wild-type pyruvate oxidases have improved activity in the conversion of pyruvate to acetyl phosphate, as compared to another wild-type or reference pyruvate oxidase. In some embodiments, the wild-type pyruvate oxidase of SEQ ID NO: 4, 6, 8, 10, or 12, has at least 5-fold improved activity in the conversion of pyruvate to acetyl phosphate, as compared to the reference pyruvate oxidase of SEQ ID NO: 2. In some embodiments, the wild-type pyruvate oxidase of SEQ ID NOs: 6, 8, or 12, has at least 50-fold improved activity in the conversion of pyruvate to acetyl phosphate, as compared to the reference pyruvate oxidase of SEQ ID NO: 2. In some embodiments, the wild-type pyruvate oxidase of SEQ ID NOs: 6 or 8, has at least 250-fold improved activity in the conversion of pyruvate to acetyl phosphate, as compared to the reference pyruvate oxidase of SEQ ID NO: 2.

[0137] The wild-type pyruvate oxidase from Jeotgalibaca sp PTS2502 was selected for evolution. The pyruvate kinase polypeptides of the present disclosure are engineered variants of SEQ ID NO: 6.

[0138] The polypeptides of the present disclosure have residue differences that result in improved properties necessary for commercial applications under industrial process conditions. Various residue differences, at both conserved and non-conserved positions, have been discovered to be related to improvements in various enzymes properties, including, among others, increased activity, increased thermostability, increase activity at high temperatures, and increased solubility or protein expression. The activity, solubility, and or thermostability of each engineered pyruvate oxidase relative to a reference polypeptide was determined as conversion of the substrates described in the Examples herein. In some embodiments, a shake flask purified enzyme (SFP) is used to assess the properties of the pyruvate oxidases, the results of which are provided in the Examples.

[0139] In some embodiments, the specific enzyme properties are associated with the residues differences as compared to SEQ ID NO: 6, 16, 56, 184, 264, 290, 400, or 696, at the residue positions indicated herein. In some embodiments, the amino acid residue difference(s) comprise substitution(s) as compared to the reference sequence. Thus, where appropriate, amino acid differencefs) can be in the form of substitution(s). In some embodiments, amino acid residue differences, e.g., substitutions, affecting polypeptide expression can be used to increase expression of the engineered pyruvate oxidases.

[0140] In light of the guidance provided herein, it is further contemplated that any of the exemplary engineered polypeptides comprising the even-numbered sequences of SEQ ID NOs: 14-636 and 692- 876 find use as the starting amino acid sequence for synthesizing other pyruvate oxidases, for example by subsequent rounds of evolution that incorporate new combinations of various amino acid differences from other polypeptides in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, and 23.2, and other residue positions described herein. Further improvements may be generated by including amino acid differences at residue positions that had been maintained as unchanged throughout earlier rounds of evolution.

[0141] In some embodiments, the engineered pyruvate oxidase comprises a polypeptide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence selected from SEQ ID NO: 6, 16, 56, 184, 264, 290, 400, or 696, or a functional fragment thereof, and one or more amino acid residue differences relative to the reference sequence.

[0142] In some embodiments, a reference sequence, as well as any specified amino acid sequence herein, can be described without the amino acid residues of a His-tag when present. For example, a polypeptide sequence of an engineered pyruvate oxidase comprises residues 8-604 of an engineered pyruvate oxidase referenced by its SEQ ID NO., where the sequence of the SEQ ID NO. includes a His-tag. It is also to be understood that the range of residues can be adapted to account for any amino acid deletions within the sequence of the pyruvate oxidase polypeptide sequence.

[0143] As such, in some embodiments, the present disclosure provides an engineered pyruvate kinase comprising a polypeptide sequence having at least at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 8-604 or 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 6, 14-636, and 692-876, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 6, 14-636, and 692-876, or a functional fragment thereof, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 8-604 or 12-604 of SEQ ID NO: 6, 16, 56, 184, 264, 290, 400, or 696, or relative to the reference sequence corresponding to SEQ ID NO: 6, 16, 56, 184, 264, 290, 400, or 696.

[0144] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, 16, 56, 184, 264, 290, 400, or 696, or to the reference sequence corresponding to SEQ ID NO: 6, 16, 56, 184, 264, 290, 400, or 696, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, 16, 56, 184, 264, 290, 400, or 696, or relative to the reference sequence corresponding to SEQ ID NO: 6, 16, 56, 184, 264, 290, 400, or 696.

[0145] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 14- 636 and 692-876, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 14-636 and 692-876, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, or relative to the reference sequence corresponding to SEQ ID NO: 6.

[0146] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696, or to the reference sequence corresponding to SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, or relative to the reference sequence corresponding to SEQ ID NO: 6.

[0147] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, or to the reference sequence corresponding to SEQ ID NO: 6, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, or relative to the reference sequence corresponding to SEQ ID NO: 6.

[0148] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least amino acid residue difference at amino acid position 14, 15, 21, 31, 32, 45, 54, 57, 61, 62, 74, 108, 117, 127, 134, 135, 139, 150, 153, 161, 163, 167, 168, 177, 183, 187, 192, 193, 197, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 214, 215, 217, 218, 220, 226,228, 233, 235, 237, 239, 242, 244, 250, 259, 302, 307, 308, 314, 317, 325, 326, 331, 336, 342, 345,351, 357, 367, 374, 377, 380, 385, 389, 393, 404, 409, 417, 432, 458, 469, 471, 480, 482, 496, 504,506, 510, 511, 512, 515, 521, 523, 524, 527, 528, 530, 536, 555, 561, 563, 565, 567, 572, 575, 576,579, 580, 585, 587, 591, 594, or 604, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, or relative to the reference sequence corresponding to SEQ ID NO: 6.

[0149] In some embodiments, the engineered pyruvate oxidase polypeptide comprises a polypeptide sequence having amino acid residue difference or amino acid residue 14A, 15R / S, 21A, 31A / G / K / S, 321, 45Y, 54V, 57A / H, 611, 62K, 74G, 108S, 117A, 127T, 134G / V, 135 A / F, 139P, 150S, 153A / T, 161V, 163H / V, 167R / S, 168H / K, 177A, 183C / M / P / S, 187V, 192S, 193A, 197K, 199H, 200D / PR, 201G / P / Q / W, 202A / P, 203C / R / Y, 204C / R / T, 205R, 206A / G / H / Q / V / Y, 2071, 208S, 209R, 210A / L / R, 214C / RR / V, 215PM, 217R, 218H, 220K, 226C, 228V, 233A, 235R, 237L, 239N, 242E / H, 244L, 250V, 259S, 302V, 307F, 3O8K, 314R, 317G / R, 325P, 326S / V, 331A, 336T, 342S, 345W, 351H / K / P / R / S, 357L, 367C, 374C, 377P, 380V, 385R, 389R, 393S, 404P, 409L / V, 417R, 432G, 458G / Q, 469W, 471R, 480N, 482S, 496L, 504C / P / V, 506A / C, 510K, 511V, 512A, 515L, 521Q, 523A / R / T, 524G, 527H / L / M, 528L, 530R, 536L / T, 555L, 561A, 563Q, 565E, 567Q / R / V, 572G / P / T, 575A / F, 576S, 579I / L / N / S / T, 580C / R / T, 585G, 587R / K, 591V, 594A / D / L / Q / S / V, or 604S, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, or relative to the reference sequence corresponding to SEQ ID NO: 6.

[0150] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence comprising at least amino acid residue difference at amino acid position 134, 153, 168, 192, 197, 242, 308, 317, 325, 331, 336, 469, 504, 510, 512, or 567, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12- 604 of SEQ ID NO: 6, or relative to the reference sequence corresponding to SEQ ID NO: 6.

[0151] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence comprising at least amino acid residue difference or amino acid residue 134G / V, 153A / T, 168H / K, 192S, 197K, 242E / H, 308K, 317G / R, 325P, 331A / S, 336T, 469W, 504V, 510K, 512A, or 567Q / R / V, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, or relative to the reference sequence corresponding to SEQ ID NO: 6.

[0152] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence comprising at least amino acid residue difference or amino acid residue 134V, 153T, 168K, 192S, 197K, 242E, 3O8K, 317R, 325P, 331A, 336T, 469W, 504P, 510K, 512A, 512A, or 567R, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, or relative to the reference sequence corresponding to SEQ ID NO: 6.

[0153] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence comprising at least amino acid residue difference, or amino acid residue N134V, L153T, R168K, T192S, R197K, R242E, N3O8K, T317R, A325P, G331A, A336T, R469W, T504P, M510K, G512A, or S567R, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, or relative to the reference sequence corresponding to SEQ ID NO: 6.

[0154] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, or to the reference sequence corresponding to SEQ ID NO: 6, and at least amino acid residue difference as compared to amino acid residues 12-604 of SEQ ID NO: 6 or compared to SEQ ID NO: 6, at amino acid position 61, 168, 202, 233, 242, 244, 308, 336, 480, 510, 512, or 528, or combinations thereof. In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, or to the reference sequence corresponding to SEQ ID NO: 6, and at least amino acid residue difference as compared to amino acid residues 12-604 of SEQ ID NO: 6 or compared to SEQ ID NO: 6, or amino acid residue 611, 168K, 202P, 233A, 242E, 244L, 3O8K, 336T, 480N, 510K, 512A, or 528L, or combinations thereof. In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 6 and one or more residue differences as compared to amino acid residues 12-604 of SEQ ID NO: 6 or compared to SEQ ID NO:6, or amino acid residue L61I, R168K, S202P, H233A, R242E, I244L, N3O8K, A336T, T480N, M510K, G512A, or I528L, or combinations thereof. In the above embodiments, the engineered pyruvate oxidase polypeptide may additionally comprise improved conversion of pyruvate to acetyl phosphate, as compared to a reference sequence of SEQ ID NO: 6.

[0155] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, or to the reference sequence corresponding to SEQ ID NO: 6, and at least amino acid residue difference as compared to amino acid residues 12-604 of SEQ ID NO: 6 or compared to SEQ ID NO: 6, at amino acid position 168, 220, 242, 308, 377, 432, or 512, or combinations thereof. In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, or to the reference sequence corresponding to SEQ ID NO: 6, and at least amino acid residue difference as compared to amino acid residues 12-604 of SEQ ID NO: 6 or compared to SEQ ID NO: 6, or amino acid residue 168K, 220K, 242E, 308K, 377P, 432G, or 512A, or combinations thereof. In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, or to the reference sequence corresponding to SEQ ID NO: 6, and at least amino acid residue difference as compared to amino acid residues 12-604 of SEQ ID NO: 6 or compared to SEQ ID NO: 6, or amino acid residue R168K, D220K, R242E, N308K, D377P, A432G, or G512 A, or combinations thereof. In the above embodiments, the engineered pyruvate oxidase polypeptide may additionally comprise improved conversion of pyruvate to acetyl phosphate, as compared to a reference sequence of SEQ ID NO: 6.

[0156] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, or to the reference sequence corresponding to SEQ ID NO: 6, and at least amino acid residue difference(s) as compared to amino acid residues 12-604 of SEQ ID NO: 6 or compared to SEQ ID NO: 6, at amino acid position(s) 54, 54 / 57, 54 / 57 / 168, 54 / 57 / 168 / 482 / 510 / 512, 54 / 57 / 336, 54 / 57 / 482, 54 / 57 / 510, 54 / 57 / 510 / 512, 54 / 168, 54 / 168 / 336 / 512, 54 / 168 / 482 / 510, 54 / 168 / 510, 54 / 168 / 510 / 512, 54 / 242 / 482 / 512, 54 / 336 / 510, 54 / 336 / 512, 54 / 482, 54 / 482 / 512 / 528, 54 / 510, 54 / 512 / 528, 57, 57 / 168 / 482 / 510 / 512, 57 / 336 / 512,57 / 482, 57 / 510, 57 / 512, 57 / 512 / 528, 61 / 202 / 308 / 469 / 536, 61 / 244, 61 / 308 / 469, 61 / 308 / 471 / 536, 61 / 471 / 536, 168 / 242 / 336 / 510 / 512, 168 / 242 / 482 / 512 / 528, 168 / 512, 202 / 244 / 308, 202 / 244 / 308 / 471, 202 / 244 / 536, 202 / 308 / 432 / 536, 202 / 308 / 469 / 471, 202 / 308 / 469 / 536, 202 / 308 / 536, 202 / 471 / 536, 202 / 536, 242 / 512 / 528, 244 / 308 / 469, 308 / 469 / 471, 336 / 482, 336 / 482 / 510 / 512, 469, 482, 482 / 510, 482 / 510 / 512 / 528, or 536. In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, to the reference sequence corresponding to SEQ ID NO: 6, and amino acid residue difference(s) as compared to amino acid residues 12-604 of SEQ ID NO: 6 or compared to SEQ ID NO: 6, or amino acid residue(s) 54V, 54V / 57A, 54V / 57A / 168K, 54V / 57A / 168K / 482S / 510K / 512A, 54V / 57A / 336T, 54V / 57A / 482S, 54V / 57A / 510K, 54V / 57A / 510K / 512A, 54V / 168K, 54V / 168K / 336T / 512A, 54V / 168K / 482S / 510K, 54V / 168K / 510K, 54V / 168K / 510K / 512A, 54V / 242E / 482S / 512A, 54V / 336T / 510K, 54V / 336T / 512A, 54V / 482S, 54V / 482S / 512A / 528L, 54V / 510K, 54V / 512A / 528L, 57 A, 57A / 168K / 482S / 510K / 512A, 57A / 336T / 512A, 57A / 482S, 57A / 510K, 57A / 512A, 57A / 512A / 528L, 57H, 61E202P / 308K / 469W / 536T, 61E244L, 61E308K / 469W, 61I7308K / 471R / 536L, 61U471R / 536L, 168K / 242E / 336T / 510K / 512A, 168K / 242E / 482S / 512A / 528L, 168K / 512A, 202P / 244L / 308K, 202P / 244L / 308K / 471R, 202P / 244L / 536L, 202P / 308K / 432G / 536L, 202P / 308K / 469W / 471R, 202P / 308K / 469W / 536T, 202P / 308K / 536L, 202P / 471R / 536L, 202P / 536L, 242H / 512A / 528L, 244L / 308K / 469W, 308K / 469W / 471R, 336T / 482S, 336T / 482S / 510K / 512A, 469W, 482S, 482S / 510K, 482S / 510K / 512A / 528L, or 536L. In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, or to the reference sequence corresponding to SEQ ID NO: 6, and at least amino acid residue difference(s) as compared to amino acid residues 12-604 of SEQ ID NO: 6 or compared to SEQ ID NO: 6, or amino acid residue(s) E54V, E54V / D57A, E54V / D57A / R168K, E54V / D57A / R168K / T482S / M510K / G512A, E54V / D57A / A336T, E54V / D57A / T482S, E54V / D57A / M510K, E54V / D57A / M510K / G512A, E54V / R168K, E54V / R168K / A336T / G512A, E54V / R168K / T482S / M510K, E54V / R168K / M510K, E54V / R168K / M510K / G512A, E54V / R242E / T482S / G512A, E54V / A336T / M510K, E54V / A336T / G512A, E54V / T482S, E54V / T482S / G512A / I528L, E54V / M510K, E54V / G512A / I528L, D57A, D57A / R168K / T482S / M510K / G512A, D57A / A336T / G512A, D57A / T482S, D57A / M510K, D57A / G512A, D57A / G512A / I528L, D57H, L611 / S202P / N308K / R469W / V536T, L61I / I244L, L61I / N308K / R469W, L61I / N308K / N471R / V536L, L61I / N471R / V536L,R168K / R242E / A336T / M510K / G512A, R168K / R242E / T482S / G512A / I528L, R168K / G512A, S202P / I244L / N308K, S202P / I244L / N308K / N471R, S202P / I244L / V536L,S202P / N308K / A432G / V536L, S202P / N308K / R469W / N471R, S202P / N308K / R469W / V536T, S202P / N308K / V536L, S202P / N471R / V536L, S202P / V536L, R242H / G512A / I528L, I244L / N308K / R469W, N308K / R469W / N471R, A336T / T482S, A336T7T482S / M510K / G512A, R469W, T482S, T482S / M510K, T482S / M510K / G512A / I528L, or V536L. In the above embodiments, the engineered pyruvate oxidase polypeptide may additionally comprise improved conversion of pyruvate to acetyl phosphate, as compared to a reference sequence of SEQ ID NO: 6.

[0157] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696, or to the reference sequence corresponding to SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696, or relative to the reference sequence corresponding to SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696.

[0158] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 14- 636 and 692-876, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 14-636 and 692-876, or a functional fragment thereof, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696, or relative to the reference sequence corresponding to SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696.

[0159] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence comprising at least amino acid residue difference at amino acid position 14, 15, 21, 31, 32, 45, 54, 57, 61, 62, 74, 108, 117, 127, 134, 135, 139, 150, 153, 161, 163, 167, 168, 177, 183, 187, 192, 193, 197, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 214, 215, 217, 218, 220, 226,228, 233, 235, 237, 239, 242, 244, 250, 259, 302, 307, 308, 314, 317, 325, 326, 331, 336, 342, 345,351, 357, 367, 374, 377, 380, 385, 389, 393, 404, 409, 417, 432, 458, 469, 471, 480, 482, 496, 504,506, 510, 511, 512, 515, 521, 523, 524, 527, 528, 530, 536, 555, 561, 563, 565, 567, 572, 575, 576,579, 580, 585, 587, 591, 594, or 604, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696, or relative to the reference sequence corresponding to SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696..

[0160] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence comprising at least amino acid residue difference, or amino acid residue 14A, 15R / S, 21 A, 31A / G / K / S, 321, 45Y, 54V, 57A / H, 611, 62K, 74G, 108S, 117A, 127T, 134G / V, 135A / F, 139P, 150S, 153A / T, 161V, 163H / V, 167R / S, 168H / K, 177A, 183C / M / P / S, 187V, 192S, 193A, 197K, 199H, 200D / I / R, 201G / P / Q / W, 202A / P, 203C / R / Y, 204C / R / T, 205R, 206A / G / H / Q / V / Y, 2071, 208S, 209R, 210A / L / R, 214C / I / R / V, 2151 / M, 217R, 218H, 220K, 226C, 228V, 233A, 235R, 237L, 239N, 242E / H, 244L, 250V, 259S, 302V, 307F, 3O8K, 314R, 317G / R, 325P, 326S / V, 331A, 336T, 342S, 345W, 351H / K / P / R / S, 357L, 367C, 374C, 377P, 380V, 385R, 389R, 393S, 404P, 409L / V, 417R, 432G, 458G / Q, 469W, 471R, 480N, 482S, 496L, 504C / P / V, 506A / C, 510K, 511V, 512A, 515L, 521Q, 523A / R / T, 524G, 527H / L / M, 528L, 530R, 536L / T, 555L, 561A, 563Q, 565E, 567Q / R / V, 572G / P / T, 575A / F, 576S, 579I / L / N / S / T, 580C / R / T, 585G, 587R / K, 591V, 594A / D / L / Q / S / V, or 604S, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696, or relative to the reference sequence corresponding to SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696.

[0161] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence comprising at least amino acid residue difference at amino acid position 134, 153, 168, 192, 197, 242, 308, 317, 325, 331, 336, 469, 504, 510, 512, or 567, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12- 604 of SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696, or relative to the reference sequence corresponding to SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696.

[0162] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence comprising at least amino acid residue difference, or amino acid residue 134G / V, 153A / T, 168H / K, 192S, 197K, 242E / H, 308K, 317G / R, 325P, 331A / S, 336T, 469W, 504V, 510K, 512A, or 567Q / R / V, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696, or relative to the reference sequence corresponding to SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696.

[0163] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 56, or to the reference sequence corresponding to SEQ ID NO: 56, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 56, or relative to the reference sequence corresponding to SEQ ID NO: 56.

[0164] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 154-178, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 154-178, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 56, or relative to the reference sequence corresponding to SEQ ID NO: 56.

[0165] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 56, or to the reference sequence corresponding to SEQ ID NO: 56, and at amino acid residue difference as compared to amino acid residues 12-604 of SEQ ID NO: 56 or compared to SEQ ID NO: 6, at amino acid position 14, 199, 201, 202, 215, 218, 496, 575, or 580, or combinations thereof. In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 56, or to the reference sequence corresponding to SEQ ID NO: 56, and at least amino acid residue difference as compared to SEQ ID NO: 56 or compared to SEQ ID NO: 56, or amino acid residue 14A, 199H, 201G, 201P, 202A, 2151, 215L, 215M, 218H, 496L, 496S, 575A, or 580R, or combinations thereof. In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 56, or to the reference sequence corresponding to SEQ ID NO: 56, and at least amino acid residue difference as compared to SEQ ID NO: 56, or amino acid residue D14A, Y199H, S201G, S201P, S202A, V215I, V215L, V215M, N218H, K496L, K496S, K575A, or K580R, or combinations thereof. In the above embodiments, the engineered pyruvate oxidase polypeptide may additionally comprise improved conversion of pyruvate to acetyl phosphate, as compared to a reference sequence of SEQ ID NO: 56.

[0166] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 16, or to the reference sequence corresponding to SEQ ID NO: 16, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acidresidues 12-604 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0167] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 180-248, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 180-248, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0168] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 16, or to the reference sequence corresponding to SEQ ID NO: 16, and at least amino acid residue difference as compared to amino acid residues 12-604 of SEQ ID NO: 16 or compared to SEQ ID NO: 16, at amino acid position 21, 183, 193, 201, 203, 204, 205, 206, 207, 209, 214, 235, 307, 325, 345, 374, 393, 510, 527, 530, 561, 563, 565, 580, 594, or 604, or combinations thereof. In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 16, or to the reference sequence corresponding to SEQ ID NO: 16, and at least amino acid residue difference as compared to SEQ ID NO: 16 or compared to SEQ ID NO: 16, or amino acid residue 21A, 183S, 193A, 201Q, 201W, 203C, 203L, 204C, 204R, 204T, 205R, 206H, 206V, 206Y, 2071, 209R, 214C, 2141, 214R, 214V, 235R, 307F, 325P, 345W, 374C, 393S, 510K, 527H, 530R, 561A, 563Q, 565E, 580C, 594Q, or 604S, or combinations thereof. In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 16, or to the reference sequence corresponding to SEQ ID NO: 16, and at least amino acid residue difference as compared to SEQ ID NO: 16 or compared to SEQ ID NO: 16, or an amino acid residue T21 A, E183S, G193A, S201 Q, S201W, A203C, A203L, I204C, I204R, I204T, N205R, E206H, E206V, E206Y, A207I, I209R, E214C, E214I, E214R, E214V, P235R, K307F, A325P, T345W, E374C, E393S, M510K, D527H, Q530R, T561A, K563Q, D565E, K580C, K594Q, or K604S, or combinations thereof. In the above embodiments, the engineeredpyruvate oxidase polypeptide may additionally comprise improved conversion of pyruvate to acetyl phosphate, as compared to a reference sequence of SEQ ID NO: 16.

[0169] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 184, or to the reference sequence corresponding to SEQ ID NO: 184, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 184, or relative to the reference sequence corresponding to SEQ ID NO: 184.

[0170] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 250-288, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 250-288, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 184, or relative to the reference sequence corresponding to SEQ ID NO: 184.

[0171] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 184, or to the reference sequence corresponding to SEQ ID NO: 184. and at least amino acid residue difference(s) as compared to amino acid residues 12-604 of SEQ ID NO: 184 or compared to SEQ ID NO: 184, at amino acid position(s) 177, 177 / 209, 177 / 209 / 336, 177 / 209 / 563, 177 / 336, 177 / 336 / 471, 177 / 336 / 563, 177 / 563, 206 / 308, 206 / 308 / 469, 206 / 308 / 536 / 591, 206 / 469, 308, 308 / 469, 308 / 469 / 536, 336 / 471, 336 / 563, 469, 469 / 536, or 563. In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 184, or to the reference sequence corresponding to SEQ ID NO: 184, and at least amino acid residue difference(s) as compared to amino acid residues 12-604 of SEQ ID NO: 184 or compared to SEQ ID NO: 184, or amino acid residue(s) 177A, 177A / 209R, 177A / 209R / 336T, 177A / 209R / 563Q, 177A / 336T, 177A / 336T / 471R, 177A / 336T / 563Q, 177A / 563Q, 206H / 308K, 206H / 308K / 469W, 206H / 308K / 536T / 591V, 206H / 469W, 308K, 308K / 469W, 308K / 469W / 536T, 336T / 471R, 336T / 563Q, 469W, 469W / 536T, or 563Q. In some embodiments, the engineered pyruvate oxidasepolypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 184, or to the reference sequence corresponding to SEQ ID NO: 184, and at least amino acid residue difference(s) as compared to amino acid residues 12-604 of SEQ ID NO: 184 or compared to SEQ ID NO: 184, or amino acid residue(s) G177A, G177A / I209R, G177A / I209R / A336T, G177A / I209R / K563Q, G177A / A336T, G177A / A336T / N471R, G177A / A336T / K563Q, G I 77A / K563Q, E206H / N308K, E206H / N308K / R469W, E206H / N308K / V536T / E591V, E206H / R469W, N308K, N308K7R469W, N308K / R469W / V536T, A336T / N471R, A336T / K563Q, R469W, R469W / V536T, or K563Q. In the above embodiments, the engineered pyruvate oxidase polypeptide may additionally comprise improved conversion of pyruvate to acetyl phosphate, as compared to a reference sequence of SEQ ID NO: 184.

[0172] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 264, or to the reference sequence corresponding to SEQ ID NO: 264, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 264, or relative to the reference sequence corresponding to SEQ ID NO: 264.

[0173] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 290-398, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 290-398, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 264, or relative to the reference sequence corresponding to SEQ ID NO: 264.

[0174] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 264, or to the reference sequence corresponding to SEQ ID NO: 264, and at least amino acid residue difference as compared to amino acid residues 12-604 of SEQ ID NO: 264 or compared to SEQ ID NO: 264, at amino acid position 135, 153, 161, or 504, or combinations thereof. In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 264, or to the reference sequence corresponding to SEQ ID NO: 264, and at least amino acid residue difference as compared to amino acid residues 12-604 of SEQ ID NO: 264 or compared to SEQ ID NO: 264, or amino acid residues 135A, 135L, 153A, 161V, or 504P, or combinations thereof. In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 264, or to the reference sequence corresponding to SEQ ID NO: 264, and at least amino acid residue difference as compared to amino acid residues 12-604 of SEQ ID NO: 264 or compared to SEQ ID NO: 264, or amino acid residue P135A, P135L, L153A, I161V, or T504P, or combinations thereof. In the above embodiments, the engineered pyruvate oxidase polypeptide may additionally comprise improved conversion of pyruvate to acetyl phosphate, as compared to a reference sequence of SEQ ID NO: 264.

[0175] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 264, or to the reference sequence corresponding to SEQ ID NO: 264, and at least amino acid residue difference as compared to amino acid residues 12-604 of SEQ ID NO: 264 or compared to SEQ ID NO: 264, at amino acid position 32, 127, 134, 135, 153, 163, 192, 197, 200, 317, 331, 385, 504, or 567, or combinations thereof. In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 264, or to the reference sequence corresponding to SEQ ID NO: 264, and at least amino acid residue difference as compared to amino acid residues 12-604 of SEQ ID NO: 264 or compared to SEQ ID NO: 264, or amino acid residue 321, 127T, 134G, 134V, 135A, 153T, 163H, 163V, 192S, 197K, 2001, 317G, 317R, 331A, 385R, 504V, 567R, or 567V, or combinations thereof. In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 264, or to the reference sequence corresponding to SEQ ID NO: 264, and at least amino acid residue difference as compared to SEQ ID NO: 264, or amino acid residue V32I, A127T, N134G, N134V, P135A, L153T, T163H, T163V, T192S, R197K, V200I, T317G, T317R, G331A, N385R, T504V, S567R, or S567V, or combinations thereof. In the above embodiments, theengineered pyruvate oxidase polypeptide may additionally comprise improved conversion of pyruvate to acetyl phosphate, as compared to a reference sequence of SEQ ID NO: 264.

[0176] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 264, or to the reference sequence corresponding to SEQ ID NO: 264, and at least amino acid residue difference as compared to amino acid residues 12-604 of SEQ ID NO: 264 or compared to SEQ ID NO: 264, at amino acid position 32, 74, 108, 117, 127, 134, 135, 139, 150, 153, 163, 177, 192, 197, 200, 226, 237, 250, 259, 302, 314, 317, 331, 385, 404, 409, 458, 504, 506, 511, 515, 523, or 567, or combinations thereof. In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 264, or to the reference sequence corresponding to SEQ ID NO: 264, and at least amino acid residue difference as compared to amino acid residues 12-604 of SEQ ID NO: 264 or compared to SEQ ID NO: 264, or amino acid residue 321, 74G, 108S, 117A, 127T, 134V, 135F, 139P, 150S, 153T, 163H, 163V, 177L, 192S, 197K, 200D, 2001, 200L, 200R, 226C, 237L, 250V, 259S, 302V, 314R, 317G, 317R, 331A, 331S, 385R, 404P, 409L, 409V, 458G, 458Q, 504C, 504S, 506A, 506C, 506L, 511V, 515L, 523A, 523R, 523T, 567Q, 567R, or 567V, or combinations thereof. In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to amino acid residues 12-603 of SEQ ID NO: 264, or to the reference sequence corresponding to SEQ ID NO: 264, and at least amino acid residue difference as compared to amino acid residues 12-604 of SEQ ID NO: 264 or compared to SEQ ID NO 264, or amino acid residue V32I, V74G, N108S, SI 17A, A127T, N134V, P135F, N139P, A150S, L153T, T163H, T163V, G177L, T192S, R197K, V200D, V200I, V200L, V200R, A226C, V237L, T250V, E259S, T302V, N314R, T317G, T317R, G331A, G331S, N385R, S404P, I409L, I409V, N458G, N458Q, T504C, T504S, V506A, V506C, V506L, I511V, Q515L, S523A, S523R, S523T, S567Q, S567R, or S567V, or combinations thereof. In the above embodiments, the engineered pyruvate oxidase polypeptide may additionally comprise improved conversion of pyruvate to acetyl phosphate, as compared to a reference sequence of SEQ ID NO: 264.

[0177] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 290, or to the referencesequence corresponding to SEQ ID NO: 290, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 290, or relative to the reference sequence corresponding to SEQ ID NO: 290.

[0178] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 400-636, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 400-636, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 290, or relative to the reference sequence corresponding to SEQ ID NO: 290.

[0179] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 290, or to the reference sequence corresponding to SEQ ID NO: 290, and at least amino acid residue difference(s) as compared to amino acid residues 12-604 of SEQ ID NO: 290 or compared to SEQ ID NO: 290, at amino acid position(s) 32 / 134 / 135 / 523 / 567, 32 / 134 / 139 / 163 / 250 / 331 / 523, 32 / 134 / 163 / 409, 32 / 134 / 331 / 567, 32 / 139 / 163 / 250, 32 / 139 / 163 / 409, 32 / 139 / 163 / 567, 32 / 139 / 250 / 567, 32 / 139 / 409, 32 / 163, 32 / 163 / 250, 32 / 163 / 250 / 567, 32 / 163 / 331, 32 / 163 / 331 / 409 / 523 / 567, 32 / 163 / 331 / 409 / 567, 32 / 163 / 409 / 567, 32 / 163 / 523 / 567, 32 / 163 / 567, 32 / 250, 32 / 250 / 409, 32 / 250 / 523 / 567, 32 / 250 / 567, 32 / 331 / 567, 32 / 409, 32 / 409 / 567, 32 / 567, 127, 127 / 153, 127 / 153 / 192 / 197, 127 / 153 / 192 / 197 / 200 / 317, 127 / 153 / 192 / 200 / 317 / 385, 127 / 153 / 192 / 504, 127 / 153 / 201 / 385, 127 / 153 / 317 / 385, 127 / 153 / 317 / 567, 127 / 192 / 197 / 200 / 385 / 504, 127 / 192 / 197 / 200 / 504, 127 / 192 / 197 / 317 / 504 / 567, 127 / 192 / 197 / 385 / 504, 127 / 192 / 197 / 504, 127 / 192 / 317 / 385 / 504, 127 / 192 / 385 / 504 / 567, 127 / 197 / 200 / 317, 127 / 200, 127 / 200 / 317 / 567, 127 / 317, 127 / 385, 127 / 385 / 567, 134 / 135 / 163 / 567, 134 / 163 / 331 / 409 / 567, 134 / 250, 134 / 409 / 567, 139, 139 / 163, 139 / 163 / 250 / 331 / 523, 139 / 163 / 331 / 523, 139 / 163 / 567, 139 / 523 / 567, 153, 153 / 192 / 197 / 200 / 385 / 567, 153 / 192 / 197 / 200 / 504, 153 / 192 / 197 / 317 / 504, 153 / 192 / 197 / 317 / 567, 153 / 192 / 200 / 504, 153 / 192 / 317 / 385, 153 / 197 / 504 / 567, 153 / 200, 153 / 317, 153 / 317 / 385, 153 / 317 / 504, 153 / 317 / 504 / 567, 153 / 385, 153 / 504, 153 / 504 / 567, 163, 163 / 250, 163 / 250 / 567, 163 / 331, 163 / 331 / 409, 163 / 331 / 409 / 567, 163 / 331 / 567, 163 / 409, 163 / 523 / 567, 163 / 567, 192 / 197 / 200, 192 / 197 / 200 / 317 / 567, 192 / 197 / 200 / 385, 192 / 197 / 200 / 385 / 567, 192 / 197 / 317, 192 / 197 / 385 / 504 / 567, 192 / 197 / 504, 192 / 200, 192 / 200 / 385, 192 / 317, 192 / 504, 197 / 200 / 567, 197 / 317, 197 / 317 / 385 / 504 / 567, 200 / 317 / 385, 200 / 385 / 567, 250, 250 / 331, 250 / 331 / 567, 250 / 523, 250 / 567, 317, 317 / 385 / 567, 317 / 567, 331 / 409, 331 / 567, 385, 385 / 567, 409 / 523, 523, 523 / 567, or 567. In some embodiments, the engineeredpyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 290, or to the reference sequence corresponding to SEQ ID NO: 290, and at least amino acid residue difference(s) as compared to amino acid residues 12-604 of SEQ ID NO: 290 or compared to SEQ ID NO: 290, or amino acid residue(s) 321 / 134 V7135A / 523R / 567V, 32I / 134V / 139P / 163H / 250V / 331A / 523R, 321 / 134 V / 163H / 409L, 32I / 134V / 331A / 567V, 32I / 139P / 163H / 250V, 32I / 139P / 163H / 409L, 32I / 139P / 163H / 567V, 321 / 139P / 250V / 567V, 321 / 139P / 409L, 32I / 163H, 32I / 163H / 331A, 32I / 163H / 331A / 409L / 567V, 32I / 163H / 409L / 567V, 321 / 163H / 523R / 567V, 32I / 163V, 32I / 163V / 250V, 32I / 163V / 250V / 567V, 32I / 163V / 331A / 409L / 523R / 567V, 32I / 163V / 567V, 32I / 250V, 32I / 250V / 409L, 32I / 250V / 523R / 567V, 32I / 250V / 567V, 321 / 331A / 567V, 32I / 409L, 32I / 409L / 567V, 32I / 567V, 127T, 127T / 153T, 127T / 153T / 192S / 197K, 127T / 153T / 192S / 197K / 200I / 317R, 127T / 153T / 192S / 200I / 317R / 385R, 127T / 153T / 192S / 504V, 127T / 153T / 201G / 385R, 127T / 153T / 317R / 385R, 127T / 153T / 317R / 567R, 127T / 192S / 197K / 200I / 385R / 504V, 127T / 192S / 197K / 200I / 504V, 127T / 192S / 197K / 317R / 504V / 567R, 127T / 192S / 197K / 385R / 504V, 127T / 192S / 197K / 504V, 127T / 192S / 317R / 385R / 504V, 127T / 192S / 385R / 504V / 567R, 127T / 197K / 200I / 317R, 127T / 200I, 127T / 200I / 317R / 567R, 127T / 317R, 127T / 385R, 127T / 385R / 567R, 134V / 135A / 163H / 567V, 134V / 163H / 331A / 409L / 567V, 134V / 250V, 134V / 409L / 567V, 139P, 139P / 163H, 139P / 163H / 250V / 331A / 523R, 139P / 163H / 331A / 523R,139P / 163 V / 567 V, 139P / 523R / 567V, 153T, 153T / 192S / 197K / 200I7385R / 567R, 153T / 192S / 197K / 200I / 504V, 153T / 192S / 197K / 317R / 504V, 153T / 192S / 197K / 317R / 567R, 153T / 192S / 200I / 504V, 153T / 192S / 317R / 385R, 153T / 197K / 504V / 567R, 153T / 200I, 153T / 317R, 153T / 317R / 385R, 153T / 317R / 504V, 153T / 317R / 504V / 567R, 153T / 385R, 153T / 504V, 153T / 504V / 567R, 163H / 331A, 163H / 331A / 409L, 163H / 331A / 567V, 163H / 409L, 163V, 163V / 250V, 163V / 250V / 567V, 163V / 331A, 163 V / 331A / 409L / 567V, 163V / 409L, 163V / 523R / 567V, 163V / 567V, 192S / 197K / 200I, 192S / 197K / 200I / 317R / 567R, 192S / 197K / 200I / 385R, 192S / 197K / 200I / 385R / 567R, 192S / 197K / 317R, 192S / 197K / 385R / 504V / 567R, 192S / 197K / 504V, 192S / 200I, 192S / 200I / 385R, 192S / 317R, 192S / 504V, 197K / 200I / 567R, 197K / 317R, 197K / 317R / 385R / 504V / 567R,2001 / 317R / 385R, 200I / 385R / 567R, 250V, 250V / 331A, 250V / 331A / 567V, 250V / 523R, 250V / 567V, 317R, 317R / 385R / 567R, 317R / 567R, 331A / 409L, 331A / 567V, 385R, 385R / 567R, 409L / 523R, 523R, 523R / 567V, or 567V. In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 290, or to the reference sequence corresponding to SEQ ID NO: 290, and at least amino acid residue difference(s) as compared to amino acid residues 12-604 of SEQ ID NO: 290 or compared to SEQ ID NO: 290, oramino acid residue(s) V32PN134V / P135A / S523R / S567V,V32PN134V / N139P / T163H / T250V / G331A / S523R, V32UN134WT163H / I409L, V32I / N134V / G331A / S567V, V32I / N139P / T163H / T250V, V32I / N139P / T163H / I409L, V32I / N139P / T163H / S567V, V32I / N139P / T250V / S567V, V32UN139P / I409L, V32I / T163H, V32I / T163H / G331A, V32I / T163H / G331A / I409L / S567V, V32I / T163H / I409L / S567V, V32I / T163H / S523R / S567V, V32I / T163V, V32PT163V / T250V, V32I / T163V / T250V / S567V, V32I / T163V / G331A / I409L / S523R / S567V, V32I / T163V / S567V, V32I / T250V, V32I / T250V / I409L, V32PT250V / S523R / S567V, V32I / T250V / S567V, V32I / G331A / S567V, V32I / I409L,V32PI409L / S567V, V32I / S567V, A127T, A127T / L153T, A127T / L153T / T192S / R197K, A127T / L153T / T192S / R197K / V200I / T317R, A127T / L153T / T192S / V200I / T317R / N385R, A127T / L153T / T192S / P5O4V, A127T / L153T / S201G / N385R, A127T / L153T / T317R / N385R, A127T7L153T / T317R / S567R, A127T / T192S / R197K / V200I / N385R / P504V,A127T / T192S / R197K / V200I / P504V, A127T / T192S / R197K / T317R / P5O4V / S567R, A127T / T192S / R197K / N385R / P5O4V, A127T / T192S / R197K / P504V,A127T / T192S / T317R / N385R / P5O4V, A127T / T192S / N385R / P504V / S567R, A127T / R197K / V200I / T317R, A127T / V200I, A127T / V200I / T 17R / S567R, A127T / T317R,A127T / N385R, A127T / N385R / S567R, N134V / P135A / T163H / S567V,N134V / T163H / G331A / I409L / S567V, N134V / T250V, N134V / I409L / S567V, N139P, N139P / T163H, N139P / T163H / T250V / G331A / S523R, N139P / T163H / G331A / S523R, N139P / T163V / S567V, N139P / S523R / S567V, L153T, L153T / T192S / R197K / V200PN385R / S567R, L153T / T192S / R197K / V200I / P504V, L153T / T192S / R197K / T317R / P504V,L153T / T192S / R197K / T317R / S567R, L153T / T192S / V200I7P504V, L153T / T192S / T317R / N385R, L153T / R197K / P504V / S567R, L153T / V200I, L153T / T317R, L153T / T317R / N385R, L153T / T317R / P504V, L153T / T317R / P504V / S567R, L153T / N385R, L153T / P5O4V, L153T / P5O4V / S567R, T163H / G331A, T163H / G331A / I409L, T163H / G331A / S567V, T163H / I409L, T163V, T163V / T250V, T163V / T250V / S567V, T163V / G331A, T163V / G331A / I4O9L / S567V, T163V / I409L, T163V / S523R / S567V, T163V / S567V, T192S / R197K / V200I,T192S / R197K / V200I / T317R / S567R, T192S / R197K / V200I / N385R, T192S / R197K / V200I / N385R / S567R, T192S / R197K / T317R, T192S / R197K / N385R / P504V / S567R, T192S / R197K / P504V, T192S / V200I, T192S / V200I / N385R, T192S / T317R, T192S / P504V,R197K / V200I / S567R, R197K / T317R, R197K / T317R / N385R / P504V / S567R, V200I7T317R / N385R, V200I / N385R / S567R, T250V, T250V / G331A, T250V / G331A / S567V, T25OV / S523R, T25OV / S567V, T317R, T317R / N385R / S567R, T317R / S567R, G331A / I409L, G331A / S567V,N385R, N385R / S567R, I409L / S523R, S523R, S523R / S567V, or S567V. In the above embodiments, the engineered pyruvate oxidase polypeptide may additionally comprise improved conversion of pyruvate to acetyl phosphate, as compared to a reference sequence of SEQ ID NO: 290.

[0180] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 400, or to the reference sequence corresponding to SEQ ID NO: 400, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 400, or relative to the reference sequence corresponding to SEQ ID NO: 400.

[0181] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 692-766, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 692-766, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 400, or relative to the reference sequence corresponding to SEQ ID NO: 400.

[0182] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 400, or to the reference sequence corresponding to SEQ ID NO: 400, and at least amino acid residue difference(s) as compared to amino acid residues 12-604 of SEQ ID NO: 400 or compared to SEQ ID NO: 400, at amino acid position(s) 32, 32 / 135, 134, 134 / 200 / 331, 134 / 331, or 331. In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 400, or to the reference sequence corresponding to SEQ ID NO: 400, and at least amino acid residue difference(s) as compared to amino acid residues 12-604 of SEQ ID NO: 400 or compared to SEQ ID NO: 400, or amino acid residue(s) 321, 321 / 135A, 134V, 134V / 200I / 331A, 134V / 331A, or 331A. In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 400, or to the reference sequence corresponding to SEQ ID NO: 400, and at least amino acid residue differences as compared to SEQ ID NO: 400, or amino acid residue(s) V32I, V32I / PI 35A, N134V, N134V / V200I / G331 A, N134V / G331 A, or G331A. In the above embodiments, theengineered pyruvate oxidase polypeptide may additionally comprise improved conversion of pyruvate to acetyl phosphate, as compared to a reference sequence of SEQ ID NO: 400.

[0183] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 400, or to the reference sequence corresponding to SEQ ID NO: 400, and at least amino acid residue difference(s) as compared to amino acid residues 12-604 of SEQ ID NO: 400 or compared to SEQ ID NO: 400, at amino acid residue position(s) 62 / 117, 117 / 139 / 331 / 523, 117 / 200 / 523, 117 / 523, 134, or 200 / 523. In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 400, or to the reference sequence corresponding to SEQ ID NO: 400, and at least amino acid residue difference(s) as compared to amino acid residue(s) 12-604 of SEQ ID NO: 400 or compared to SEQ ID NO: 400, or amino acid residue(s) 62K / 117A, 117A / 139P / 331A / 523R, 117A / 200I / 523T, 117A / 523T, 134G, or 200E523T. In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 400, or to the reference sequence corresponding to SEQ ID NO: 400, and at least amino acid residue difference(s) as compared to amino acid residues 12-604 of SEQ ID NO: 400 or compared to SEQ ID NO: 400, or amino acid residue(s) Q62K / S117A, S117A / N139P / G331A / S523R, S117A / V200I / S523T, S117A / S523T, N134G, or V200I / S523T. In the above embodiments, the engineered pyruvate oxidase polypeptide may additionally comprise improved conversion of pyruvate to acetyl phosphate, as compared to a reference sequence of SEQ ID NO: 400.

[0184] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 400, or to the reference sequence corresponding to SEQ ID NO: 400, and at least amino acid residue difference as compared to amino acid residues 12-604 of SEQ ID NO: 400 or compared to SEQ ID NO: 400, at amino acid position 31, 45, 167, 208, 210, 217, 228, 351, 357, 367, 380, or 579, or combinations thereof. In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequencecorresponding to amino acid residues 12-604 of SEQ ID NO: 400, or to the reference sequence corresponding to SEQ ID NO: 400, and at least amino acid residue difference as compared to amino acid residues 12-604 of SEQ ID NO: 400 or compared to SEQ ID NO: 400, or amino acid residue 31A, 31G, 31K, 31S, 45Y, 167R, 167S, 208S, 210A, 210L, 210R, 217R, 228V, 351H, 351K, 351P, 351R, 35 IS, 357L, 367C, 380V, 5791, 579L, 579N, 579S, or 579T, or combinations thereof. In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 400, or to the reference sequence corresponding to SEQ ID NO: 400, and at least amino acid residue difference as compared to amino acid residues 12- 604 of SEQ ID NO: 400 or compared to SEQ ID NO: 400, or an amino acid residue D31A, D31G, D31K, D31S, P45Y, K167R, K167S, D208S, D210A, D210L, D210R, N217R, I228V, A351H, A351K, A351P, A351R, A351S, V357L, M367C, L38OV, E579I, E579L, E579N, E579S, or E579T, or combinations thereof. In the above embodiments, the engineered pyruvate oxidase polypeptide may additionally comprise improved conversion of pyruvate to acetyl phosphate, as compared to a reference sequence of SEQ ID NO: 400.

[0185] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 696, or to the reference sequence corresponding to SEQ ID NO: 696, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 696, or relative to the reference sequence corresponding to SEQ ID NO: 696.

[0186] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 768-876, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 768-876, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 696, or relative to the reference sequence corresponding to SEQ ID NO: 696.

[0187] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 696, or to the referencesequence corresponding to SEQ ID NO: 696, and at least amino acid residue difference as compared to amino acid residues 12-604 of SEQ ID NO: 696 or compared to SEQ ID NO: 696, at amino acid position 15, 203, 206, 239, 326, 417, 524, 527, 555, 572, 587, or 594, or combinations thereof. In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 696, or to the reference sequence corresponding to SEQ ID NO: 696, and at least amino acid residue difference as compared to amino acid residues 12-604 of SEQ ID NO: 696 or compare to SEQ ID NO: 696, or amino acid residue 15R, 203Y, 206G, 206S, 239N, 326S, 326V, 417R, 524G, 527M, 555L, 572G, 572T, 587R, or 594L, or combinations thereof. In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 696, or to the reference sequence corresponding to SEQ ID NO: 696, and at least amino acid residue difference as compared to amino acid residues 12-604 of SEQ ID NO: 696 or compared to SEQ ID NO: 696, or amino acid residue G15R, A203Y, E206G, E206S, E239N, D326S, D326V, K417R, R524G, D527M, R555L, E572G, E572T, V587R, or K594L, or combinations thereof. In the above embodiments, the engineered pyruvate oxidase polypeptide may additionally comprise improved conversion of pyruvate to acetyl phosphate, as compared to a reference sequence of SEQ ID NO: 696.

[0188] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 696, or to the reference sequence corresponding to SEQ ID NO: 696, and at least amino acid residue difference as compared to amino acid residues 12-604 of SEQ ID NO: 696 or compared to SEQ ID NO: 696, at amino acid position 15, 168, 183, 187, 203, 206, 214, 389, 417, 521, 527, 572, 575, 580, 585, 587, or 594, or combinations thereof. In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 696, or to the reference sequence corresponding to SEQ ID NO: 696, and at least amino acid residue difference as compared to amino acid residues 12-604 of SEQ ID NO: 696 or compared to SEQ ID NO: 696, or amino acid residue 15R, 168H, 183C, 183M, 183P, 187V, 203R, 203Y, 206G, 214V, 389R, 417R, 52 IQ, 527L, 527M, 572G, 572P, 572T, 575F, 580T, 585G, 587R, 594 A, 594D, 594S, or 594V, or combinations thereof. In some embodiments, the engineered pyruvate oxidase polypeptide comprisesan amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 696, or to the reference sequence corresponding to SEQ ID NO: 696, and at least amino acid residue difference as compared to amino acid residues 12-604 of SEQ ID NO: 696 or compared to SEQ ID NO: 696, or amino acid residue G15R, R168H, E183C, E183M, E183P, D187V, A203R, A203Y, E206G, E214V, K389R, K417R, T521Q, D527L, D527M, E572G, E572P, E572T, K575F, K580T, E585G, V587R, K594A, K594D, K594S, or K594V, or combinations thereof. In the above embodiments, the engineered pyruvate oxidase polypeptide may additionally comprise improved conversion of pyruvate to acetyl phosphate, as compared to a reference sequence of SEQ ID NO: 696.

[0189] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 696, or to the reference sequence corresponding to SEQ ID NO: 696, and at least amino acid residue difference(s) as compared to amino acid residues 12-604 of SEQ ID NO: 696 or compared to SEQ ID NO: 696, at amino acid position(s) 15, 203 / 206, 203 / 206 / 521, 203 / 521, 206, 206 / 342 / 521, 206 / 342 / 576, 206 / 521, 206 / 585 / 587, 342 / 576, 342 / 576 / 580, 342 / 585 / 587, or 576. In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 696, or to the reference sequence corresponding to SEQ ID NO: 696, and at least amino acid residue difference(s) as compared to amino acid residues 12-604 of SEQ ID NO: 696 or compared to SEQ ID NO: 696, or amino acid residue(s) 15S, 203R / 206Q / 521Q, 203Y / 206Q, 203Y / 521Q, 206A / 521Q, 206Q, 206Q / 342S / 521Q, 206Q / 342S / 576S, 206Q / 585G / 587K, 342S / 576S, 342S / 576S / 580T, 342S / 585G / 587K, or 576S. In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 696, or to the reference sequence corresponding to SEQ ID NO: 696, and at least amino acid residue difference(s) as compared to amino acid residues 12-604 of SEQ ID NO: 696 or compared to SEQ ID NO: 696, or amino acid rcsiduc(s) G15S, A203R / E206Q / T521Q, A203Y / E206Q, A203Y / T521Q, E206A / T521Q, E206Q, E206Q / E342S / T521Q, E206Q / E342S / A576S, E206Q / E585G / V587K, E342S / A576S, E342S / A576S / K580T, E342S / E585G / V587K, or A576S. In the above embodiments, the engineered pyruvate oxidase polypeptide may additionally compriseimproved conversion of pyruvate to acetyl phosphate, as compared to a reference sequence of SEQ ID NO: 696.

[0190] In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 696, or to the reference sequence corresponding to SEQ ID NO: 696, and at least amino acid residue difference(s) as compared to amino acid residues 12-604 of SEQ ID NO: 696 or compared to SEQ ID NO: 696, at amino acid position(s) 15, 15 / 183, 15 / 389, 203 / 206, 203 / 206 / 342 / 576, 203 / 206 / 521, 203 / 206 / 576, 206, 206 / 342, 206 / 521, 342 / 576, 576, or 576 / 580. In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 696, or to the reference sequence corresponding to SEQ ID NO: 696, and at least amino acid residue difference(s) as compared to amino acid residues 12-604 of SEQ ID NO: 696 or compared to SEQ ID NO: 696, or amino acid residue(s) 15R / 183M, 15R / 389R, 15S, 15S / 183M, 203R / 206Q / 342S / 576S, 203R / 206Q / 521Q, 203R / 206Q / 576S, 203Y / 206Q, 206A, 206Q / 342S, 206Q / 521Q, 342S / 576S, 576S, or 576S / 580T. In some embodiments, the engineered pyruvate oxidase polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 696, or to the reference sequence correponding to SEQ ID NO: 696, and at least amino acid residue difference(s) as compared to amino acid residues 12-604 of SEQ ID NO: 696 or compared to SEQ ID NO: 696, or amino acid residue(s) G15R / E183M, G15R / K389R, G15S, G15S / E183M, A203R / E206Q / E342S / A576S, A203R / E206Q / T521Q, A203R / E206Q / A576S, A203Y / E206Q, E206A, E206Q / E342S, E206Q / T521Q, E342S / A576S, A576S, or A576S / K580T. In the above embodiments, the engineered pyruvate oxidase polypeptide may additionally comprise improved conversion of pyruvate to acetyl phosphate, as compared to a reference sequence of SEQ ID NO: 696.

[0191] In some embodiments, the engineered pyruvate oxidase, comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to an amino acid sequence corresponding to amino acid residues 12-604 of one of the even-numbered sequences in the range of SEQ ID NOs: 14-636 and 692-876, or an amino acid sequence corresponding to one of the even-numbered sequences in the range of SEQ ID NOs: 14-636 and 692-876, as provided in the Examples.

[0192] In some embodiments, the engineered pyruvate oxidase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to an amino acid sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696, or an amino acid sequence corresponding to SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696.

[0193] In some embodiments, the engineered pyruvate oxidase with improved properties has an amino acid sequence comprising amino acid residues 12-604 of an even-numbered SEQ ID NO. in the range of SEQ ID NOs: 14-636 and 692-876, or a sequence comprising an even-numbered SEQ ID NO. in the range of SEQ ID NOs: 14-636 and 692-876.

[0194] In some embodiments, the engineered pyruvate oxidase with improved properties has an amino acid sequence comprising amino acid residues 12-604 of SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696, or a sequence comprising SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696.

[0195] As noted above, the engineered pyruvate oxidase polypeptides are also capable of converting substrates (pyruvate, phosphate, and oxygen) to products (acetyl phosphate, hydrogen peroxide, and carbon dioxide). In some embodiments, the engineered pyruvate oxidase polypeptide is capable of converting the substrate compounds to the product compound with at least 1.1 fold, 1.2 fold, 1.3 fold, 1 .4 fold, 1 .5 fold, 1 .6 fold, 1 .7 fold, 2 fold, 2.8 fold, 4 fold, 9.5 fold, 20 fold, 25 fold, 50 fold, 100 fold, or more activity relative to the activity of a reference polypeptide of SEQ ID NO: 6, 16, 56, 184, 264, 400, or 696.

[0196] In some embodiments, the engineered pyruvate oxidase polypeptide is thermostable and capable of converting the substrate compounds to the product compound after incubation at a high temperature of 44 °C, 48 °C, 50 °C, 52 °C, 53 °C, 55 °C, 56 °C, 62 °C or higher with at least 1.1 fold, 1.2 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 2.4 fold, 3 fold, 3.3 fold, 4.7 fold, 5.2 fold, 6 fold, 9 fold, 10.2 fold, 16.3 fold, 26.1 fold, 30 fold, 50 fold, 100 fold, or more activity relative to the activity of a reference polypeptide of SEQ ID NO: 6, 16, 184, 264, 290, 400, or 696.

[0197] In some embodiments, the engineered pyruvate oxidase capable of converting the substrate compounds to the product compounds with at least 2 fold the activity relative to SEQ ID NO: 6, comprises an amino acid sequence comprising amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 14-18, 44-92, and 154-876, or a sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOs: 14-18, 44-92, and 154-876.

[0198] In some embodiments, the engineered pyruvate oxidase capable of converting the substrate compounds to the product compounds with at least 2.8 fold the activity relative to SEQ ID NO: 6, comprises an amino acid sequence comprising amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 14-18, 44-80, 90, 92, and 154-876, or a sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOs: 14-18, 44-80, 90, 92, and 154-876, and comprises at least amino acid residue difference 336T, 512A, or 308K.

[0199] In some embodiments, the engineered pyruvate oxidase has an amino acid sequence comprising one or more residue differences as compared to SEQ ID NO: 6, that increases soluble expression or isolated protein yield of the engineered pyruvate oxidase in a bacterial host cell, particularly in E. coli, as compared to a wild-type or engineered reference pyruvate oxidase, and comprises an amino acid sequence comprising amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 14-636 and 692-876, or a sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOs: 14-636 and 692-876.

[0200] In some embodiments, the engineered pyruvate oxidase has an amino acid sequence comprising one or more residue differences as compared to SEQ ID NO: 6 that increases activity of the engineered pyruvate oxidase one or more substrates, as compared to a wild-type or engineered reference pyruvate oxidase and comprises an amino acid sequence comprising amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 14-636 and 692-876, or a sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOs: 14-636 and 692-876.

[0201] In addition to the residue positions specified above, any of the engineered pyruvate oxidase polypeptides disclosed herein can further comprise other residue differences relative to SEQ ID NO: 6, 16, 56, 184, 264, 290, 400, or 696, at other residue positions (i.e., residue positions other than those included herein). Residue differences at these other residue positions can provide for additional variations in the amino acid sequence without adversely affecting the ability of the polypeptide to carry out the conversion of substrate to product. Accordingly, in some embodiments, in addition to the amino acid residue differences present in any one of the engineered pyruvate oxidase polypeptides selected from the even-numbered sequences in the range of SEQ ID NOs: 14-636 and 692-876, the sequence can further comprise 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-100, or 1-150 residue differences at other amino acid residue positions as compared to the SEQ ID NO: 6. In some embodiments, the number of amino acid residue differences as compared to the reference sequence can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, 50, 100, or 150 residue positions. In some embodiments, the number of amino acid residue differences as compared to the reference sequence can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, or 25 residue positions. The residue differences at these other positions can be conservative changes or non-conservative changes. In some embodiments, the residue differences can comprise conservative substitutions and non-conservative substitutions as compared to the pyruvate oxidase polypeptide of SEQ ID NO: 6, 16, 56, 184, 264, 290, 400, or 696.

[0202] In some embodiments, the present invention also provides engineered polypeptides that comprise a fragment of any one of the engineered pyruvate oxidase polypeptides described herein that retains the functional activity and / or improved property of that engineered pyruvate oxidase.Accordingly, in some embodiments, the present invention provides a polypeptide fragment capable of converting substrate to product under suitable reaction conditions, wherein the fragment comprises at least about 90%, 95%, 96%, 97%, 98%, or 99% of a full-length or truncated amino acid sequence of an engineered pyruvate oxidase of the present invention, such as an exemplary pyruvate oxidase polypeptide sequence selected from the even-numbered SEQ ID NOs. in the range of SEQ ID NOs: 14-636 and 692-876. In some embodiments, the engineered pyruvate oxidase can have an amino acid sequence comprising a deletion in any one of the pyruvate oxidase polypeptide sequences described herein, such as the exemplary engineered polypeptides of the even-numbered SEQ ID NOs. in the range of SEQ ID NOs: 14-636 and 692-876.

[0203] Thus, for each and every embodiment of the engineered pyruvate oxidase polypeptides of the invention, the amino acid sequence can comprise deletions of one or more amino acids, 2 or more amino acids, 3 or more amino acids, 4 or more amino acids, 5 or more amino acids, 6 or more amino acids, 8 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids, up to 20% of the total number of amino acids, or up to 30% of the total number of amino acids of the pyruvate oxidase polypeptides, where the associated functional activity and / or improved properties of the engineered pyruvate oxidases described herein are maintained. In some embodiments, the deletions can comprise 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45, or 1-50 amino acid residues. In some embodiments, the number of deletions can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, or 50 amino acid residues. In some embodiments, the deletions can comprise deletions of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, or 25 amino acid residues.

[0204] In some embodiments, the engineered pyruvate oxidase polypeptide described herein can have an amino acid sequence comprising an insertion as compared to any one of the engineered pyruvate oxidase polypeptides described herein, such as the exemplary engineered polypeptides of the even-numbered sequences in the range of SEQ ID NOs: 14-636 and 692-876. Thus, for each and every embodiment of the pyruvate oxidase polypeptides of the invention, the insertions can comprise one or more amino acids, 2 or more amino acids, 3 or more amino acids, 4 or more amino acids, 5 or more amino acids, 6 or more amino acids, 8 or more amino acids, 10 or more amino acids, 15 or more amino acids, 20 or more amino acids, 30 or more amino acids, 40 or more amino acids, or 50 or more amino acids, where the associated functional activity and / or improved properties of the engineered pyruvate oxidase described herein is maintained. The insertions can be to amino or carboxy terminus, or internal portions of the pyruvate oxidase polypeptide.

[0205] In some embodiments, the engineered pyruvate oxidase described herein can have an amino acid sequence comprising a sequence selected from the even-numbered SEQ ID NOs. in the range of SEQ ID NOs: 14-636 and 692-876, and optionally one or several (e.g., up to 3, 4, 5, or up to 10) amino acid residue deletions, insertions and / or substitutions. In some embodiments, the amino acid sequence has optionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45, 1-50, 1-75, 1-100, or 1-150 amino acid residue deletions, insertions and / or substitutions. In some embodiments, the amino acid sequence has optionally around 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acid residue deletions, insertions and / or substitutions. In some embodiments, the substitutions can be conservative or non-conservative substitutions.

[0206] In the above embodiments, the suitable reaction conditions determining activity of the engineered polypeptides are provided as described in the Examples herein.

[0207] In some embodiments, the polypeptides of the present invention are fusion polypeptides in which the engineered polypeptides are fused to other polypeptides, such as, by way of example and not limitation, antibody tags (e.g., myc epitope), purification sequences (e.g., His tags for binding to metals), and cell localization signals (e.g., secretion signals). Thus, the engineered polypeptides described herein can be used with or without fusions to other polypeptides.

[0208] It is to be understood that the polypeptides described herein are not restricted to the genetically encoded amino acids. In addition to the genetically encoded amino acids, the polypeptides described herein may be comprised, either in whole or in part, of naturally occurring and / or synthetic non-encoded amino acids. Certain commonly encountered non-encoded amino acids of which the polypeptides described herein may be comprised include, but are not limited to: the D-stereoisomers of the genetically-encoded amino acids; 2,3-diaminopropionic acid (Dpr); a-aminoisobutyric acid (Aib): E- aminohexanoic acid (Aha); 8-aminovaleric acid (Ava); N-methylglycine or sarcosine (MeGly or Sar); ornithine (Orn); citrulline (Cit); t-butylalanine (Bua); t-butylglycine (Bug); N- methylisoleucine (Melle); phenylglycine (Phg); cyclohexylalanine (Cha); norleucine (Nle); naphthylalanine (Nal); 2-chlorophenylalanine (Ocf); 3-chlorophenylalanine (Mcf);4-chlorophenylalanine (Pcf); 2-fluorophenylalanine (Off); 3 -fluorophenylalanine (Mff); 4-fluorophenylalanine (Pff); 2-bromophenylalanine (Obf); 3 -bromophenylalanine (Mbf); 4- bromophenylalanine (Pbf); 2-methylphenylalanine (Omf); 3-methylphenylalanine (Mmf); 4- methylphenylalanine (Pmf); 2-nitrophenylalanine (Onf); 3-nitrophenylalanine (Mnf); 4- nitrophenylalanine (Pnf); 2-cyanophenylalanine (Ocf); 3-cyanophenylalanine (Mcf); 4- cyanophenylalanine (Pcf); 2-trifluoromethylphenylalanine (Otf); 3-trifluoromethylphenylalanine (Mtf); 4-trifluoromethylphenylalanine (Ptf); 4-aminophenylalanine (Paf); 4-iodophenylalanine (Pif); 4- aminomethylphenylalanine (Pamf); 2,4-dichlorophenylalanine (Opef); 3,4-dichlorophenylalanine(Mpcf); 2,4-difluorophenylalanine (Opff); 3,4-difluorophenylalanine (Mpff); pyrid-2-ylalanine (2pAla); pyrid-3-ylalanine (3pAla); pyrid-4-ylalanine (4pAla); naphth- 1 -ylalanine (InAla); naphth-2- ylalanine (2nAla); thiazolylalanine (taAla); benzothienylalanine (bAla); thienylalanine (tAla); furylalanine (fAla); homophenylalanine (hPhe); homotyrosine (hTyr); homotryptophan (hTrp); pentafluorophenylalanine (5ff); styrylkalanine (sAla); authrylalanine (aAla); 3,3-diphenylalanine (Dfa); 3-amino-5-phenypentanoic acid (Afp); penicillamine (Pen); l,2,3,4-tetrahydroisoquinoline-3- carboxylic acid (Tic); P-2-thienylalanine (Thi); methionine sulfoxide (Mso); N(w)-nitroarginine (nArg); homolysine (hLys); phosphonomethylphenylalanine (pmPhe); phosphoserine (pSer); phosphothreonine (pThr); homoaspartic acid (hAsp); homoglutanic acid (hGlu); l-aminocyclopent-(2 or 3)-ene-4 carboxylic acid; pipecolic acid (PA), azetidine-3-carboxylic acid (ACA); 1- aminocyclopentane-3-carboxylic acid; allylglycine (aGly); propargylglycine (pgGly); homoalanine (hAla); norvaline (nVal); homoleucine (hLeu), homovaline (hVal); homoisoleucine (hlle); homoarginine (hArg); Nacetyl- lysine (AcLys); 2,4-diaminobutyric acid (Dbu); 2,3-diaminobutyric acid (Dab); N-methyl valine (MeVal); homocysteine (hCys); homoserine (hSer); hydroxyproline (Hyp) and homoproline (hPro). Additional non-encoded amino acids of which the polypeptides described herein may be comprised will be apparent to those of skill in the art (See e.g., the various amino acids provided in Fasman, CRC Practical Handbook of Biochemistry and Molecular Biology, CRC Press, Boca Raton, FL, pp. 3-70 (1989), and the references cited therein, all of which are incorporated by reference). These amino acids may be in either the L or D configuration.

[0209] Those of skill in the art will recognize that amino acids or residues bearing side chain protecting groups may also comprise the polypeptides described herein. Non-limiting examples of such protected amino acids, which in this case belong to the aromatic category, include (protecting groups listed in parentheses), but are not limited to: Arg(tos), Cys(methylbenzyl), Cys (nitropyridinesulfenyl), Glu(S-benzylester), Gln(xanthyl), Asn(N-8-xanthyl), His(bom), His(benzyl), His(tos), Lys(fmoc), Lys(tos), Ser(O-benzyl), Thr (O-benzyl) and Tyr(O-benzyl).

[0210] Non-encoding amino acids that are conformationally constrained of which the polypeptides described herein may be composed include, but are not limited to, N-methyl amino acids (L configuration); l-aminocyclopent-(2 or 3)-ene-4-carboxylic acid; pipecolic acid; azetidine-3- carboxylic acid; homoproline (hPro); and l-aminocyclopentane-3-carboxylic acid.

[0211] In some embodiments, the engineered polypeptides can be in various forms, for example, such as an isolated preparation, as a substantially purified enzyme, whole cells transformed with gene(s) encoding the enzyme, and / or as cell extracts and / or lysates of such cells. The enzymes can be lyophilized, spray-dried, precipitated, or be in the form of a crude paste, as further discussed below.

[0212] In some embodiments, the engineered polypeptides can be in the form of a biocatalytic composition. In some embodiments, the biocatalytic composition comprises (a) a means forconversion of pyruvate to acetyl phosphate, and (b) a suitable cofactor. The suitable cofactor may be thiamine pyrophosphate (TPP), flavin adenine dinucleotide (FAD), or another suitable cofactor.

[0213] In some embodiments, the biocatalytic composition comprises (a) an engineered pyruvate oxidase having an amino acid sequence selected from an even-numbered SEQ ID NO. of SEQ ID NOs: 14-636 and 692-876, and (b) a suitable cofactor. In some embodiments, the suitable cofactor comprises thiamine pyrophosphate (TPP), flavin adenine dinucleotide (FAD), or another suitable cofactor.

[0214] In some embodiments, the biocatalytic composition comprises (a) an engineered pyruvate oxidase having an amino acid sequence selected from an even-numbered SEQ ID NO. of SEQ ID NOs: 14-636 and 692-876, and (b) a substrate. In some embodiments, the substrate comprises pyruvate, oxygen, phosphate, or another suitable substrate.

[0215] In some embodiments, the biocatalytic composition comprises (a) an engineered pyruvate oxidase having an amino acid sequence selected from an even-numbered SEQ ID NO. of SEQ ID NOs: 14-636 and 692-876, and (b) a second enzyme. In some embodiments, the second enzyme comprises an enzyme that uses acetyl phosphate as a substrate. In some embodiments, the second enzyme comprises acetate kinase or any other enzyme that utilizes acetyl phosphate. In some embodiments, the second enzyme degrades hydrogen peroxide, such as a catalase.

[0216] In some embodiments, the biocatalytic composition further comprises one or more additional enzymes, wherein the one or more additional enzymes are selected from adenylate kinase, adenosine kinase, terminal nucleotidyl transferase, 3’O-kinase, and inorganic pyrophosphatase. In some embodiments, the one or more additional enzymes generate NDP from substrate NTP, and / or are used in polynucleotide synthesis, either template dependent or template independent polymerases.

[0217] In some embodiments, the polypeptides described herein are provided in the form of kits. The enzymes in the kits may be present individually or as a plurality of enzymes. The kits can further include reagents for carrying out the enzymatic reactions, substrates for assessing the activity of enzymes, as well as reagents for detecting the products. The kits can also include reagent dispensers and instructions for use of the kits.

[0218] In some embodiments, the kits of the present invention include arrays comprising a plurality of different pyruvate oxidase polypeptides at different addressable positions, wherein the different polypeptides are different variants of a reference sequence, each having at least one different improved enzyme property. In some embodiments, a plurality of polypeptides immobilized on solid supports are configured on an array at various locations, addressable for robotic delivery of reagents, or by detection methods and / or instruments. The array can be used to test a variety of substrate compounds for conversion by the polypeptides. Such arrays comprising a plurality of engineered polypeptides and methods of their use are known in the art (See e.g., W02009 / 008908).Polynucleotides Encoding Engineered Pyruvate Oxidases, Expression Vectors and Host Cells

[0219] In another aspect, the present invention provides recombinant polynucleotides encoding the engineered pyruvate oxidase polypeptides described herein. In some embodiments, the recombinant polynucleotides may be operatively linked to one or more heterologous regulatory sequences that control gene expression to create a recombinant polynucleotide capable of expressing the polypeptide. Expression constructs containing a heterologous polynucleotide encoding the engineered pyruvate oxidase are introduced into appropriate host cells to express the corresponding pyruvate oxidase polypeptide.

[0220] As will be apparent to the skilled artisan, availability of a protein sequence and the knowledge of the codons corresponding to the various amino acids provide a description of all the polynucleotides capable of encoding the subject polypeptides. The degeneracy of the genetic code, where the same amino acids are encoded by alternative or synonymous codons, allows an extremely large number of nucleic acids to be made, all of which encode the improved pyruvate oxidase enzymes. Thus, having knowledge of a particular amino acid sequence, those skilled in the art could make any number of different nucleic acids by simply modifying the sequence of one or more codons in a way which does not change the amino acid sequence of the protein. In this regard, the present invention specifically contemplates each and every possible variation of polynucleotides that could be made encoding the polypeptides described herein by selecting combinations based on the possible codon choices, and all such variations are to be considered specifically disclosed for any polypeptide described herein, including the amino acid sequences presented in Tables 7.2, 8.2, 9.2, 10.2, 11.2, 12.2, 13.2, 14.2, 15.2, 16.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, and 23.2, and disclosed in the sequence listing incorporated by reference herein as the even-numbered SEQ ID NOs. in the range of SEQ ID NOs: 14-636 and 692-876.

[0221] In various embodiments, the codons are preferably selected to fit the host cell in which the protein is being produced. For example, preferred codons used in bacteria are used to express the gene in bacteria; preferred codons used in yeast are used for expression in yeast; and preferred codons used in mammals are used for expression in mammalian cells. In some embodiments, all codons need not be replaced to optimize the codon usage of the pyruvate oxidase since the natural sequence will comprise preferred codons and because use of preferred codons may not be required for all amino acid residues. Consequently, codon optimized polynucleotides encoding the pyruvate oxidase enzymes may contain preferred codons at about 40%, 50%, 60%, 70%, 80%, or greater than 90% of codon positions of the full-length coding region.

[0222] In some embodiments, the recombinant polynucleotide comprises a codon optimized nucleotide sequence encoding the pyruvate oxidase polypeptide amino acid sequence, as represented by SEQ ID NO: 6, 16, 56, 184, 264, 290, 400, or 696. In some embodiments, the polynucleotide has anucleic acid sequence comprising at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the codon optimized nucleic acid sequences encoding the even-numbered sequences in the range of SEQ ID NOs: 14-636 and 692-876.

[0223] In some embodiments, the recombinant polynucleotide encodes a pyruvate oxidase polypeptide capable of converting one or more substrates to product with improved properties as compared to SEQ ID NO: 6, 16, 56, 184, 264, 290, 400, or 696, wherein the polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQ ID NO: 6, 16, 56, 184, 264, 290, 400, or 696.

[0224] In some embodiments, as described above, the polynucleotide encodes an engineered pyruvate oxidase polypeptide with improved properties as compared to SEQ ID NO: 6, 16, 56, 184, 264, 290, 400, or 696, wherein the polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a reference sequence selected from SEQ ID NO: 6, 16, 56, 184, 264, 290, 400, or 696, and one or more residue differences as compared to SEQ ID NO: 6, 16, 56, 184, 264, 290, 400, or 696, wherein the sequence is selected from the even-numbered sequences in the range of SEQ ID NOs: 14-636 and 692-876. In some embodiments, the reference amino acid sequence is selected from the even-numbered sequences in the range of SEQ ID NOs: 14- 636 and 692-876. In some embodiments, the reference amino acid sequence is SEQ ID NO: 6, 16, 56, 184, 264, 290, 400, or 696.

[0225] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered pyruvate oxidase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 6, 14-636, and 692-876, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 6, 14-636, and 692-876, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, 16, 56, 184, 264, 290, 400, or 696, or relative to the reference sequence corresponding to SEQ ID NO: 6, 16, 56, 184, 264, 290, 400, or 696.

[0226] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered pyruvate oxidase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acidresidues 12-604 of SEQ ID NO: 6, 16, 56, 184, 264, 290, 400, or 696, or to a reference sequence corresponding to SEQ ID NO: 6, 16, 56, 184, 264, 290, 400, or 696, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, 16, 56, 184, 264, 290, 400, or 696, or relative to the reference sequence corresponding to SEQ ID NO: 6, 16, 56, 184, 264, 290, 400, or 696.

[0227] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered pyruvate oxidase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 14-636 and 692-876, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 14-636 and 692-876, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, or relative to the reference sequence corresponding to SEQ ID NO: 6.

[0228] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered pyruvate oxidase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, or to a reference sequence corresponding to SEQ ID NO: 6, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, or relative to the reference sequence corresponding to SEQ ID NO: 6.

[0229] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered pyruvate oxidase comprising an amino acid sequence comprising at least amino acid residue difference at amino acid position 14, 15, 21, 31, 32, 45, 54, 57, 61, 62, 74, 108, 117, 127, 134, 135, 139, 150, 153, 161, 163, 167, 168, 177, 183, 187, 192, 193, 197, 199, 200, 201,202, 203, 204, 205, 206, 207, 208, 209, 210, 214, 215, 217, 218, 220, 226, 228, 233, 235, 237, 239,242, 244, 250, 259, 302, 307, 308, 314, 317, 325, 326, 331, 336, 342, 345, 351, 357, 367, 374, 377,380, 385, 389, 393, 404, 409, 417, 432, 458, 469, 471, 480, 482, 496, 504, 506, 510, 511, 512, 515,521, 523, 524, 527, 528, 530, 536, 555, 561, 563, 565, 567, 572, 575, 576, 579, 580, 585, 587, 591,594, or 604, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, or relative to the reference sequence corresponding to SEQ ID NO: 6.

[0230] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered pyruvate oxidase comprising an amino acid sequence comprising at leastamino acid residue difference at amino acid position 134, 153, 168, 192, 197, 242, 308, 317, 325, 331, 336, 469, 504, 510, 512, or 567, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, or relative to the reference sequence corresponding to SEQ ID NO: 6.

[0231] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered pyruvate oxidase comprising an amino acid sequence comprising at least amino acid residue difference at amino acid position 61, 168, 202, 233, 242, 244, 308, 336, 480, 510, 512, or 528, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, or relative to the reference sequence corresponding to SEQ ID NO: 6.

[0232] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered pyruvate oxidase comprising an amino acid sequence comprising at least amino acid residue difference at amino acid position 168, 220, 242, 308, 377, 432, or 512, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, or relative to the reference sequence corresponding to SEQ ID NO: 6.

[0233] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered pyruvate oxidase comprising an amino acid sequence comprising at least amino acid residue difference(s) at amino acid position(s) 54, 54 / 57, 54 / 57 / 168, 54 / 57 / 168 / 482 / 510 / 512, 54 / 57 / 336, 54 / 57 / 482, 54 / 57 / 510, 54 / 57 / 510 / 512, 54 / 168, 54 / 168 / 336 / 512, 54 / 168 / 482 / 510, 54 / 168 / 510, 54 / 168 / 510 / 512, 54 / 242 / 482 / 512, 54 / 336 / 510, 54 / 336 / 512, 54 / 482, 54 / 482 / 512 / 528, 54 / 510, 54 / 512 / 528, 57, 57 / 168 / 482 / 510 / 512, 57 / 336 / 512, 57 / 482, 57 / 510, 57 / 512, 57 / 512 / 528, 61 / 202 / 308 / 469 / 536, 61 / 244, 61 / 308 / 469, 61 / 308 / 471 / 536, 61 / 471 / 536, 168 / 242 / 336 / 510 / 512, 168 / 242 / 482 / 512 / 528, 168 / 512, 202 / 244 / 308, 202 / 244 / 308 / 471, 202 / 244 / 536, 202 / 308 / 432 / 536, 202 / 308 / 469 / 471, 202 / 308 / 469 / 536, 202 / 308 / 536, 202 / 471 / 536, 202 / 536, 242 / 512 / 528, 244 / 308 / 469, 308 / 469 / 471, 336 / 482, 336 / 482 / 510 / 512, 469, 482, 482 / 510, 482 / 510 / 512 / 528, or 536, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, or relative to the reference sequence corresponding to SEQ ID NO: 6.

[0234] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered pyruvate oxidase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696, or to the reference sequence corresponding to SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696, wherein the amino acid sequencecomprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696, or relative to the reference sequence corresponding to SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696.

[0235] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered pyruvate oxidase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 14-636 and 692-876, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 14-636 and 692-876, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696, or relative to the reference sequence corresponding to SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696.

[0236] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered pyruvate oxidase comprising an amino acid sequence comprising at least amino acid residue difference at amino acid position 14, 15, 21, 31, 32, 45, 54, 57, 61, 62, 74, 108, 117, 127, 134, 135, 139, 150, 153, 161, 163, 167, 168, 177, 183, 187, 192, 193, 197, 199, 200, 201,202, 203, 204, 205, 206, 207, 208, 209, 210, 214, 215, 217, 218, 220, 226, 228, 233, 235, 237, 239,242, 244, 250, 259, 302, 307, 308, 314, 317, 325, 326, 331, 336, 342, 345, 351, 357, 367, 374, 377,380, 385, 389, 393, 404, 409, 417, 432, 458, 469, 471, 480, 482, 496, 504, 506, 510, 511, 512, 515,521, 523, 524, 527, 528, 530, 536, 555, 561, 563, 565, 567, 572, 575, 576, 579, 580, 585, 587, 591,594, or 604, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696, or relative to the reference sequence corresponding to SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696.

[0237] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered pyruvate oxidase comprising an amino acid sequence comprising at least amino acid residue difference at amino acid position 134, 153, 168, 192, 197, 242, 308, 317, 325, 331, 336, 469, 504, 510, 512, or 567, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696, or relative to the reference sequence corresponding to SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696.

[0238] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered pyruvate oxidase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 56, or to the reference sequence corresponding to SEQ ID NO: 56, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 56, or relative to the reference sequence corresponding to SEQ ID NO: 56.

[0239] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered pyruvate oxidase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 154-178, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 154-178, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 56, or relative to the reference sequence corresponding to SEQ ID NO: 56.

[0240] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered pyruvate oxidase comprising an amino acid sequence comprising at least amino acid residue difference at amino acid position 14, 199, 201, 202, 215, 218, 496, 575, or 580, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 56, or relative to the reference sequence corresponding to SEQ ID NO: 56.

[0241] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered pyruvate oxidase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 16, or to the reference sequence corresponding to SEQ ID NO: 16, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0242] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered pyruvate oxidase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 180-248, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 180-248, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the referencesequence corresponding to amino acid residues 12-604 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0243] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered pyruvate oxidase comprising an amino acid sequence comprising at least amino acid residue difference at amino acid position 21, 183, 193, 201, 203, 204, 205, 206, 207, 209, 214, 235, 307, 325, 345, 374, 393, 510, 527, 530, 561, 563, 565, 580, 594, or 604, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

[0244] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered pyruvate oxidase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 184, or to the reference sequence corresponding to SEQ ID NO: 184, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 184, or relative to the reference sequence corresponding to SEQ ID NO: 184.

[0245] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered pyruvate oxidase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 250-288, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 250-288, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 184, or relative to the reference sequence corresponding to SEQ ID NO: 184.

[0246] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered pyruvate oxidase comprising an amino acid sequence comprising at least amino acid residue difference(s) at amino acid position(s) 177, 177 / 209, 177 / 209 / 336, 177 / 209 / 563, 177 / 336, 177 / 336 / 471, 177 / 336 / 563, 177 / 563, 206 / 308, 206 / 308 / 469, 206 / 308 / 536 / 591, 206 / 469, 308, 308 / 469, 308 / 469 / 536, 336 / 471, 336 / 563, 469, 469 / 536, and 563, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 184, or relative to the reference sequence corresponding to SEQ ID NO: 184.

[0247] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered pyruvate oxidase comprising an amino acid sequence having at least 70%,75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 264, or to the reference sequence corresponding to SEQ ID NO: 264, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 264, or relative to the reference sequence corresponding to SEQ ID NO: 264.

[0248] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered pyruvate oxidase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 290-398, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 290-398, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 264, or relative to the reference sequence corresponding to SEQ ID NO: 264.

[0249] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered pyruvate oxidase comprising an amino acid sequence comprising at least amino acid residue difference at amino acid position 135, 153, 161, or 504, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 264, or relative to the reference sequence corresponding to SEQ ID NO: 264.

[0250] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered pyruvate oxidase comprising an amino acid sequence comprising at least amino acid residue difference at amino acid position 32, 127, 134, 135, 153, 163, 192, 197, 200, 317, 331, 385, 504, or 567, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 264, or relative to the reference sequence corresponding to SEQ ID NO: 264.

[0251] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered pyruvate oxidase comprising an amino acid sequence comprising at least amino acid residue difference at amino acid position 32, 74, 108, 117, 127, 134, 135, 139, 150, 153, 163, 177, 192, 197, 200, 226, 237, 250, 259, 302, 314, 317, 331, 385, 404, 409, 458, 504, 506, 511, 515, 523, or 567, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 264, or relative to the reference sequence corresponding to SEQ ID NO: 264.

[0252] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered pyruvate oxidase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 290, or to the reference sequence corresponding to SEQ ID NO: 290, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 290, or relative to the reference sequence corresponding to SEQ ID NO: 290.

[0253] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered pyruvate oxidase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 400-636, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 400-636, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 290, or relative to the reference sequence corresponding to SEQ ID NO: 290.

[0254] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered pyruvate oxidase comprising an amino acid sequence comprising at least amino acid residue difference(s) at amino acid position(s) 32 / 134 / 135 / 523 / 567, 32 / 134 / 139 / 163 / 250 / 331 / 523, 32 / 134 / 163 / 409, 32 / 134 / 331 / 567, 32 / 139 / 163 / 250, 32 / 139 / 163 / 409, 32 / 139 / 163 / 567, 32 / 139 / 250 / 567, 32 / 139 / 409, 32 / 163, 32 / 163 / 250, 32 / 163 / 250 / 567, 32 / 163 / 331, 32 / 163 / 331 / 409 / 523 / 567, 32 / 163 / 331 / 409 / 567, 32 / 163 / 409 / 567, 32 / 163 / 523 / 567, 32 / 163 / 567, 32 / 250, 32 / 250 / 409, 32 / 250 / 523 / 567, 32 / 250 / 567, 32 / 331 / 567, 32 / 409, 32 / 409 / 567, 32 / 567, 127, 127 / 153, 127 / 153 / 192 / 197, 127 / 153 / 192 / 197 / 200 / 317, 127 / 153 / 192 / 200 / 317 / 385, 127 / 153 / 192 / 504, 127 / 153 / 201 / 385, 127 / 153 / 317 / 385, 127 / 153 / 317 / 567, 127 / 192 / 197 / 200 / 385 / 504, 127 / 192 / 197 / 200 / 504, 127 / 192 / 197 / 317 / 504 / 567, 127 / 192 / 197 / 385 / 504, 127 / 192 / 197 / 504, 127 / 192 / 317 / 385 / 504, 127 / 192 / 385 / 504 / 567, 127 / 197 / 200 / 317, 127 / 200, 127 / 200 / 317 / 567, 127 / 317, 127 / 385, 127 / 385 / 567, 134 / 135 / 163 / 567, 134 / 163 / 331 / 409 / 567, 134 / 250, 134 / 409 / 567, 139, 139 / 163, 139 / 163 / 250 / 331 / 523, 139 / 163 / 331 / 523, 139 / 163 / 567, 139 / 523 / 567, 153, 153 / 192 / 197 / 200 / 385 / 567, 153 / 192 / 197 / 200 / 504, 153 / 192 / 197 / 317 / 504, 153 / 192 / 197 / 317 / 567, 153 / 192 / 200 / 504, 153 / 192 / 317 / 385, 153 / 197 / 504 / 567, 153 / 200, 153 / 317, 153 / 317 / 385, 153 / 317 / 504, 153 / 317 / 504 / 567, 153 / 385, 153 / 504, 153 / 504 / 567, 163, 163 / 250, 163 / 250 / 567, 163 / 331, 163 / 331 / 409, 163 / 331 / 409 / 567, 163 / 331 / 567, 163 / 409, 163 / 523 / 567, 163 / 567, 192 / 197 / 200, 192 / 197 / 200 / 317 / 567, 192 / 197 / 200 / 385, 192 / 197 / 200 / 385 / 567, 192 / 197 / 317, 192 / 197 / 385 / 504 / 567, 192 / 197 / 504, 192 / 200, 192 / 200 / 385, 192 / 317, 192 / 504, 197 / 200 / 567, 197 / 317, 197 / 317 / 385 / 504 / 567, 200 / 317 / 385, 200 / 385 / 567, 250,250 / 331, 250 / 331 / 567, 250 / 523, 250 / 567, 317, 317 / 385 / 567, 317 / 567, 331 / 409, 331 / 567, 385, 385 / 567, 409 / 523, 523, 523 / 567, or 567, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 290, or relative to the reference sequence corresponding to SEQ ID NO: 290.

[0255] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered pyruvate oxidase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 400, or to the reference sequence corresponding to SEQ ID NO: 400, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 400, or relative to the reference sequence corresponding to SEQ ID NO: 400.

[0256] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered pyruvate oxidase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 692-766, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 692-766, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 400, or relative to the reference sequence corresponding to SEQ ID NO: 400.

[0257] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered pyruvate oxidase comprising an amino acid sequence comprising at least amino acid residue difference(s) at amino acid position(s) 32, 32 / 135, 134, 134 / 200 / 331, 134 / 331, or 331, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 400, or relative to the reference sequence corresponding to SEQ ID NO: 400.

[0258] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered pyruvate oxidase comprising an amino acid sequence comprising at least amino acid residue difference(s) at amino acid position(s) 62 / 117, 117 / 139 / 331 / 523, 117 / 200 / 523, 117 / 523, 134, or 200 / 523, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 400, or relative to the reference sequence corresponding to SEQ ID NO: 400.

[0259] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered pyruvate oxidase comprising an amino acid sequence comprising at leastamino acid residue difference at amino acid position 31, 45, 167, 208, 210, 217, 228, 351, 357, 367, 380, or 579, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 400, or relative to the reference sequence corresponding to SEQ ID NO: 400.

[0260] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered pyruvate oxidase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 696, or to the reference sequence corresponding to SEQ ID NO: 696, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 696, or relative to the reference sequence corresponding to SEQ ID NO: 696.

[0261] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered pyruvate oxidase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 768-876, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 768-876, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 696, or relative to the reference sequence corresponding to SEQ ID NO: 696.

[0262] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered pyruvate oxidase comprising an amino acid sequence comprising at least amino acid residue difference at amino acid position 15, 203, 206, 239, 326, 417, 524, 527, 555, 572, 587, or 594, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 696, or relative to the reference sequence corresponding to SEQ ID NO: 696.

[0263] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered pyruvate oxidase comprising an amino acid sequence comprising at least amino acid residue difference at amino acid position 15, 168, 183, 187, 203, 206, 214, 389, 417, 521, 527, 572, 575, 580, 585, 587, or 594, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 696, or relative to the reference sequence corresponding to SEQ ID NO: 696.

[0264] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered pyruvate oxidase comprising an amino acid sequence comprising at leastamino acid residue difference(s) at amino acid position(s) 15, 203 / 206, 203 / 206 / 521, 203 / 521, 206, 206 / 342 / 521, 206 / 342 / 576, 206 / 521, 206 / 585 / 587, 342 / 576, 342 / 576 / 580, 342 / 585 / 587, or 576, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 696, or relative to the reference sequence corresponding to SEQ ID NO: 696.

[0265] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered pyruvate oxidase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 6, 14-636 and 692-876, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 6, 14-636 and 692-876.

[0266] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered pyruvate oxidase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696, or to a reference sequence corresponding to SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696.

[0267] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered pyruvate oxidase comprising an amino acid sequence comprising amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 14-636 and 692-876, or a sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOs: 14-636 and 692-876.

[0268] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding an engineered pyruvate oxidase comprising an amino acid sequence comprising amino acid residues 12-604 of SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696, or a sequence comprising SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696.

[0269] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34-1812 of SEQ ID NO: 5, 15, 55, 183, 263, 289, 399, or 695, or to a reference polynucleotide sequence of SEQ ID NO: 5, 15, 55, 183, 263, 289, 399, or 695, wherein the recombinant polynucleotide encodes a polypeptide having pyruvate oxidase activity.

[0270] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotidesequence corresponding to nucleotide residues 34-1812 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 13-635 and 691-875, or to a reference polynucleotide sequence corresponding to an odd- numbered SEQ ID NO. of SEQ ID NOs: 13-635 and 691-875, wherein the recombinant polynucleotide encodes a polypeptide having pyruvate oxidase activity.

[0271] In some embodiments, the recombinant polynucleotide comprises a nucleic acid sequence comprising at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the codon optimized nucleic acid sequences in the odd-numbered SEQ ID NOs. in the range of SEQ ID NOs: 13-635 and 691-875.

[0272] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising nucleotide residues 34-1812 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 13-635 and 691-875, or a polynucleotide sequence comprising an odd-numbered SEQ ID NO. of SEQ ID NOs: 13-635 and 691-875.

[0273] In some embodiments, the codon optimized sequences of the odd-numbered SEQ ID NOs. in the range of SEQ ID NOs: 13-635 and 691-875, enhance expression of the encoded pyruvate oxidase, providing preparations of enzyme capable of converting substrate to product.

[0274] In some embodiments, the recombinant polynucleotides are capable of hybridizing under highly stringent conditions to a reference sequence selected from the odd-numbered sequences in SEQ ID NOs: 13-635, 691-875, or a complement thereof, and encode a pyruvate oxidase.

[0275] In some embodiments, the polynucleotides are capable of hybridizing under highly stringent conditions to a reference polynucleotide sequence selected from the odd-numbered sequences in the range of SEQ ID NOs: 13-635, 691-875 or a complement thereof and encode a pyruvate oxidase polypeptide with one or more of the improved properties described herein. In some embodiments, the polynucleotide capable of hybridizing under highly stringent conditions encodes a pyruvate oxidase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 6, 16, 56, 184, 264, 290, 400, or 696, that has an amino acid sequence comprising one or more residue differences as compared to SEQ ID NO: 6, 16, 56, 184, 264, 290, 400, or 696, as described above and in the Examples, below.

[0276] In some embodiments, the polynucleotide capable of hybridizing under highly stringent conditions encodes an engineered pyruvate oxidase polypeptide with improved properties comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 6, 16, 56, 184, 264, 290, 400, or 696. In some embodiments, the polynucleotides encode the polypeptides described herein but have at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity at the nucleotide level to a reference polynucleotide encoding the engineered pyruvate oxidase. In some embodiments, the reference polynucleotide sequence is selected from SEQ ID NOs:5, 15, 55, 183, 263, 289, 399, and 695.

[0277] In some embodiments, the polynucleotide capable of hybridizing under highly stringent conditions encodes an engineered pyruvate oxidase polypeptide with improved properties comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 6, 16, 56, 184, 264, 290, 400, or 696.

[0278] In some embodiments, the polynucleotides encode the polypeptides described herein but have at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity at the nucleotide level to a reference polynucleotide encoding the engineered pyruvate oxidase. In some embodiments, the reference polynucleotide sequence is selected from SEQ ID NOs: 5, 15, 55, 183, 263, 289, 399, and 695.

[0279] In some embodiments, an isolated polynucleotide encoding any of the engineered pyruvate oxidase polypeptides provided herein is manipulated in a variety of ways to provide for expression of the polypeptide. In some embodiments, the polynucleotides encoding the polypeptides are provided as expression vectors where one or more control sequences is present to regulate the expression of the polynucleotides and / or polypeptides. Manipulation of the isolated polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides and nucleic acid sequences utilizing recombinant DNA methods are well known in the art.

[0280] In some embodiments, the control sequences include, among other sequences, promoters, leader sequences, polyadenylation sequences, propeptide sequences, signal peptide sequences, and transcription terminators.

[0281] In some embodiments, suitable promoters are selected based on the host cell selection. For bacterial host cells, suitable promoters for directing transcription of the nucleic acid constructs of the present disclosure, include, but are not limited to promoters obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and prokaryotic beta-lactamase gene (see, e.g., Villa-Kamaroff et ah, Proc. Natl Acad. Sci. USA, 1978, 75:3727-3731), as well as the tac promoter (see, e.g., DeBoer et al., Proc. Natl Acad. Sci. USA, 1983, 80:21-25). Exemplary promoters for filamentous fungal host cells, include, but are not limited to promoters obtained from the genes forAspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha- amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (see, e.g., WO 96 / 00787), as well as the NA2-tpi promoter (a hybrid of the promoters from the genes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase), and mutant, truncated, and hybrid promoters thereof. Exemplary yeast cell promoters can be from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GALI), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde- 3-phosphate dehydrogenase (ADH2 / GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are known in the art (see, e.g., Romanos et al., Yeast, 1992, 8:423-488). Exemplary promoters for use in insect cells include, but are not limited to, polyhedrin, plO, ELT, OpIE2, and hr5 / iel promoters. Exemplary promoters for use in mammalian cells include, but are not limited to, those from cytomegalovirus (CMV), chicken (Lactin promoter fused with the CMV enhancer, Simian vacuolating virus 40 (SV40), from Homo sapiens phosphoglycerate kinase, beta actin, elongation factor- la or glyceraldehyde-3-phosphate dehydrogenase, and from Gallus [Lactin.

[0282] In some embodiments, the control sequence is a suitable transcription terminator sequence (i.e., a sequence recognized by a host cell to terminate transcription). In some embodiments, the terminator sequence is operably linked to the 3' terminus of the nucleic acid sequence encoding the pyruvate oxidase polypeptide. Any suitable terminator which is functional in the host cell of choice finds use in the present invention. For bacterial expression, the transcription terminators can be a Rho-dependent terminators that rely on a Rho transcription factor, or a Rho-independent, or intrinsic terminators, which do not require a transcription factor. Exemplary bacterial transcription terminators are described in Peters et al., J Mol Biol., 2011, 412(5):793-813. Exemplary transcription terminators for filamentous fungal host cells can be obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease. Exemplary terminators for yeast host cells can be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are known in the art (see, e.g., Romanos et aL, Yeast, 1992, 8(6):423-88). Exemplary terminators for insect cells and mammalian cells include, but are not limited to, those from cytomegalovirus (CMV), Simian virus 40 (SV40), from Homo sapiens growth hormone hGH, from bovine growth hormone BGH, and from human or rabbit beta globulin.

[0283] In some embodiments, the control sequence is a suitable leader sequence, a non-translated region of an mRNA that is used for translation by the host cell. In some embodiments, the leader sequence is operably linked to the 5' terminus of the nucleic acid sequence encoding the pyruvate oxidase polypeptide. Any suitable leader sequence that is functional in the host cell of choice find use in the present invention. Exemplary leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, and Aspergillus nidulans triose phosphate isomerase. Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alphafactor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP). Suitable leaders for mammalian host cells include but are not limited to the 5 -UTR element present in orthopoxvirus mRNA.

[0284] In some embodiments, the control sequence is a polyadenylation sequence (i.e., a sequence operably linked to the 3' terminus of the nucleic acid sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA). Any suitable polyadenylation sequence which is functional in the host cell of choice finds use in the present invention. Exemplary poly adenylation sequences for filamentous fungal host cells include, but are not limited to the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase. Useful poly adenylation sequences for yeast host cells are known (see, e.g., Guo and Sherman, Mol. Cell. Biol., 1995, 15:5983-5990). Useful polyadenylation and 3’ UTR sequences for insect and mammalian host cells include, but are not limited to, OpIE2 polyA sequence, D. melanogaster metallothionein (Mt) polyA signal sequence, D. melanogaster alcohol dehydrogenase (adh), SV40 polyA signal sequence, and the 3'-UTRs of a- and 0-globin mRNAs harboring sequence elements that increase the stability and translation of mRNA.

[0285] In some embodiments, the control sequence is a signal peptide coding region that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell's secretory pathway. In some embodiments, the 5’ end of the coding sequence of the nucleic acid sequence inherently contains a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region that encodes the secreted polypeptide. Alternatively, in some embodiments, the 5’ end of the coding sequence contains a signal peptide coding region that is foreign to the coding sequence. Any suitable signal peptide coding region which directs the expressed polypeptide into the secretory pathway of a host cell of choice finds use for expression of the engineered polypeptide(s). Effective signal peptide coding regions for bacterial host cells are the signal peptide coding regions include, but are not limited to those obtained from the genes for Bacillus NC1B 11837 maltogenic amylase, Bacillus stearothermophilus alphaamylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillusstearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are known in the art (see, e.g., Simonen and Palva, Microbiol. Rev., 1993, 57:109-137). In some embodiments, effective signal peptide coding regions for filamentous fungal host cells include, but are not limited to the signal peptide coding regions obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase. Useful signal peptides for yeast host cells include, but are not limited to those from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Useful signal peptides for insect and mammalian host cells include but are not limited to, those from the genes for immunoglobulin gamma (IgG) and the signal peptide in a human secreted protein, such as human beta-galactosidase polypeptide.

[0286] In some embodiments, the control sequence is a propeptide coding region that codes for an amino acid sequence positioned at the amino terminus of a polypeptide. The resultant polypeptide is referred to as a “proenzyme,” “propolypeptide,” or “zymogen,” in some cases). A propolypeptide can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding region includes but is not limited to the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophila lactase (See e.g., WO 95 / 33836). Where both signal peptide and propeptide regions are present at the amino terminus of a polypeptide, the propeptide region is positioned next to the amino terminus of a polypeptide and the signal peptide region is positioned next to the amino terminus of the propeptide region.

[0287] In some embodiments, regulatory sequences are also utilized. These sequences facilitate the regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those that cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. In prokaryotic host cells, suitable regulatory sequences include, but are not limited to the lac, tac, and trp operator systems. In yeast host cells, suitable regulatory systems include, but are not limited to the ADH2 system or GALI system. In filamentous fungi, suitable regulatory sequences include, but are not limited to the TAKA alpha-amylase promoter, Aspergillus niger glucoamylase promoter, and Aspergillus oryzae glucoamylase promoter. Exemplary inducible promoters regulated by exogenous agents include the zinc-inducible sheep metallothionine (MT) promoter, dexamethasone (Dex)- inducible promoter, mouse mammary tumor virus (MMTV) promoter; ecdysone insect promoter, tetracycline-inducible promoter system, RU486-inducible promoter system, and the rapamycin- inducible promoter system.

[0288] The present invention also provides recombinant expression vectors comprising a recombinant polynucleotide encoding an engineered pyruvate oxidase polypeptide, and one or more expression regulating regions such as a promoter and a terminator, a replication origin, etc., depending on the type of hosts into which they are to be introduced. In some embodiments, the various nucleic acid and control sequences described above are combined together to produce a recombinant expression vector which includes one or more convenient restriction sites to allow for insertion or substitution of the nucleic acid sequence encoding the variant pyruvate oxidase polypeptide at such sites. Alternatively, the polynucleotide sequence(s) of the present invention are expressed by inserting the polynucleotide sequence or a nucleic acid construct comprising the polynucleotide sequence into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

[0289] The recombinant expression vector may be any vector (e.g., a plasmid or virus), that can be conveniently subjected to recombinant DNA procedures and can result in the expression of the variant pyruvate oxidase polynucleotide sequence. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids.

[0290] In some embodiments, the expression vector is an autonomously replicating vector (i.e., a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, such as a plasmid, an extra-chromosomal element, a minichromosome, or an artificial chromosome). The vector may contain any means for assuring self-replication. In some alternative embodiments, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used.

[0291] In some embodiment, the recombinant polynucleotides may be provided on a non-replicating expression vector or plasmid. In some embodiments, the non-replicating expression vector or plasmid can be based on viral vectors defective in replication (see, e.g., Travieso et al., npj Vaccines, 2022, Vol. 7, Article 75).

[0292] In some embodiments, the expression vector preferably contains one or more selectable markers, which permit easy selection of transformed cells. A “selectable marker” is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophy, and the like. Examples of bacterial selectable markers include but are not limited to the dal genes from Bacillus subtilis or Bacillus lichenifonnis, or markers, which confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Suitable markersfor yeast host cells include, but are not limited to ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungal host cell include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferases), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. In another aspect, the present invention provides a host cell comprising a polynucleotide encoding at least one engineered pyruvate oxidase polypeptide of the present invention, the polynucleotide being operatively linked to one or more control sequences for expression of the engineered pyruvate oxidase enzyme(s) in the host cell.

[0293] In another aspect, the present disclosure provides a host cell comprising a recombinant polynucleotide encoding an engineered pyruvate oxidase polypeptide described herein, the polynucleotide(s) being operably linked to one or more control sequences for expression of the engineered pyruvate oxidase enzyme(s) in the host cell. Host cells suitable for use in expressing the polypeptides encoded by the expression vectors of the present invention are known in the art and include but are not limited to, bacterial cells, such as E. coli, B. subtilis, Vibrio fluvialis, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, BHK, 293, and Bowes melanoma cells; and plant cells. Exemplary host cells also include various Escherichia coli strains (e.g., W3110 (AfhuA) and BL21).

[0294] In some embodiments, the host cell strain comprises a knockout of one or more genes, in particular phosphatase genes. In some embodiments, the host cell comprises a knockout or single gene deletion of E. coli genes aphA, surE, phoA, and / or cpdB, as described below in the Examples. In some embodiments, the host cell comprising a knockout of one or more phosphatase genes has increased production of the product and / or decreased de-phosphorylation of the product or substrate.

[0295] Accordingly, in another aspect, the present invention provides methods for producing the engineered pyruvate oxidase polypeptides, where the methods comprise culturing a host cell capable of expressing a polynucleotide encoding the engineered pyruvate oxidase polypeptide under conditions suitable for expression of the polypeptide. In some embodiments, the method further comprises isolating the engineered pyruvate oxidase polypeptides from the culture and / or host cells. In some embodiments, the method further comprises purifying the expressed pyruvate oxidase polypeptide, as described herein.

[0296] Suitable culture media and growth conditions for the above-described host cells are well- known in the art. Polynucleotides for expression of the pyruvate oxidase polypeptides may be introduced into the host cells by various methods known in the art. Techniques include, among others,electroporation, biolistic particle bombardment, liposome mediated transfection, calcium chloride transfection, and protoplast fusion.

[0297] In some embodiments, recovery of the engineered pyruvate oxidase enzymes from the cells and / or the culture medium, and purification of the expressed pyruvate oxidase can use any one or more of the known techniques for protein isolation and purification, including, among others, lysozyme treatment, sonication, filtration, salting-out, ultra-centrifugation, and chromatography. Suitable solutions for lysing and the high efficiency extraction of proteins from bacteria, such as E. coli, are commercially available (e.g., CelLytic B™, Sigma-Aldrich, St. Louis MO).

[0298] Chromatographic techniques for isolation of the pyruvate oxidase polypeptide include, among others, reverse phase chromatography high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, and affinity chromatography. Conditions for purifying a particular enzyme will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc., and will be apparent to those having skill in the art.

[0299] In some embodiments, affinity techniques may be used to isolate the improved pyruvate oxidase enzymes. For affinity chromatography purification, any antibody which specifically binds the pyruvate oxidase polypeptide may be used. For the production of antibodies, various host animals, including but not limited to rabbits, mice, rats, etc., may be immunized by injection with a pyruvate oxidase polypeptide, or a fragment thereof. The pyruvate oxidase polypeptide or fragment may be attached to a suitable carrier, such as BSA, by means of a side chain functional group or linkers attached to a side chain functional group. In some embodiments, the affinity purification can use a specific ligand bound by the pyruvate oxidase or dye affinity column (See e.g., EP0641862;Stellwagen, “Dye Affinity Chromatography,” In Current Protocols in Protein Science, Unit 9.2-9.2.16 (2001)).

[0300] The engineered pyruvate oxidases with the properties disclosed herein can be obtained by subjecting the polynucleotide encoding the naturally occurring or engineered pyruvate oxidase polypeptide to mutagenesis and / or directed evolution methods known in the art, and as described herein. An exemplary directed evolution technique is mutagenesis and / or DNA shuffling (See e.g., Stemmer, Proc. Natl. Acad. Sci. USA, 1994, 91:10747-10751; WO 95 / 22625; WO 97 / 0078; WO 97 / 35966; WO 98 / 27230; WO 00 / 42651; WO 01 / 75767 and U.S. Pat. 6,537,746). Other directed evolution procedures that can be used include, among others, staggered extension process (StEP), in vitro recombination (See e.g., Zhao et al., Nat. Biotechnol., 1998, 16:258-261), mutagenic PCR (See e.g., Caldwell et al., PCR Methods AppL, 1994, 3:S136-S140), and cassette mutagenesis (See e.g., Black et al., Proc. Natl. Acad. Sci. USA, 1996, 93:3525-3529).

[0301] For example, mutagenesis and directed evolution methods can be readily applied to polynucleotides to generate variant libraries that can be expressed, screened, and assayed.Mutagenesis and directed evolution methods are known in the art (See e.g., US Patent Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, 5,837,458, 5,928,905, 6,096,548, 6,117,679, 6,132,970, 6,165,793, 6,180,406, 6,251,674, 6,265,201, 6,277,638, 6,287,861, 6,287,862, 6,291,242, 6,297,053, 6,303,344, 6,309,883, 6,319,713, 6,319,714, 6,323,030, 6,326,204, 6,335,160, 6,335,198, 6,344,356, 6,352,859, 6,355,484, 6,358,740, 6,358,742, 6,365,377, 6,365,408, 6,368,861, 6,372,497, 6,337,186, 6,376,246, 6,379,964, 6,387,702, 6,391,552, 6,391,640, 6,395,547, 6,406,855, 6,406,910, 6,413,745, 6,413,774, 6,420,175, 6,423,542, 6,426,224, 6,436,675, 6,444,468, 6,455,253, 6,479,652, 6,482,647, 6,483,011, 6,484,105, 6,489,146, 6,500,617, 6,500,639, 6,506,602, 6,506,603, 6,518,065, 6,519,065, 6,521,453, 6,528,311, 6,537,746, 6,573,098, 6,576,467, 6,579,678, 6,586,182, 6,602,986, 6,605,430, 6,613,514, 6,653,072, 6,686,515, 6,703,240, 6,716,631, 6,825,001, 6,902,922, 6,917,882, 6,946,296, 6,961,664, 6,995,017, 7,024,312, 7,058,515, 7,105,297, 7,148,054, 7,220,566, 7,288,375, 7,384,387, 7,421,347, 7,430,477, 7,462,469, 7,534,564, 7,620,500, 7,620,502, 7,629,170, 7,702,464, 7,747,391, 7,747,393, 7,751,986, 7,776,598, 7,783,428, 7,795,030, 7,853,410, 7,868,138, 7,783,428, 7,873,477, 7,873,499, 7,904,249, 7,957,912, 7,981,614, 8,014,961, 8,029,988, 8,048,674, 8,058,001, 8,076,138, 8,108,150, 8,170,806, 8,224,580, 8,377,681, 8,383,346, 8,457,903, 8,504,498, 8,589,085, 8,762,066, 8,768,871, 9,593,326, and all related US, as well as PCT and non-US counterparts; Ling et al., Anal. Biochem., 1997, 254(2): 157-78; Dale et al., Meth. Mol. Biol., 1996, 57:369-74; Smith, Ann. Rev. Genet., 1985, 19:423-462; Botstein et al., Science, 1985, 229: 1193-1201; Carter, 1986, Biochem. J., 237: 1-7;Kramer et al., Cell, 1984, 38:879-887; Wells et al., Gene, 1985, 34:315-323; Minshull et al., Curr. Op. Chem. BioL, 1999, 3:284-290; Christians et al., Nat. Biotechnol., 1999, 17:259-264; Crameri et al., Nature, 1998, 391:288-291; Crameri, et al., Nat. Biotechnol., 1997, 15:436-438; Zhang et al., Proc. Nat. Acad. Sci. U.S.A., 1997, 94:4504-4509; Crameri et al., Nat. Biotechnol., 1996, 14:315-319; Stcmmcr, 1994, Nature, 370:389-391; Stcmmcr, Proc. Nat. Acad. Sci. USA, 1994, 91:10747-10751; WO 95 / 22625; WO 97 / 0078; WO 97 / 35966; WO 98 / 27230; WO 00 / 42651; WO 01 / 75767; and WO 2009 / 152336, all of which are incorporated herein by reference).

[0302] In some embodiments, the enzyme clones obtained following mutagenesis treatment are screened by subjecting the enzymes to a defined temperature (or other assay conditions, such as testing the enzyme’s activity over a broad range of substrates) and measuring the amount of enzyme activity remaining after heat treatments or other assay conditions. Clones containing a polynucleotide encoding a pyruvate oxidase polypeptide are then sequenced to identify the nucleotide sequence changes (if any) and used to express the enzyme in a host cell. Measuring enzyme activity from the expression libraries can be performed using any suitable method known in the art (e.g., standard biochemistry techniques, such as HPLC analysis) and as provided in the Examples.

[0303] In some embodiments, the clones obtained following mutagenesis treatment can be screened for engineered pyruvate oxidases having one or more desired improved enzyme properties (e.g., improved regioselectivity). Measuring enzyme activity from the expression libraries can beperformed using the standard biochemistry techniques, such as HPLC analysis, LC-MS analysis, RapidFire-MS analysis, and / or capillary electrophoresis analysis.

[0304] When the sequence of the engineered polypeptide is known, the polynucleotides encoding the enzyme can be prepared by standard solid-phase methods, according to known synthetic methods. In some embodiments, fragments of up to about 100 bases can be individually synthesized, then joined (e.g., by enzymatic or chemical ligation methods, or polymerase mediated methods) to form any desired continuous sequence. For example, polynucleotides and oligonucleotides encoding portions of the pyruvate oxidase can be prepared by chemical synthesis as known in the art (e.g., the classical phosphoramidite method of Beaucage et al., 1981, Tet. Lett. 22: 1859-69, or the method described by Matthes et al., 1984, EMBO J. 3:801-05) as typically practiced in automated synthetic methods. According to the phosphoramidite method, oligonucleotides are synthesized (e.g., in an automatic DNA synthesizer), purified, annealed, ligated and cloned in appropriate vectors. In addition, essentially any nucleic acid can be obtained from any of a variety of commercial sources. In some embodiments, additional variations can be created by synthesizing oligonucleotides containing deletions, insertions, and / or substitutions, and combining the oligonucleotides in various permutations to create engineered pyruvate oxidases with improved properties.

[0305] Accordingly, in some embodiments, a method for preparing the engineered pyruvate oxidase polypeptide comprises: (a) synthesizing a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to an amino acid sequence corresponding to amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 14-636 and 692-876, or to an amino acid sequence corresponding to an even- numbered SEQ ID NO. of SEQ ID NOs: 14-636 and 692-876, and having one or more residue differences as compared to residues 12-604 of SEQ ID NO: 14-636 and 692-876, or compared to SEQ ID NO: 6, 16, 56, 184, 264, 290, 400, or 696; and (b) expressing the pyruvate oxidase polypeptide encoded by the polynucleotide.

[0306] In some embodiments of the method, the polynucleotide encodes an engineered pyruvate oxidase that has optionally one or several (e.g., up to 3, 4, 5, or up to 10) amino acid residue deletions, insertions and / or substitutions. In some embodiments, the amino acid sequence has optionally 1-2, 1- 3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45, 1- 50, 1-75, 1-100, or 1-150 amino acid residue deletions, insertions and / or substitutions. In some embodiments, the amino acid sequence has optionally around 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acid residue deletions, insertions and / or substitutions. In some embodiments, the substitutions can be conservative or non-conservative substitutions.Methods of Using the Engineered Pyruvate Oxidase Enzymes

[0307] In a further aspect, the present disclosure provides methods to generate acetyl phosphate from pyruvate using a pyruvate oxidase. In some embodiments, the present disclosure provides pyruvate oxidase enzymes for use in the method for the conversion of pyruvate, oxygen, and phosphate to acetyl phosphate, hydrogen peroxide, and carbon dioxide. In some embodiments, the pyruvate oxidase is used in coupled reaction as a phosphate recycling enzyme. A non-limiting example of the use of pyruvate oxidase coupled with acetate kinase during NTP is depicted in Scheme 1, above.

[0308] In some embodiments, the pyruvate oxidase is further coupled with a catalase enzyme to degrade hydrogen peroxide to water and oxygen.

[0309] In any of the above embodiments, the method may further comprise a source of phosphate from a salt or buffer, such as potassium phosphate, sodium phosphate, combinations of potassium phosphate and sodium phosphate, or other phosphate buffers.

[0310] In particular, the engineered pyruvate oxidase polypeptides of the present disclosure have been engineered for increased activity, thermostability, and activity at high temperatures. A variety of suitable reaction conditions are known to those skilled in the art, including the reaction conditions detailed in the Examples. A variety of methods of generating acetyl phosphate are possible using the enzymes, substrates, and cofactors described herein. These embodiments are intended to be nonlimiting; the present disclosure contemplates methods comprising every combination of enzymes, substrates, and cofactors.

[0311] In some embodiments, a method of producing acetyl phosphate comprises contacting an engineered pyruvate oxidase with pyruvate under suitable reaction conditions that cause the production of acetyl phosphate.

[0312] In certain embodiments, the present disclosure provides a method of producing acetyl phosphate, the method comprising (i) providing an engineered pyruvate oxidase comprising at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to an amino acid sequence corresponding to amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 14-636 and 692-876, or to an amino acid sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 14-636 and 692-876, and (ii) contacting the engineered pyruvate oxidase with pyruvate, under suitable reaction conditions, such that acetyl phosphate is produced. In some embodiments, the method comprises (i) providing an engineered pyruvate oxidase comprising at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to an amino acid sequence corresponding to amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 14-636 and 692-876, and (ii) contacting the engineered pyruvate oxidase with pyruvate, under suitable reaction conditions, such that acetyl phosphate is produced,wherein said engineered pyruvate oxidase converts said pyruvate to acetyl phosphate with a conversion rate that is at least 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 2 fold, 2.8 fold, 4 fold, 9.5 fold, 20 fold, 25 fold, 50 fold, 100 fold, or more increased, as compared to a wild type or reference pyruvate oxidase of SEQ ID NO: 6, 56, 264, 400, or 696.

[0313] In certain embodiments, the present disclosure provides a method of producing acetyl phosphate, the method comprising (i) providing an engineered pyruvate oxidase comprising at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to an amino acid sequence corresponding to amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 14-636 and 692-876, and (ii) contacting the engineered pyruvate oxidase with pyruvate, under suitable reaction conditions, such that acetyl phosphate is produced. In some embodiments, the method comprises (i) providing an engineered pyruvate oxidase comprising at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to an amino acid sequence corresponding to amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 14-636 and 692-876, or to an amino acid sequence corresponding to an even- numbered SEQ ID NO. of SEQ ID NOs: 14-636 and 692-876, and comprising a substitution of 336T, 512A, or 308 K, or combinations thereof, wherein said positions are numbered with reference to SEQ ID NO: 6, and (ii) contacting the engineered pyruvate oxidase with pyruvate, under suitable reaction conditions, such that acetyl phosphate is produced, wherein said engineered pyruvate oxidase converts said pyruvate to acetyl phosphate with a conversion rate that is at least 2.8 fold or more increased, as compared to the pyruvate oxidase of SEQ ID NO: 6.

[0314] In certain embodiments, the present disclosure provides a method of producing acetyl phosphate, the method comprising (i) providing an engineered pyruvate oxidase comprising at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to an amino acid sequence corresponding to amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 14-636 and 692-876, or to an amino acid sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 14-636 and 692-876, and (ii) contacting the engineered pyruvate oxidase with pyruvate, under suitable reaction conditions, such that acetyl phosphate is produced. In some embodiments, the method comprises (i) providing an engineered pyruvate oxidase comprising at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to an amino acid sequence corresponding to amino acid residues 1 -604 of SEQ ID NO: 6, or and an amino acid sequence corresponding to SEQ ID NO: 6, and (ii) contacting the engineered pyruvate oxidase with pyruvate, under suitable reaction conditions, such that acetyl phosphate is produced, wherein said engineered pyruvate oxidase converts said pyruvate to acetyl phosphate with aconversion rate that is at least 2 fold, or more increased, as compared to the reference pyruvate oxidase of SEQ ID NO: 6.

[0315] In any of the above embodiments of the method, the pyruvate oxidase can exhibit increased activity, increased activity on non-natural substrates, increased selectivity, increased substrate promiscuity, decreased product inhibition, and / or decreased byproduct formation, as compared to a wild-type or reference pyruvate oxidase.

[0316] In some embodiments, the enzymes described herein find use in processes for conversion of one or more suitable substrates to a product.

[0317] In the embodiments provided herein and illustrated in the Examples, various ranges of suitable reaction conditions that can be used in the processes, include but are not limited to, substrate loading, co-substrate loading, pH, temperature, buffer, solvent system, cofactor, polypeptide loading, and reaction time. Further suitable reaction conditions for carrying out the process for biocatalytic conversion of substrate compounds to product compounds using the enzymes described herein can be determined in view of the guidance provided herein by routine experimentation that includes, but is not limited to, contacting the enzymes and one or more substrate compounds under experimental reaction conditions of concentration, pH, temperature, and solvent conditions, and detecting the product compound. The reaction conditions described herein are examples only. The present disclosure contemplates any suitable reaction conditions that may find use in the methods described herein.

[0318] The substrate compound in the reaction mixtures can be varied, taking into consideration, for example, the desired amount of product compound, the effect of each substrate concentration on enzyme activity, stability of enzyme under reaction conditions, and the percent conversion of each substrate to product. In some embodiments, the suitable reaction conditions comprise a substrate compound loading of about 0.1 mM to 5000 mM, about 1 mM to 4000 mM, about 2 mM to 3000 mM, about 5 mM to 2000 mM, about 10 mM to 1000 mM, about 15 mM to 800 mM, about 20 mM to 500 mM, about 30 mM to 400 mM, about 40 mM to 300 mM, about 50 mM to 200 mM, or about 60 mM to 100 mM. In some embodiments, the suitable reaction conditions comprise a substrate compound loading of at least about 0.1 mM to 5 mM, 5 mM to 10 mM, 10 mM to 30 mM, 30 mM to 50 mM, 50 mM to 100 mM, 100 mM to 1000 mM, 1000 mM to 5000 mM, or greater.

[0319] In some embodiments, the suitable reaction conditions comprise a substrate compound loading of about 0.5 to about 50 g / L, about 1 to about 40 g / L, about 5 to about 35 g / L, about 10 to about 30 g / L, or about 15 to about 25 g / L. In some embodiments, the suitable reaction conditions comprise a substrate compound loading of at least about 0.5 to about 25 g / L, about 1 to about 25 g / L, about 5 to about 25 g / L, about 10 to about 25 g / L, or about 20 to about 25 g / L. In some embodiments, the suitable reaction conditions comprise a substrate compound loading of at least about 0.5 g / L, atleast about 1 g / L, at least about 5 g / L, at least about 10 g / L, at least about 15 g / L, at least about 20 g / L, at least about 25 g / L, at least about 30 g / L, at least about 35 g / L, at least about 40 g / L, at least about 50 g / L, or even greater.

[0320] In carrying out the synthesis processes described herein, the engineered polypeptides may be added to the reaction mixture in the form of a purified enzyme, partially purified enzyme, whole cells transformed with gene(s) encoding the enzyme, as cell extracts and / or lysates of such cells, and / or as an enzyme immobilized on a solid support. Whole cells transformed with gene(s) encoding the enzyme(s) or cell extracts, lysates thereof, and isolated enzymes may be employed in a variety of different forms, including solid (e.g., lyophilized, spray-dried, and the like) or semisolid (e.g., a crude paste). The cell extracts or cell lysates may be partially purified by precipitation (ammonium sulfate, polyethyleneimine, heat treatment or the like, followed by a desalting procedure prior to lyophilization (e.g., ultrafiltration, dialysis, etc.). Any of the enzyme preparations (including whole cell preparations) may be stabilized by crosslinking using known crosslinking agents, such as, for example, glutaraldehyde or immobilization to a solid phase (e.g., Eupergit C, and the like).

[0321] The gene(s) encoding the polypeptides can be transformed into host cell separately or together into the same host cell. For example, in some embodiments one set of host cells can be transformed with gene(s) encoding one polypeptide and another set can be transformed with gene(s) encoding another polypeptide. Both sets of transformed cells can be utilized together in the reaction mixture in the form of whole cells, or in the form of lysates or extracts derived therefrom. In other embodiments, a host cell can be transformed with gene(s) encoding multiple polypeptides. In some embodiments the polypeptides can be expressed in the form of secreted polypeptides and the culture medium containing the secreted polypeptides can be used for the synthesis reaction.

[0322] In some embodiments, the improved activity of the engineered pyruvate oxidase polypeptides disclosed herein provides for processes wherein higher percentage conversion can be achieved with lower concentrations of the engineered polypeptide. In some embodiments of the process, the suitable reaction conditions comprise an engineered polypeptide amount of about 1% (w / w), 2% (w / w), 5% (w / w), 10% (w / w), 20% (w / w), 30% (w / w), 40% (w / w), 50% (w / w), 75% (w / w), 100% (w / w) or more of substrate compound loading.

[0323] In some embodiments, the engineered pyruvate oxidase polypeptide is present at a molar ratio of engineered polypeptide to substrate of about 50 to 1, 25 to 1, 10 to 1, 5 to 1, 1 to 1, 1 to 5, 1 to 10, 1 to 25 or 1 to 50. In some embodiments, the engineered polypeptide is present at a molar ratio of engineered polypeptide to substrate from a range of about 50 to 1 to a range of about 1 to 50.

[0324] In some embodiments, the engineered polypeptide is present at about 0.01 g / L to about 50 g / L; about 0.01 to about 0.1 g / L; about 0.05 g / L to about 50 g / L; about 0.1 g / L to about 40 g / L; about 1 g / L to about 40 g / L; about 2 g / L to about 40 g / L; about 5 g / L to about 40 g / L; about 5 g / L to about30 g / L; about 0.1 g / L to about 10 g / L; about 0.5 g / L to about 10 g / L; about 1 g / L to about 10 g / L; about 0.1 g / L to about 5 g / L; about 0.5 g / L to about 5 g / L; or about 0.1 g / L to about 2 g / L. In some embodiments, the pyruvate oxidase polypeptide is present at about 0.01 g / L, 0.05 g / L, 0.1 g / L, 0.2 g / L, 0.5 g / L, 1, 2 g / L, 5 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, or 50 g / L.

[0325] During the course of the reaction, the pH of the reaction mixture may change. The pH of the reaction mixture may be maintained at a desired pH or within a desired pH range. This may be done by the addition of an acid or a base, before and / or during the course of the reaction. Alternatively, the pH may be controlled by using a buffer. Accordingly, in some embodiments, the reaction condition comprises a buffer. Suitable buffers to maintain desired pH ranges are known in the art and include, by way of example and not limitation, borate, potassium phosphate, 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), acetate, triethanolamine, and 2-amino- 2-hydroxymethyl-propane-l,3-diol (Tris), and the like. In some embodiments, the buffer, when used, is present at a concentration of about 1 mM-500 mM, about 5 mM to 450 mM, about 10 mM to 400 mM, about 20 mM to 350 mM, about 30 mM to 300 mM, about 40 mM to 200 mM, or about 50 mM to 100 mM. In some embodiments, the buffer is present at about 1 mM, about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM or about 500 mM. In some embodiments, the reaction conditions comprise water as a suitable solvent with no buffer present.

[0326] In some embodiments of the process, the reaction conditions comprise a divalent metal cofactor. Suitable divalent metal cofactors include magnesium (Mg+2), manganese (Mn+2), nickel (Ni+2), and cobalt (Co+2). In some embodiments, the divalent metal cofactor may be present as a salt, such as, magnesium chloride (MgCh). In some embodiments, the divalent metal is present at a concentration of about 0.5 mM to about 5 mM, about 1 mM to about 50 mM, or about 5 mM to about 100 mM. In some embodiments, the divalent metal is present at about 0.5 mM, about 1 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, or about 100 mM, or greater.

[0327] In some embodiments of the process, a cofactor may be added to the reaction mixture and / or to the growth medium for the engineered polypeptides. In some embodiments, the cofactor is thiamine pyrophosphate or flavin adenine dinucleotide. In some embodiments, thiamin or another derivative is used.

[0328] In the embodiments of the process, the reaction conditions comprise a suitable pH. The desired pH or desired pH range can be maintained by use of an acid or base, an appropriate buffer, or a combination of buffering and acid or base addition. The pH of the reaction mixture can be controlled before and / or during the course of the reaction. In some embodiments, the suitable reactionconditions comprise a solution pH from about 4 to about 10, pH from about 5 to about 10, pH from about 5 to about 9, pH from about 6 to about 9, pH from about 6 to about 8. In some embodiments, the reaction conditions comprise a solution pH of about 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10.

[0329] In the embodiments of the processes herein, a suitable temperature is used for the reaction conditions, for example, taking into consideration the increase in reaction rate at higher temperatures, and the activity of the enzyme during the reaction time period. Accordingly, in some embodiments, the suitable reaction conditions comprise a temperature of about 10 °C to about 95 °C, about 10 °C to about 75 °C, about 15 °C to about 95 °C, about 20 °C to about 95 °C, about 20 °C to about 65 °C, about 25 °C to about 70 °C, or about 50 °C to about 70 °C. In some embodiments, the suitable reaction conditions comprise a temperature of about 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, 75 °C, 80 °C, 85 °C, 90 °C or 95 °C. In some embodiments, the temperature during the enzymatic reaction can be maintained at a specific temperature throughout the course of the reaction. In some embodiments, the temperature during the enzymatic reaction can be adjusted over a temperature profile during the course of the reaction.

[0330] In some embodiments, the processes of the invention are carried out in a solvent. Suitable solvents include water, aqueous buffer solutions, organic solvents, polymeric solvents, and / or cosolvent systems, which generally comprise aqueous solvents, organic solvents and / or polymeric solvents. The aqueous solvent (water or aqueous co-solvent system) may be pH-buffered or unbuffered. In some embodiments, the processes using the engineered pyruvate oxidase polypeptides can be carried out in an aqueous co-solvent system comprising an organic solvent (e.g., ethanol, isopropanol (IP A), dimethyl sulfoxide (DMSO), dimethylformamide (DMF) ethyl acetate, butyl acetate, 1 -octanol, heptane, octane, methyl t butyl ether (MTBE), toluene, and the like), ionic or polar solvents (e.g., 1-ethyl 4 methylimidazolium tetrafluoroborate, l-butyl-3-methylimidazolium tetrafluoroborate, 1 -butyl 3 methylimidazolium hexafluorophosphate, glycerol, polyethylene glycol, and the like). In some embodiments, the co-solvent can be a polar solvent, such as a polyol, dimethylsulfoxide (DMSO), or lower alcohol. The non-aqueous co- solvent component of an aqueous co-solvent system may be miscible with the aqueous component, providing a single liquid phase, or may be partly miscible or immiscible with the aqueous component, providing two liquid phases.Exemplary aqueous co-solvent systems can comprise water and one or more co-solvents selected from an organic solvent, polar solvent, and polyol solvent. In general, the co-solvent component of an aqueous co-solvent system is chosen such that it does not adversely inactivate the enzymes under the reaction conditions. Appropriate co-solvent systems can be readily identified by measuring the enzymatic activity of the specified enzymes with a defined substrate of interest in the candidate solvent system, utilizing an enzyme activity assay, such as those described herein.

[0331] In some embodiments of the process, the suitable reaction conditions comprise an aqueous cosolvent, where the co-solvent comprises DMSO at about 1% to about 50% (v / v), about 1 to about 40% (v / v), about 2% to about 40% (v / v), about 5% to about 30% (v / v), about 10% to about 30% (v / v), or about 10% to about 20% (v / v). In some embodiments of the process, the suitable reaction conditions can comprise an aqueous co-solvent comprising ethanol at about 1% (v / v), about 5% (v / v), about 10% (v / v), about 15% (v / v), about 20% (v / v), about 25% (v / v), about 30% (v / v), about 35% (v / v), about 40% (v / v), about 45% (v / v), or about 50% (v / v).

[0332] In some embodiments, the reaction conditions comprise a surfactant for stabilizing or enhancing the reaction. Surfactants can comprise non-ionic, cationic, anionic and / or amphiphilic surfactants. Exemplary surfactants, include by way of example and not limitation, nonyl phenoxy polyethoxylethanol (NP40), TRITON™ X-100 polyethylene glycol tert-octylphenyl ether, polyoxyethylene-stearylamine, cetyltrimethylammonium bromide, sodium oleylamidosulfate, polyoxyethylene-sorbitanmonostearate, hexadecyldimethylamine, etc. Any surfactant that may stabilize or enhance the reaction may be employed. The concentration of the surfactant to be employed in the reaction may be generally from 0.1 to 50 mg / ml, particularly from 1 to 20 mg / ml.

[0333] In some embodiments, the reaction conditions include an antifoam agent, which aids in reducing or preventing formation of foam in the reaction solution, such as when the reaction solutions are mixed or sparged. Anti-foam agents include non-polar oils (e.g., minerals, silicones, etc.), polar oils (e.g., fatty acids, alkyl amines, alkyl amides, alkyl sulfates, etc.), and hydrophobic (e.g., treated silica, polypropylene, etc.), some of which also function as surfactants. Exemplary anti-foam agents include Y-30® (Dow Corning), poly-glycol copolymers, oxy / ethoxylated alcohols, and polydimethylsiloxanes. In some embodiments, the anti-foam can be present at about 0.001% (v / v) to about 5% (v / v), about 0.01% (v / v) to about 5% (v / v), about 0.1% (v / v) to about 5% (v / v), or about 0.1% (v / v) to about 2% (v / v). In some embodiments, the anti-foam agent can be present at about 0.001% (v / v), about 0.01% (v / v), about 0.1% (v / v), about 0.5% (v / v), about 1% (v / v), about 2% (v / v), about 3% (v / v), about 4% (v / v), or about 5% (v / v) or more as desirable to promote the reaction.

[0334] The quantities of reactants used in the synthesis reaction will generally vary depending on the quantities of product desired, and concomitantly the amount of substrates employed. Those having ordinary skill in the art will readily understand how to vary these quantities to tailor them to the desired level of productivity and scale of production.

[0335] In some embodiments, the order of addition of reactants is not critical. The reactants may be added together at the same time to a solvent (e.g., monophasic solvent, biphasic aqueous co-solvent system, and the like), or alternatively, some of the reactants may be added separately, and some together at different time points. For example, the cofactor, co-substrate and substrate may be added first to the solvent.

[0336] The solid reactants (e.g., enzyme, salts, etc.) may be provided to the reaction in a variety of different forms, including powder (e.g., lyophilized, spray dried, and the like), solution, emulsion, suspension, and the like. The reactants can be readily lyophilized or spray dried using methods and equipment that are known to those having ordinary skill in the art. For example, the protein solution can be frozen at -80 °C in small aliquots, then added to a pre-chilled lyophilization chamber, followed by the application of a vacuum.

[0337] For improved mixing efficiency when an aqueous co-solvent system is used, the polypeptide(s), and co-substrate may be added and mixed into the aqueous phase first. The substrate may be added and mixed in, followed by the organic phase or the substrate may be dissolved in the organic phase and mixed in. Alternatively, the substrate may be premixed in the organic phase, prior to addition to the aqueous phase.

[0338] The processes of the present invention are generally allowed to proceed until further conversion of substrate to product does not change significantly with reaction time (e.g., less than 10% of substrate being converted, or less than 5% of substrate being converted). In some embodiments, the reaction is allowed to proceed until there is complete or near complete conversion of substrate to product. Transformation of substrate to product can be monitored using known methods by detecting substrate and / or product, with or without derivatization. Suitable analytical methods include gas chromatography, HPLC, MS, and the like. In some embodiments, after suitable conversion to product, the reactants are separated from the product and additional reactants are added.

[0339] Any of the processes disclosed herein using the polypeptides for the preparation of products can be carried out under a range of suitable reaction conditions, including but not limited to ranges of substrates, temperature, pH, solvent system, substrate loading, polypeptide loading, cofactor loading, and reaction time. In one example, the suitable reaction conditions for the conversion of pyruvate to acetyl phosphate comprise: (a) sodium pyruvate substrate loading of about 25-5000 mM; (b) about 0.01 g / L to 5 g / L engineered pyruvate oxidase polypeptide; (c) 1-100 mM MgCb; (e) 5 to 100 mM of buffer, e.g., Tris-HCl buffer; (f) 10-5000 mM potassium phosphate (K2HPO4); (g) pH at 6-9; and (h) temperature of about 15 °C to 70 °C. In one example, the suitable reaction conditions for the conversion of an NTP to an NQP comprise: (a) substrate loading of about 50 mM sodium pyruvate;(b) about 0.01 g / L to 5 g / L engineered pyruvate oxidase polypeptide; (c) 10 mM MgCh; (e) 50 mM of buffer, e.g., Tris-HCl buffer; (f) 50 mM potassium phosphate (K2HPO4); (g) pH 8; and (h) temperature of about 40 °C. In some embodiments, the enzyme loading is between 1-30% w / w. In some embodiments, additional reaction components or additional techniques carried out to supplement the reaction conditions. These can include taking measures to stabilize or prevent inactivation of the enzyme, reduce product inhibition, shift reaction equilibrium to formation of the desired product.

[0340] Accordingly, it is further contemplated that any of the methods of using the polypeptides of the present invention can be carried out using the polypeptides bound or immobilized on a solid support.

[0341] Methods of enzyme immobilization are well-known in the art. The engineered polypeptides can be bound non-covalently or covalently. Various methods for conjugation and immobilization of enzymes to solid supports (e.g., resins, membranes, beads, glass, etc.) are well known in the art (See e.g., Yi et al., Proc. Biochem., 2007, 42(5): 895-898; Martin et al., AppL Microbiol. Biotechnol., 2007, 76(4): 843-851; Koszelewski et al., J. Mol. Cat. B: Enzymatic, 2010. 63: 39-44; Truppo et al., Org. Proc. Res. Dev., published online: dx.doi.org / 10.1021 / op200157c; Hermanson, Bioconjugate Techniques, 2nded., Academic Press, Cambridge, MA (2008); Mateo et al., Biotechnol. Prog., 2002, 18(3):629-34; and “Bioconjugation Protocols: Strategies and Methods,” In Methods in Molecular Biology, Niemeyer (ed.), Humana Press, New York, NY (2004); the disclosures of each which are incorporated by reference herein). Solid supports useful for immobilizing the engineered pyruvate oxidase of the present invention include but are not limited to beads or resins comprising polymethacrylate with epoxide functional groups, polymethacrylate with amino epoxide functional groups, styrene / DVB copolymer or polymethacrylate with octadecyl functional groups. Exemplary solid supports useful for immobilizing the engineered pyruvate oxidase polypeptides of the present invention include, but are not limited to, EnginZyme (including, EziG-1, EziG-1, and EziG-3), chitosan beads, Eupergit C, and SEPABEADs (Mitsubishi) (including EC-EP, EC-HFA / S, EXA252, EXE119 and EXE120).

[0342] In further embodiments, any of the above-described processes for the conversion of one or more substrate compounds to product compound can further comprise one or more steps selected from: extraction; isolation; purification; and crystallization of product compound. Methods, techniques, and protocols for extracting, isolating, purifying, and / or crystallizing the product from biocatalytic reaction mixtures produced by the above disclosed processes are known to the ordinary artisan and / or accessed through routine experimentation. Additionally, illustrative methods are provided in the Examples below.

[0343] Various features and embodiments of the invention are illustrated in the following representative examples, which are intended to be illustrative, and not limiting.EXAMPLES

[0344] The following Examples, including experiments and results achieved, are provided for illustrative purposes only and are not to be construed as limiting the present invention. Indeed, there are various suitable sources for many of the reagents and equipment described below. It is not intended that the present invention be limited to any particular source for any reagent or equipment item.

[0345] In the experimental disclosure below, the following abbreviations apply: M (molar); mM (millimolar), uM and p,M (micromolar); nM (nanomolar); mol (moles); gm and g (gram); mg (milligrams); ug and pg (micrograms); L and 1 (liter); ml and mL (milliliter); cm (centimeters); mm (millimeters); uni and pii] (micrometers); sec. (seconds); min(s) (minute(s)); h(s) and hr(s) (hour(s)); U (units); MW (molecular weight); rpm (rotations per minute); psi and PSI (pounds per square inch); °C (degrees Celius); RT and rt (room temperature); CV (coefficient of variability); CAM and cam (chloramphenicol); PMBS (polymyxin B sulfate); IPTG (isopropyl [3-D-l-thiogalactopyranoside); LB (lysogeny broth); TB (terrific broth); SFP (shake flask powder); CDS (coding sequence); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); nt (nucleotide; polynucleotide); aa (amino acid; polypeptide); E. coli W3110 (commonly used laboratory E. coli strain, available from the Coli Genetic Stock Center [CGSC], New Haven, CT); HTP (high throughput); HPLC (high pressure liquid chromatography); HPLC-UV (HPLC-Ultraviolet Visible Detector); 1H NMR (proton nuclear magnetic resonance spectroscopy); FIOPC (fold improvements over positive control); Sigma and Sigma- Aldrich (Sigma- Aldrich, St. Louis, MO; Difco (Difco Laboratories, BD Diagnostic Systems, Detroit, MI); Microfluidics (Microfluidics, Westwood, MA); Life Technologies (Life Technologies, a part of Fisher Scientific, Waltham, MA); Amresco (Amresco, LLC, Solon, OH); Carbosynth (Carbosynth, Ltd., Berkshire, UK); Varian (Varian Medical Systems, Palo Alto, CA); Agilent (Agilent Technologies, Inc., Santa Clara, CA); Infers (Infers USA Inc., Annapolis Junction, MD); and Thermotron (Thermotron, Inc., Holland, MI).Example 1Pyruvate oxidase (POx) selection, plasmid construction, and directed evolution

[0346] Synthetic genes encoding an N-terminal or C-terminal 6-histidine tagged version of multiple wild-type (WT) pyruvate oxidase (POx) enzymes were cloned into the pCKl 10900 vector system (See e.g., US Pat. No. 9,714,437, which is hereby incorporated by reference in its entirety) and subsequently expressed in an E. coli strain derived from W3110.

[0347] Cells transformed with the POx expression constructs were grown at shake-flask scale using IPTG induction as described below in Example 3. Cells were then lysed in 20 mM Triethanolamine buffer (TEoA; pH 7.5) supplemented with 1 mM thiamine pyrophosphate (TPP), clarified, and the soluble fractions were purified as described in Example 3. Purified POx enzymes were used in reactions containing AdoK SEQ ID NO: 638, AdyK SEQ ID NO: 652, and AcK SEQ ID NO: 666 using 10 mM guanosine (G) as a substrate as described in Example 4. Samples were analyzed by HPLC as described in Example 5. Pyruvate oxidase activity levels are summarized in Table 1.1, show fold improvement in activity relative to the pyruvate oxidase from Bifidobacterium mongoliense (UniProt ID: A0A087C4V4; SEQ ID NO: 2).Example 2POx expression and lysate processing for high throughput (HTP) screeningHigh Throughput (HTP) Growth of POx Enzyme and Variants

[0348] Transformed E. coli cells were selected by plating onto LB agar plates containing 1 % glucose and 30 pg / mL chloramphenicol. After overnight incubation at 37 °C, colonies were placed into the wells of 96-well shallow flat bottom NUNC™ (Thermo-Scientific) plates filled with 180 pl / well LB medium supplemented with 1 % glucose and 30 pg / mL chloramphenicol. The cultures were allowed to grow overnight for 18-20 hours in a shaker (200 rpm, 30 °C, and 85% relative humidity; Kuhner). Overnight growth samples (20 pL) were transferred into Costar 96-well deep plates filled with 380 pL of Terrific Broth supplemented with 30 g / mL chloramphenicol. The plates were incubated for 120 minutes in a shaker (250 rpm, 30 C, and 85% relative humidity; Kuhner) until the OD600 reached between 0.4-0.8. The cells were then induced with 40 pL of 10 mM IPTG in sterile water and incubated overnight for 18-20 hours in a shaker (250 rpm, 30 C, and 85% relative humidity; Kuhner). The cells were pelleted (4,000 rpm for 20 min), the supernatants were discarded, and the cells were frozen at -80 °C prior to analysis.Thermal lysis of HTP Cell Pellets with Lysozyme

[0349] For lysis, 400 pL lysis buffer containing 50 mM triethanolamine buffer, pH 7.5, and 0.1 g / L lysozyme were added to the cell pellet in each well. The cells were shaken vigorously at room temperature for 5 minutes on a bench top shaker. An aliquot of the re-suspended cells (5-100 uL) was transferred to a 96-well format 200 pL BioRad PCR plate, diluting to 100 uL in lysis buffer, if necessary, then briefly spun-down prior to 1 h heat treatment at the temperature indicated, typically 45-60 °C. Following heat-treatment, the cell debris was pelleted by centrifugation (4,000 rpm at 4 °C for 10 min), and clear supernatants were then used in biocatalytic reactions to determine their activity levels.Example 3Shake Flask Expression and Purification of POxShake Flask Expression

[0350] Selected HTP cultures grown as described above were plated onto LB agar plates with 1 % glucose and 30 pg / mL chloramphenicol and grown overnight at 37 °C. A single colony from each culture was transferred to 5 mL of LB broth with 1% glucose and 30 pg / mL chloramphenicol. The cultures were grown for 20 h at 30 °C, 250 rpm, and subcultured at a dilution of approximately 1:50 into 250 mL of Terrific Broth with 30 pg / mL of chloramphenicol, to a final ODeoo of about 0.05. The cultures were incubated for approximately 195 min at 30 °C, 250 rpm, to an ODeoo of about 0.6, and then induced with the addition of IPTG at a final concentration of 1 mM. The induced cultures were incubated for 20 h at30 °C, 250 rpm. Following this incubation period, the cultures were centrifuged at 4,000 rpm for 10 min. The culture supernatant was discarded, and the pellets were resuspended in 35 mL of 20 mM triethanolamine, pH 7.5. This cell suspension was chilled in an ice bath and lysed using a Microfluidizer cell disruptor (Microfluidics M-110L). The crude lysate was pelleted by centrifugation (10,000 rpm for 60 min at 4 °C), and the supernatant was then filtered through a 0.2 pm PES membrane to further clarify the lysate.Purification of POx from Shake Flask Lysates

[0351] POx lysates were supplemented with l / 50thvolume of SF elution buffer (50 mM Tris-HCl, 500 mM NaCl, 250 mM imidazole, 0.02% v / v Triton X- 100 reagent) per well. Lysates were then purified using an AKTA Start purification system and a 5 mL HisTrap FF column (GE Healthcare). The SF wash buffer comprised 50 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, 0.02% v / v Triton X-100 reagent.

[0352] Elution fractions containing protein were identified by UV absorption (A280) and pooled, then dialyzed overnight in dialysis buffer (20 mM Tris-HCl, pH 7.4, 100 mM KC1, 0.1 mM EDTA, and 50% glycerol) in a 3.5K Slide- A-Lyzer™ dialysis cassette (Thermo Fisher) for buffer exchange. POx concentrations in the preparations were measured by absorption at 280 nm.Example 4Biosynthetic cascade reactions for production of nucleotide triphosphates (NTPs)NTP biosynthetic reaction setup

[0353] Reactions were performed in 96-well format 200 uL BioRad PCR plates or 384-well format 40 pL BioRad PCR plates. POx variants were assayed in the presence of adenosine kinase (AdoK), adenylate kinase (AdyK) and acetate kinase (AcK) variants to enable direct conversion of nucleosides to the corresponding triphosphate using acetyl phosphate generated by POx, as depicted in Scheme 1, above.

[0354] The reactions were set up as follows: (i) all reaction components, except for the nucleoside substrate, phosphate, pyruvate, and the POx lysate, were premixed in a single solution and were aliquoted into each well of the reaction plates, (ii) POx lysate solution was then added into the wells, and (iii) aliquots of a phosphate / pyruvate solution in water and the substrate nucleoside in DMSO was added to initiate the reaction. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 10 °C prior to analysis.Example 5 High-performance liquid chromatography (HPLC) analysis of phosphorylated productsSample preparation and reaction analysis using HPLC:

[0355] The nucleoside substrates, along with their respective 5 ’-monophosphate (NMP), 5’- diphosphate (NDP), and 5 ’-triphosphate (NTP) products produced using reactions set up as described in Example 4 were analyzed using HPLC. Mobile phases consisted of 50 mM potassium phosphate (pH 7) with 2 mM tetrabutylammonium hydrogen sulfate (Solvent A), acetonitrile (solvent B), and water (Solvent C). Products were detected by UV absorption at 254 nm. In some instances, a Zorbax RR StableBond Aq, 3.0x150 mm, 3.5 pm (Agilent, #863954-314) column was used. In other instances, a Zorbax RR StableBond Aq, 3.0x100 mm, 3.5 pm (Agilent, #861954-314) column was used, while in other instances, a Zorbax RR StableBond Aq, 2.1x50 mm, 3.5 pm (Agilent, #871700- 914) column was used.Example 6Analysis of NTP production in biosynthetic cascade using capillary electrophoresisCoupling of NTP biosynthetic reactions to TnT for CE:

[0356] For high-throughput (HTP) determination of NTP yield, NTP biosynthetic cascade reactions were terminated with either a heatkill at 95 °C for 2 minutes or by dilution with 75% methanol. Samples were then diluted into a coupling reaction, wherein the NTP yield was determined by the amount of product generated by a terminal nucleotidyl transferase (TnT)- catalyzed addition of the product NTP to an oligonucleotide substrate. The reaction contained 20 mM triethanolamine (TEoA), 0.25 mM C0O2, 0.001 units of inorganic pyrophosphatase (New England Biolabs), 4 pM TnT SEQ ID NO: 670 (PCT / US2023 / 076667), 12.375 pM unlabeled oligonucleotide, and 0.125 pM 5’-6-FAM- labeled oligonucleotide. Reactions were carried out at 50 °C for 60 minutes, followed by 2 minutes at 95 °C.Sample preparation for reaction analysis using CE:

[0357] For analysis of reaction samples, capillary electrophoresis was performed using either an ABI 3500XL Genetic Analyzer (ThermoFisher) or a SeqStudio™ Flex Genetic Analyzer (ThermoFisher). Reactions (1 pL) were quenched by the additions of 19 pL of 1 mM aqueous ethylenediaminetetraacetic acid (EDTA) Quenched reactions were diluted to 1.25 nM oligonucleotide,and a 2-LI L aliquot of this solutions was transferred to a new 96- well MicroAmp Optical PCR plate or a 384-well Micro Amp Optical PCR plate containing 18 pL Hi-Di™ Formamide (ThermoFisher) containing the Alexa633 size standard. The ABI 3500XL and SeqStudio™ Flex were configured with POP6 polymer, 50 cm capillaries, and a 55 °C oven temperature. Pre -run settings were 18 kV for 50 sec. Injection was 10 kV for 2 sec, and the run settings were 19 kV for 620-640 sec. FAM-labeled oligo substrates and products were identified by their sizes relative to the sizing ladder.Example 7Activity improvements over SEQ ID NO: 6 in the conversion of nucleosides to nucleotidesHTP Screening for Improved POx Variants

[0358] SEQ ID NO: 6 was selected as the parent POx enzyme. Libraries of genes were produced from the parent gene using various techniques (e.g. saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 7.1.

[0359] Reactions were performed as described in Example 4 using conditions summarized in Table 7.1. Data were collected using the TnT-coupled reaction and CE assay described in Example 6.

[0360] Activity relative to SEQ ID NO: 6 (activity FIOP) was calculated based on the percentage of extension products observed for the variant compared with the percentage observed with SEQ ID NO: 6 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 7.2.Example 8Stability improvements over SEQ ID NO: 6 in the conversion of nucleosides to nucleotidesHTP Screening for Improved POx Variants

[0361] SEQ ID NO: 6 was selected as the parent POx enzyme. Libraries of genes were produced from the parent gene using various techniques (e.g. saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 8.1.

[0362] Reactions were performed as described in Example 4 using conditions summarized in Table 8.1. Data were collected using the TnT-coupled reaction and CE assay described in Example 6.

[0363] Stability relative to SEQ ID NO: 6 (Stability FIOP) was calculated based on the percentage of extension products observed for the variant compared with the percentage observed with SEQ ID NO: 6 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 8.2.Example 9Activity improvements over SEQ ID NO: 6 in the conversion of nucleosides to nucleotidesHTP Screening for Improved POx Variants

[0364] SEQ ID NO: 6 was selected as the parent POx enzyme. Libraries of genes were produced from the parent gene using various techniques (e.g. saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 9.1.

[0365] Reactions were performed as described in Example 4 using conditions summarized in Table 9.1. Data were collected using the TnT-coupled reaction and CE assay described in Example 6.

[0366] Activity relative to SEQ ID NO: 6 (activity FIOP) was calculated based on the percentage of extension products observed for the variant compared with the percentage observed with SEQ ID NO: 6 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 9.2.Example 10Activity improvements over SEQ ID NO: 56 in the conversion of nucleosides to nucleotidesHTP Screening for Improved POx Variants

[0367] SEQ ID NO: 56 was selected as the parent POx enzyme. Libraries of genes were produced from the parent gene using various techniques (e.g. saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 10.1.

[0368] Reactions were performed as described in Example 4 using conditions summarized in Table 10.1. Data were collected using the TnT-coupled reaction and CE assay described in Example 6.

[0369] Activity relative to SEQ ID NO: 56 (activity FIOP) was calculated based on the percentage of extension products observed for the variant compared with the percentage observed with SEQ ID NO: 56 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 10.2.Example 11Stability improvements over SEQ ID NO: 16 in the conversion of nucleosides to nucleotidesHTP Screening for Improved POx Variants

[0370] SEQ ID NO: 16 was selected as the parent POx enzyme. Libraries of genes were produced from the parent gene using various techniques (e.g. saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 11.1.

[0371] Reactions were performed as described in Example 4 using conditions summarized in Table 11.1. Data were collected using the TnT-coupled reaction and CE assay described in Example 6.

[0372] Stability relative to SEQ ID NO: 16 (stability FIOP) was calculated based on the percentage of extension products observed for the variant compared with the percentage observed with SEQ ID NO: 16 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results arc shown in Table 11.2.Example 12Stability improvements over SEQ ID NO: 184 in the conversion of nucleosides to nucleotidesHTP Screening for Improved POx Variants

[0373] SEQ ID NO: 184 was selected as the parent POx enzyme. Libraries of genes were produced from the parent gene using various techniques (e.g. saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 12.1.

[0374] Reactions were performed as described in Example 4 using conditions summarized in Table 12.1. Data were collected using the TnT-coupled reaction and CE assay described in Example 6.

[0375] Stability relative to SEQ ID NO: 184 (Stability FIOP) was calculated based on the percentage of extension products observed for the variant compared with the percentage observed with SEQ ID NO: 184 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 12.2.Example 13Activity improvements over SEQ ID NO: 264 in the conversion of nucleosides to nucleotidesHTP Screening for Improved POx Variants

[0376] SEQ ID NO: 264 was selected as the parent POx enzyme. Libraries of genes were produced from the parent gene using various techniques (e.g. saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 13.1.

[0377] Reactions were performed as described in Example 4 using conditions summarized in Table 13.1. Data were collected using the TnT-coupled reaction and CE assay described in Example 6.

[0378] Activity relative to SEQ ID NO: 264 (activity FIOP) was calculated based on the percentage of extension products observed for the variant compared with the percentage observed with SEQ ID NO: 264 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 13.2.Example 14Activity improvements over SEQ ID NO: 264 in the conversion of nucleosides to nucleotidesHTP Screening for Improved POx Variants

[0379] SEQ ID NO: 264 was selected as the parent POx enzyme. Libraries of genes were produced from the parent gene using various techniques (e.g. saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 14.1.

[0380] Reactions were performed as described in Example 4 using conditions summarized in Table14.1. Data were collected using the TnT-coupled reaction and CE assay described in Example 6.

[0381] Activity relative to SEQ ID NO: 264 (activity FIOP) was calculated based on the percentage of extension products observed for the variant compared with the percentage observed with SEQ IDNO: 264 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 14.2.Example 15Stability improvements over SEQ ID NO: 264 in the conversion of nucleosides to nucleotidesHTP Screening for Improved POx Variants

[0382] SEQ ID NO: 264 was selected as the parent POx enzyme. Libraries of genes were produced from the parent gene using various techniques (e.g. saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 15.1.

[0383] Reactions were performed as described in Example 4 using conditions summarized in Table 15.1. Data were collected using the TnT-coupled reaction and CE assay described in Example 6.

[0384] Stability relative to SEQ ID NO: 264 (Stability FIOP) was calculated based on the percentage of extension products observed for the variant compared with the percentage observed with SEQ IDNO: 264 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 15.2.Example 16Stability improvements over SEQ ID NO: 290 in the conversion of nucleosides to nucleotidesHTP Screening for Improved POx Variants

[0385] SEQ ID NO: 290 was selected as the parent POx enzyme. Libraries of genes were produced from the parent gene using various techniques (e.g. saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 16.1.

[0386] Reactions were performed as described in Example 4 using conditions summarized in Table16.1. Data were collected using the TnT-coupled reaction and CE assay described in Example 6.

[0387] Stability relative to SEQ ID NO: 290 (Stability FIOP) was calculated based on the percentage of extension products observed for the variant compared with the percentage observed with SEQ ID NO: 290 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 16.2.Example 17Activity improvements over SEQ ID NO: 400 in the conversion of nucleosides to nucleotidesHTP Screening for Improved POx Variants

[0388] SEQ ID NO: 400 was selected as the parent POx enzyme. Libraries of genes were produced from the parent gene using various techniques (e.g. saturation mutagenesis and recombination ofpreviously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 17.1.

[0389] Reactions were performed as described in Example 4 using conditions summarized in Table 17.1. Data were collected using the TdT-coupled reaction and CE assay described in Example 6.

[0390] Activity relative to SEQ ID NO: 400 (activity FIOP) was calculated based on the percentage of extension products observed for the variant compared with the percentage observed with SEQ ID NO: 400 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 17.2.Example 18Stability improvements over SEQ ID NO: 400 in the conversion of nucleosides to nucleotidesHTP Screening for Improved POx Variants

[0391] SEQ ID NO: 400 was selected as the parent POx enzyme. Libraries of genes were produced from the parent gene using various techniques (e.g. saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 18.1.

[0392] Reactions were performed as described in Example 4 using conditions summarized in Table18.1. Data were collected using the TdT-coupled reaction and CE assay described in Example 6.

[0393] Stability relative to SEQ ID NO: 400 (Stability FIOP) was calculated based on the percentage of extension products observed for the variant compared with the percentage observed with SEQ ID NO: 400 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 18.2.Example 19Stability improvements over SEQ ID NO: 400 in the conversion of nucleosides to nucleotidesHTP Screening for Improved POx Variants

[0394] SEQ ID NO: 400 was selected as the parent POx enzyme. Libraries of genes were produced from the parent gene using various techniques (e.g. saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 19.1.

[0395] Reactions were performed as described in Example 4 using conditions summarized in Table 19.1. Data were collected using the TdT-coupled reaction and CE assay described in Example 6.

[0396] Stability relative to SEQ ID NO: 400 (Stability FIOP) was calculated based on the percentage of extension products observed for the variant compared with the percentage observed with SEQ IDNO: 400 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 19.2.Example 20Activity improvements over SEQ ID NO: 696 in the conversion of nucleosides to nucleotidesHTP Screening for Improved POx Variants

[0397] SEQ ID NO: 696 was selected as the parent POx enzyme. Libraries of genes were produced from the parent gene using various techniques (e.g. saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 20.1.

[0398] Reactions were performed as described in Example 4 using conditions summarized in Table 20.1. Data were collected using the TdT-coupled reaction and CE assay described in Example 6.

[0399] Activity relative to SEQ ID NO: 696 (Activity FIOP) was calculated based on the percentage of extension products observed for the variant compared with the percentage observed with SEQ ID NO: 696 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 20.2.Example 21Stability improvements over SEQ ID NO: 696 in the conversion of nucleosides to nucleotidesHTP Screening for Improved POx Variants

[0400] SEQ ID NO: 696 was selected as the parent POx enzyme. Libraries of genes were produced from the parent gene using various techniques (e.g. saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 21.1.

[0401] Reactions were performed as described in Example 4 using conditions summarized in Tabic 21.1. Data were collected using the TdT-couplcd reaction and CE assay described in Example 6.

[0402] Stability relative to SEQ ID NO: 696 (Stability FIOP) was calculated based on the percentage of extension products observed for the variant compared with the percentage observed with SEQ ID NO: 696 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 21.2.Example 22Stability improvements over SEQ ID NO: 696 in the conversion of nucleosides to nucleotidesHTP Screening for Improved POx Variants

[0403] SEQ ID NO: 696 was selected as the parent POx enzyme. Libraries of genes were produced from the parent gene using various techniques (e.g. saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 22.1.

[0404] Reactions were performed as described in Example 4 using conditions summarized in Table 22.1. Data were collected using the TdT-coupled reaction and CE assay descri...

Claims

1. CLAIMSWhat is claimed is:

1. An engineered pyruvate oxidase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 6, 14-636 and 692- 876, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 6, 14-636 and 692-876, or a functional fragment thereof, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, 16, 56, 184, 264, 290, 400, or 696, or relative to a reference sequence corresponding to SEQ ID NO: 6, 16, 56, 184, 264, 290, 400, or 696.

2. The engineered pyruvate oxidase of Claim 1, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, 16, 56, 184, 264, 290, 400, or 696, or to a reference sequence corresponding to SEQ ID NO: 6, 16, 56, 184, 264, 290, 400, or 696, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, 16, 56, 184, 264, 290, 400, or 696, or relative to the reference sequence corresponding to SEQ ID NO: 6, 16, 56, 184, 264, 290, 400, or 696.

3. The engineered pyruvate oxidase of Claim 1, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 14- 636 and 692-876, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 14-636 and 692-876, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, or relative to the reference sequence corresponding to SEQ ID NO: 6.

4. The engineered pyruvate oxidase of Claim 1, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696, or to a reference sequence corresponding to SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the referencesequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, or relative to the reference sequence corresponding to SEQ ID NO: 6.

5. The engineered pyruvate oxidase of Claim 1, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, or to a reference sequence corresponding to SEQ ID NO: 6, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, or relative to the reference sequence corresponding to SEQ ID NO: 6.

6. The engineered pyruvate oxidase of any one of Claims 1-5, wherein the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference at amino acid position 14, 15, 21, 31, 32, 45, 54, 57, 61, 62, 74, 108, 117, 127, 134, 135, 139, 150, 153, 161, 163, 167, 168, 177, 183, 187, 192, 193, 197, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208,209, 210, 214, 215, 217, 218, 220, 226, 228, 233, 235, 237, 239, 242, 244, 250, 259, 302, 307, 308,314, 317, 325, 326, 331, 336, 342, 345, 351, 357, 367, 374, 377, 380, 385, 389, 393, 404, 409, 417,432, 458, 469, 471, 480, 482, 496, 504, 506, 510, 511, 512, 515, 521, 523, 524, 527, 528, 530, 536,555, 561, 563, 565, 567, 572, 575, 576, 579, 580, 585, 587, 591, 594, or 604, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, or relative to the reference sequence corresponding to SEQ ID NO: 6.

7. The engineered pyruvate oxidase of any one of Claims 1-6, wherein the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference, or amino acid residue 14A, 15R / S, 21A, 31A / G / K / S, 321, 45Y, 54V, 57A / H, 611, 62K, 74G, 108S, 117A, 127T, 134G / V, 135 A / F, 139P, 150S, 153A / T, 161V, 163H / V, 167R / S, 168H / K, 177A, 183C / M / P / S, 187V, 192S, 193 A, 197K, 199H, 200D / I / R, 201G / P / Q / W, 202A / P, 203C / R / Y, 204C / R / T, 205R, 206A / G / H / Q / V / Y, 2071, 208S, 209R, 210A / L / R, 214C / I / R / V, 2151 / M, 217R, 218H, 220K, 226C, 228V, 233 A, 235R, 237L, 239N, 242E / H, 244L, 250V, 259S, 302V, 307F, 3O8K, 314R, 317G / R, 325P, 326S / V, 331A, 336T, 342S, 345W, 351H / K / P / R / S, 357L, 367C, 374C, 377P, 380V, 385R, 389R, 393S, 404P, 409L / V, 417R, 432G, 458G / Q, 469W, 471R, 480N, 482S, 496L, 504C / P / V, 506A / C, 510K, 511V, 512A, 515L, 521Q, 523A / R / T, 524G, 527H / L / M, 528L, 53OR, 536L / T, 555L, 561A, 563Q, 565E, 567Q / R / V, 572G / P / T, 575A / F, 576S, 579I / L / N / S / T, 580C / R / T, 585G, 587R / K, 591V, 594A / D / L / Q / S / V, or 604S, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, or relative to the reference sequence corresponding to SEQ ID NO: 6.

8. The engineered pyruvate oxidase of any one of Claims 1-6, wherein the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference at amino acid position 134, 153, 168, 192, 197, 242, 308, 317, 325, 331, 336, 469, 504, 510, 512, or 567, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, or relative to the reference sequence corresponding to SEQ ID NO: 6.

9. The engineered pyruvate oxidase of any one of Claims 1-6 and 8, wherein the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference, or amino acid residue 134G / V, 153A / T, 168H / K, 192S, 197K, 242E / H, 308K, 317G / R, 325P, 331A / S, 336T, 469W, 504V, 510K, 512A, or 567Q / R / V, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, or relative to the reference sequence corresponding to SEQ ID NO: 6.

10. The engineered pyruvate oxidase of any one of Claims 1-6 and 8, wherein the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference, or amino acid residue 134V, 153T, 168K, 192S, 197K, 242E, 308K, 317R, 325P, 331A, 336T, 469W, 504P, 510K, 512A, 512A, or 567R, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, or relative to the reference sequence corresponding to SEQ ID NO: 6.

11. The engineered pyruvate oxidase of any one of Claims 1-6, wherein the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference at amino acid position 61, 168, 202, 233, 242, 244, 308, 336, 480, 510, 512, or 528, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, or relative to the reference sequence corresponding to SEQ ID NO: 6.

12. The engineered pyruvate oxidase of any one of Claims 1 -6 and 1 1 , wherein the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference 611, 168K, 202P, 233A, 242E, 244L, 308K, 336T, 480N, 510K, 512A, or 528L, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, or relative to the reference sequence corresponding to SEQ ID NO: 6.

13. The engineered pyruvate oxidase of any one of Claims 1-6, wherein the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference at amino acid position 168, 220, 242, 308, 377, 432, or 512, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, or relative to the reference sequence corresponding to SEQ ID NO: 6.

14. The engineered pyruvate oxidase of any one of Claims 1-6 and 13, wherein the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference, or amino acid residue 168K, 220K, 242E, 308K, 377P, 432G, or 512A, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, or relative to the reference sequence corresponding to SEQ ID NO: 6.

15. The engineered pyruvate oxidase of any one of Claims 1-6, wherein the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference(s) at amino acid position(s) 54, 54 / 57, 54 / 57 / 168, 54 / 57 / 168 / 482 / 510 / 512, 54 / 57 / 336, 54 / 57 / 482, 54 / 57 / 510, 54 / 57 / 510 / 512, 54 / 168, 54 / 168 / 336 / 512, 54 / 168 / 482 / 510, 54 / 168 / 510, 54 / 168 / 510 / 512, 54 / 242 / 482 / 512, 54 / 336 / 510, 54 / 336 / 512, 54 / 482, 54 / 482 / 512 / 528, 54 / 510, 54 / 512 / 528, 57, 57 / 168 / 482 / 510 / 512, 57 / 336 / 512, 57 / 482, 57 / 510, 57 / 512, 57 / 512 / 528, 61 / 202 / 308 / 469 / 536, 61 / 244, 61 / 308 / 469, 61 / 308 / 471 / 536, 61 / 471 / 536, 168 / 242 / 336 / 510 / 512, 168 / 242 / 482 / 512 / 528, 168 / 512, 202 / 244 / 308, 202 / 244 / 308 / 471, 202 / 244 / 536, 202 / 308 / 432 / 536, 202 / 308 / 469 / 471, 202 / 308 / 469 / 536, 202 / 308 / 536, 202 / 471 / 536, 202 / 536, 242 / 512 / 528, 244 / 308 / 469, 308 / 469 / 471, 336 / 482, 336 / 482 / 510 / 512, 469, 482, 482 / 510, 482 / 510 / 512 / 528, or 536, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12- 604 of SEQ ID NO: 6, or relative to the reference sequence corresponding to SEQ ID NO: 6.

16. The engineered pyruvate oxidase of any one of Claims 1-6 and 15, wherein the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference(s), or amino acid residue(s) 54V, 54V / 57A, 54V / 57A / 168K, 54V / 57A / 168K / 482S / 510K / 512A, 54V / 57 A / 336T, 54V / 57A / 482S, 54V / 57A / 510K, 54V / 57A / 510K / 512A, 54V / 168K, 54V / 168K / 336T / 512A, 54V / 168K / 482S / 510K, 54V / 168K / 510K, 54V / 168K / 510K / 512A, 54V / 242E / 482S / 512A, 54V / 336T / 510K, 54V / 336T / 512A, 54V / 482S, 54V / 482S / 512A / 528L, 54V / 510K, 54V / 512A / 528L, 57A, 57A / 168K / 482S / 510K / 512A, 57A / 336T / 512A, 57A / 482S, 57A / 510K, 57A / 512A, 57A / 512A / 528L, 57H, 61I / 202P / 308K / 469W / 536T, 61I / 244L, 61V308K / 469W, 61I / 308K / 471R / 536L, 61I / 471R / 536L, 168K / 242E / 336T / 510K / 512A, 168K / 242E / 482S / 512A / 528L, 168K / 512A, 202P / 244L / 308K, 202P / 244L / 308K / 471R, 202P / 244L / 536L, 202P / 308K / 432G / 536L, 202P / 308K / 469W / 471R, 202P / 308K / 469W / 536T, 202P / 308K / 536L, 202P / 471R / 536L, 202P / 536L, 242H / 512A / 528L, 244L / 308K / 469W, 308K / 469W / 471R, 336T / 482S, 336T / 482S / 510K / 512A, 469W, 482S, 482S / 510K, 482S / 510K / 512A / 528L, or 536L, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 6, or relative to the reference sequence corresponding to SEQ ID NO: 6.

17. The engineered pyruvate oxidase of Claim 1, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequencecorresponding to amino acid residues 12-604 of SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696, or to the reference sequence corresponding to SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696, or relative to the reference sequence corresponding to SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696.

18. The engineered pyruvate oxidase of Claim 1, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 14- 636 and 692-876, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 14-636 and 692-876, or a functional fragment thereof, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696, or relative to the reference sequence corresponding to SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696.

19. The engineered pyruvate oxidase of Claim 17 or 18, wherein the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference at amino acid position 14, 15, 21, 31, 32, 45, 54, 57, 61, 62, 74, 108, 117, 127, 134, 135, 139, 150, 153, 161, 163, 167, 168, 177, 183, 187, 192, 193, 197, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210,214, 215, 217, 218, 220, 226, 228, 233, 235, 237, 239, 242, 244, 250, 259, 302, 307, 308, 314, 317,325, 326, 331, 336, 342, 345, 351, 357, 367, 374, 377, 380, 385, 389, 393, 404, 409, 417, 432, 458,469, 471, 480, 482, 496, 504, 506, 510, 511, 512, 515, 521, 523, 524, 527, 528, 530, 536, 555, 561,563, 565, 567, 572, 575, 576, 579, 580, 585, 587, 591, 594, or 604, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696, or relative to the reference sequence corresponding to SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696.

20. The engineered pyruvate oxidase of any one of Claims 17-19, wherein the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference, or amino acid residue 14A, 15R / S, 21A, 31A / G / K / S, 321, 45Y, 54V, 57A / H, 611, 62K, 74G, 108S, 117A, 127T, 134G / V, 135 A / F, 139P, 150S, 153A / T, 161V, 163H / V, 167R / S, 168H / K, 177A, 183C / M / P / S, 187V, 192S, 193 A, 197K, 199H, 200D / I / R, 201G / P / Q / W, 202A / P, 203C / R / Y, 204C / R / T, 205R, 206A / G / H / Q / V / Y, 2071, 208S, 209R, 210A / L / R, 214C / I / R / V, 2151 / M, 217R, 218H, 220K, 226C, 228V, 233 A, 235R, 237L, 239N, 242E / H, 244L, 250V, 259S, 302V, 307F, 3O8K, 314R, 317G / R, 325P, 326S / V, 331A, 336T, 342S, 345W, 351H / K / P / R / S, 357L, 367C, 374C, 377P, 380V, 385R, 389R, 393S, 404P, 409L / V, 417R, 432G, 458G / Q, 469W, 471R, 480N, 482S, 496L, 504C / P / V, 506A / C, 510K, 511V, 512A, 515L, 521Q, 523A / R / T, 524G, 527H / L / M, 528L, 53OR, 536L / T, 555L,561 A, 563Q, 565E, 567Q / R / V, 572G / P / T, 575A / F, 576S, 579I / L / N / S / T, 580C / R / T, 585G, 587R / K, 591V, 594A / D / L / Q / S / V, or 604S, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696, or relative to the reference sequence corresponding to SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696.

21. The engineered pyruvate oxidase of any one of Claim 17-19, wherein the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference at amino acid position 134, 153, 168, 192, 197, 242, 308, 317, 325, 331, 336, 469, 504, 510, 512, or 567, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696, or relative to the reference sequence corresponding to SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696.

22. The engineered pyruvate oxidase of any one of Claim 17-19, wherein the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference, or amino acid residue 134G / V, 153A / T, 168H / K, 192S, 197K, 242E / H, 308K, 317G / R, 325P, 331A / S, 336T, 469W, 504V, 510K, 512A, or 567Q / R / V, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696, or relative to the reference sequence corresponding to SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696.

23. The engineered pyruvate oxidase of Claim 1, 17, or 18, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 56 and 154-178, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 56 and 154-178, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 56, or relative to the reference sequence corresponding to SEQ ID NO: 56.

24. The engineered pyruvate oxidase of Claim 23, wherein the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference at amino acid position 14, 199, 201, 202, 215, 218, 496, 575, or 580, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 56, or relative to the reference sequence corresponding to SEQ ID NO: 56.

25. The engineered pyruvate oxidase of Claim 23 or 24, wherein the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference, or amino acid residue 14A, 199H, 201G, 201P, 202A, 2151, 215L, 215M, 218H, 496L, 496S, 575A, or 580R, or combinations thereof, wherein the amino acid positions are relative to the reference sequencecorresponding to amino acid residues 12-604 of SEQ ID NO: 56, or relative to the reference sequence corresponding to SEQ ID NO: 56.

26. The engineered pyruvate oxidase of Claim 1, 17, or 18, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 16 and 180-248, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 16 and 180-248, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

27. The engineered pyruvate oxidase of Claim 26, wherein the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference at amino acid position 21, 183, 193, 201, 203, 204, 205, 206, 207, 209, 214, 235, 307, 325, 345, 374, 393, 510, 527, 530, 561, 563, 565, 580, 594, or 604, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

28. The engineered pyruvate oxidase of Claim 26 or 27, wherein the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference, or amino acid residue 21A, 183S, 193A, 201Q, 201W, 203C, 203L, 204C, 204R, 204T, 205R, 206H, 206V, 206Y, 2071, 209R, 214C, 2141, 214R, 214V, 235R, 307F, 325P, 345W, 374C, 393S, 510K, 527H, 530R, 561A, 563Q, 565E, 580C, 594Q, and 604S, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 16, or relative to the reference sequence corresponding to SEQ ID NO: 16.

29. The engineered pyruvate oxidase of Claim 1, 17, or 18, comprising an amino acid sequence having at least 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 184 and 250-288, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 184 and 250-288, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 184, or relative to the reference sequence corresponding to SEQ ID NO: 184.

30. The engineered pyruvate oxidase of Claim 29, wherein the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference at amino acid position of 177, 177 / 209, 177 / 209 / 336, 177 / 209 / 563, 177 / 336, 177 / 336 / 471, 177 / 336 / 563, 177 / 563, 206 / 308, 206 / 308 / 469, 206 / 308 / 536 / 591, 206 / 469, 308, 308 / 469, 308 / 469 / 536, 336 / 471, 336 / 563,469, 469 / 536, and 563, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 184, or relative to the reference sequence corresponding to SEQ ID NO: 184.

31. The engineered pyruvate oxidase of Claim 29 or 30, wherein the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference(s) or amino acid residue(s) 177 A, 177A / 209R, 177A / 209R / 336T, 177A / 209R / 563Q, 177A / 336T, 177A / 336T / 471R, 177A / 336T / 563Q, 177 A / 563Q, 206H / 308K, 206H / 308K / 469W, 206H / 308K / 536T / 591V, 206H / 469W, 3O8K, 308K / 469W, 308K / 469W / 536T, 336T / 471R, 336T / 563Q, 469W, 469W / 536T, or 563Q, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 184, or relative to the reference sequence corresponding to SEQ ID NO: 184.

32. The engineered pyruvate oxidase of Claim 1, 17, or 18, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 264 and 290-398, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 264 and 290-398, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 264, or relative to the reference sequence corresponding to SEQ ID NO: 264.

33. The engineered pyruvate oxidase of Claim 32, wherein the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference at amino acid position 135, 153, 161, or 504, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 264, or relative to the reference sequence corresponding to SEQ ID NO: 264.

34. The engineered pyruvate oxidase of Claim 32 or 33, wherein the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference, or amino acid residue 135A, 135L, 153A, 161V, or 504P, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 264, or relative to the reference sequence corresponding to SEQ ID NO: 264.

35. The engineered pyruvate oxidase of Claim 32, wherein the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference at amino acid position 32, 127, 134, 135, 153, 163, 192, 197, 200, 317, 331, 385, 504, or 567, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 264, or relative to the reference sequence corresponding to SEQ ID NO: 264.

36. The engineered pyruvate oxidase of Claim 32 or 35, wherein the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference, or amino acid residue 321, 127T, 134G, 134V, 135A, 153T, 163H, 163V, 192S, 197K, 2001, 317G, 317R, 331A, 385R, 504V, 567R, or 567V, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 264, or relative to the reference sequence corresponding to SEQ ID NO: 264.

37. The engineered pyruvate oxidase of Claim 32, wherein the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference at amino acid position 32, 74, 108, 117, 127, 134, 135, 139, 150, 153, 163, 177, 192, 197, 200, 226, 237, 250, 259, 302, 314, 317, 331, 385, 404, 409, 458, 504, 506, 511, 515, 523, or 567, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 264, or relative to the reference sequence corresponding to SEQ ID NO: 264.

38. The engineered pyruvate oxidase of Claim 32 or 37, wherein the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference, or amino acid residue 321, 74G, 108S, 117A, 127T, 134V, 135F, 139P, 150S, 153T, 163H, 163V, 177L, 192S, 197K, 200D, 2001, 200L, 200R, 226C, 237L, 250V, 259S, 302V, 314R, 317G, 317R, 331A, 331S, 385R, 404P, 409L, 409V, 458G, 458Q, 504C, 504S, 506A, 506C, 506L, 511V, 515L, 523 A, 523R, 523T, 567Q, 567R, and 567V, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 264, or relative to the reference sequence corresponding to SEQ ID NO: 264.

39. The engineered pyruvate oxidase of Claim 1, 17, or 18, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 290 and 400-636, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 290 and 400-636, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 290, or relative to the reference sequence corresponding to SEQ ID NO: 290.

40. The engineered pyruvate oxidase of Claim 39, wherein the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference(s) at amino acid position(s) 32 / 134 / 135 / 523 / 567, 32 / 134 / 139 / 163 / 250 / 331 / 523, 32 / 134 / 163 / 409, 32 / 134 / 331 / 567, 32 / 139 / 163 / 250, 32 / 139 / 163 / 409, 32 / 139 / 163 / 567, 32 / 139 / 250 / 567, 32 / 139 / 409, 32 / 163, 32 / 163 / 250, 32 / 163 / 250 / 567, 32 / 163 / 331, 32 / 163 / 331 / 409 / 523 / 567, 32 / 163 / 331 / 409 / 567, 32 / 163 / 409 / 567, 32 / 163 / 523 / 567, 32 / 163 / 567, 32 / 250, 32 / 250 / 409, 32 / 250 / 523 / 567, 32 / 250 / 567, 32 / 331 / 567, 32 / 409,32 / 409 / 567, 32 / 567, 127, 127 / 153, 127 / 153 / 192 / 197, 127 / 153 / 192 / 197 / 200 / 317, 127 / 153 / 192 / 200 / 317 / 385, 127 / 153 / 192 / 504, 127 / 153 / 201 / 385, 127 / 153 / 317 / 385, 127 / 153 / 317 / 567, 127 / 192 / 197 / 200 / 385 / 504, 127 / 192 / 197 / 200 / 504, 127 / 192 / 197 / 317 / 504 / 567, 127 / 192 / 197 / 385 / 504, 127 / 192 / 197 / 504, 127 / 192 / 317 / 385 / 504, 127 / 192 / 385 / 504 / 567, 127 / 197 / 200 / 317, 127 / 200, 127 / 200 / 317 / 567, 127 / 317, 127 / 385, 127 / 385 / 567, 134 / 135 / 163 / 567, 134 / 163 / 331 / 409 / 567, 134 / 250, 134 / 409 / 567, 139, 139 / 163, 139 / 163 / 250 / 331 / 523, 139 / 163 / 331 / 523, 139 / 163 / 567, 139 / 523 / 567, 153, 153 / 192 / 197 / 200 / 385 / 567, 153 / 192 / 197 / 200 / 504, 153 / 192 / 197 / 317 / 504, 153 / 192 / 197 / 317 / 567, 153 / 192 / 200 / 504, 153 / 192 / 317 / 385, 153 / 197 / 504 / 567, 153 / 200, 153 / 317, 153 / 317 / 385, 153 / 317 / 504, 153 / 317 / 504 / 567, 153 / 385, 153 / 504, 153 / 504 / 567, 163, 163 / 250, 163 / 250 / 567, 163 / 331, 163 / 331 / 409, 163 / 331 / 409 / 567, 163 / 331 / 567, 163 / 409, 163 / 523 / 567, 163 / 567, 192 / 197 / 200, 192 / 197 / 200 / 317 / 567, 192 / 197 / 200 / 385, 192 / 197 / 200 / 385 / 567, 192 / 197 / 317, 192 / 197 / 385 / 504 / 567, 192 / 197 / 504, 192 / 200, 192 / 200 / 385, 192 / 317, 192 / 504, 197 / 200 / 567, 197 / 317, 197 / 317 / 385 / 504 / 567, 200 / 317 / 385, 200 / 385 / 567, 250, 250 / 331, 250 / 331 / 567, 250 / 523, 250 / 567, 317, 317 / 385 / 567, 317 / 567, 331 / 409, 331 / 567, 385, 385 / 567, 409 / 523, 523, 523 / 567, or 567, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 290, or relative to the reference sequence corresponding to SEQ ID NO: 290.

41. The engineered pyruvate oxidase of Claim 39 or 40, wherein the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference of 32I / 134V / 135A / 523R / 567V, 32I / 134V / 139P / 163H / 250V / 331A / 523R, 32I / 134V / 163H / 409L, 321 / 134 V / 331A / 567V, 32I / 139P / 163H / 250V, 32I / 139P / 163H / 409L, 32I / 139P / 163H / 567V, 321 / 139P / 250V / 567V, 321 / 139P / 409L, 32I / 163H, 32I / 163H / 331A, 32I / 163H / 331A / 409L / 567V, 32I / 163H / 409L / 567V, 32I / 163H / 523R / 567V, 32V163V, 32I / 163V / 250V, 321 / 163 V / 250V / 567V, 32I / 163V / 331A / 409L / 523R / 567V, 32I / 163V / 567V, 32I / 250V, 32I / 250V / 409L, 32I / 250V / 523R / 567V, 32I / 250V / 567V, 321 / 331 A / 567V, 32I / 409L, 32I / 409L / 567V, 321 / 567 V, 127T, 127T / 153T, 127T / 153T / 192S / 197K, 127T / 153T / 192S / 197K / 200I / 317R, 127T / 153T / 192S / 200E317R / 385R, 127T / 153T / 192S / 504V, 127T / 153T / 201G / 385R, 127T / 153T / 317R / 385R, 127T / 153T / 317R / 567R, 127T / 192S / 197K / 200I / 385R / 504V, 127T / 192S / 197K / 200P504V, 127T / 192S / 197K / 317R / 504V / 567R, 127T / 192S / 197K / 385R / 504V, 127T / 192S / 197K / 504V, 127T / 192S / 317R / 385R / 504V, 127T / 192S / 385R / 504V / 567R, 127T / 197K / 200I / 317R, 127T / 200I, 127T / 200I / 317R / 567R, 127T / 317R, 127T / 385R, 127T / 385R / 567R, 134V / 135A / 163H / 567V, 134V / 163H / 331A / 409L / 567V, 134V / 250V, 134V / 409L / 567V, 139P, 139P / 163H, 139P / 163H / 250V / 331A / 523R, 139P / 163H / 331A / 523R, 139P / 163 V / 567 V, 139P / 523R / 567V, 153T, 153T / 192S / 197K / 200P385R / 567R, 153T / 192S / 197K / 200I / 504V, 153T / 192S / 197K / 317R / 504V, 153T / 192S / 197K / 317R / 567R, 153T / 192S / 200I / 504V, 153T / 192S / 317R / 385R, 153T / 197K / 504V / 567R, 153T / 200I, 153T / 317R, 153T / 317R / 385R, 153T / 317R / 504V, 153T / 317R / 504V / 567R, 153T / 385R, 153T / 504V,153T / 504V / 567R, 163H / 331A, 163H / 331A / 409L, 163H / 331A / 567V, 163H / 409L, 163V, 163V / 250V, 163V / 250V / 567V, 163V / 331A, 163 V / 331A / 409L / 567V, 163V / 409L, 163V / 523R / 567V, 163V / 567V, 192S / 197K / 200I, 192S / 197K / 200I / 317R / 567R, 192S / 197K / 200I7385R, 192S / 197K / 200I / 385R / 567R, 192S / 197K / 317R, 192S / 197K / 385R / 504V / 567R, 192S / 197K / 504V, 192S / 2001, 192S / 200I / 385R, 192S / 317R, 192S / 504V, 197K / 200I / 567R, 197K / 317R, 197K / 317R / 385R / 504V / 567R,2001 / 317R / 385R, 200I / 385R / 567R, 250V, 250V / 331A, 250V / 331A / 567V, 250V / 523R, 250V / 567V, 317R, 317R / 385R / 567R, 317R / 567R, 331A / 409L, 331A / 567V, 385R, 385R / 567R, 409L / 523R, 523R, 523R / 567V, or 567V, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 290.

42. The engineered pyruvate oxidase of Claim 1, 17, or 18, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 400 and 692-766, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 400 and 692-766, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 400, or relative to the reference sequence corresponding to SEQ ID NO: 400.

43. The engineered pyruvate oxidase of Claim 42, wherein the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference(s) at amino acid position(s) 32, 32 / 135, 134, 134 / 200 / 331, 134 / 331, or 331, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12- 604 of SEQ ID NO: 400, or relative to the reference sequence corresponding to SEQ ID NO: 400.

44. The engineered pyruvate oxidase of Claim 42 or 43, wherein the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference(s), or amino acid residue(s) 321, 32I / 135A, 134V, 134V / 200E331A, 134V / 331A, or 331A, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 400, or relative to the reference sequence corresponding to SEQ ID NO: 400.

45. The engineered pyruvate oxidase of Claim 42, wherein the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference(s) at amino acid position(s) 62 / 117, 117 / 139 / 331 / 523, 117 / 200 / 523, 117 / 523, 134, or 200 / 523, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 400, or relative to the reference sequence corresponding to SEQ ID NO: 400.

46. The engineered pyruvate oxidase of Claim 42 or 45, wherein the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference(s), or amino acid residue(s) 62K / 117A, 117A / 139P / 331A / 523R, 117A / 200I / 523T, 117A / 523T, 134G, or 200P523T, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 400, or relative to the reference sequence corresponding to SEQ ID NO: 400.

47. The engineered pyruvate oxidase of Claim 42, wherein the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference at amino acid position 31, 45, 167, 208, 210, 217, 228, 351, 357, 367, 380, or 579, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 400, or relative to the reference sequence corresponding to SEQ ID NO: 400.

48. The engineered pyruvate oxidase of Claim 42 or 47, wherein the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference, or amino acid residue 31A, 31G, 31K, 31S, 45Y, 167R, 167S, 208S, 210A, 210L, 210R, 217R, 228V, 351H, 351K, 351P, 351R, 351S, 357L, 367C, 380V, 5791, 579L, 579N, 579S, or 579T, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 400, or relative to the reference sequence corresponding to SEQ ID NO: 400.

49. The engineered pyruvate oxidase of Claim 1, 17, or 18, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 696 and 768-876, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 696 and 768-876, wherein the amino acid sequence comprises one or more amino acid residue differences relative to the reference sequence corresponding to amino acid residues 12- 604 of SEQ ID NO: 696, or relative to the reference sequence corresponding to SEQ ID NO: 696.

50. The engineered pyruvate oxidase of Claim 49, wherein the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference at amino acid position 15, 203, 206, 239, 326, 417, 524, 527, 555, 572, 587, or 594, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 696, or relative to the reference sequence corresponding to SEQ ID NO: 696.

51. The engineered pyruvate oxidase of Claim 49 or 50, wherein the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference, or amino acid residue 15R, 203Y, 206G, 206S, 239N, 326S, 326V, 417R, 524G, 527M, 555L, 572G, 572T, 587R, or594L, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 696, or relative to the reference sequence corresponding to SEQ ID NO: 696.

52. The engineered pyruvate oxidase of Claim 49, wherein the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference at amino acid position 15, 168, 183, 187, 203, 206, 214, 389, 417, 521, 527, 572, 575, 580, 585, 587, or 594, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 696, or relative to the reference sequence corresponding to SEQ ID NO: 696.

53. The engineered pyruvate oxidase of Claim 49 or 52 wherein the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference 15R, 168H, 183C, 183M, 183P, 187V, 203R, 203Y, 206G, 214V, 389R, 417R, 521Q, 527L, 527M, 572G, 572P, 572T, 575F, 580T, 585G, 587R, 594A, 594D, 594S, or 594V, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 696, or relative to the reference sequence corresponding to SEQ ID NO: 696.

54. The engineered pyruvate oxidase of Claim 49, wherein the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference(s) at amino acid position(s) 15, 203 / 206, 203 / 206 / 521, 203 / 521, 206, 206 / 342 / 521, 206 / 342 / 576, 206 / 521, 206 / 585 / 587, 342 / 576, 342 / 576 / 580, 342 / 585 / 587, or 576, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 696, or relative to the reference sequence corresponding to SEQ ID NO: 696.

55. The engineered pyruvate oxidase of Claim 49 or 54, wherein the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference^!, or amino acid residue(s) 15S, 203R / 206Q / 521Q, 203Y / 206Q, 203Y / 521Q, 206A / 521Q, 206Q, 206Q / 342S / 521Q, 206Q / 342S / 576S, 206Q / 585G / 587K, 342S / 576S, 342S / 576S / 580T, 342S / 585G / 587K, or 576S, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 696, or relative to the reference sequence corresponding to SEQ ID NO: 696.

56. The engineered pyruvate oxidase of Claim 49, wherein the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference(s) at amino acid position(s) 15, 15 / 183, 15 / 389, 203 / 206, 203 / 206 / 342 / 576, 203 / 206 / 521, 203 / 206 / 576, 206, 206 / 342, 206 / 521, 342 / 576, 576, or 576 / 580, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 696, or relative to the reference sequence corresponding to SEQ ID NO: 696.

57. The engineered pyruvate oxidase of Claim 49 or 56, wherein the amino acid sequence of the engineered pyruvate oxidase comprises at least amino acid residue difference(s), or amino acid residue(s) 15R / 183M, 15R / 389R, 15S, 15S / 183M, 203R / 206Q / 342S / 576S, 203R / 206Q / 521Q, 203R / 206Q / 576S, 203Y / 206Q, 206A, 206Q / 342S, 206Q / 521Q, 342S / 576S, 576S, or 576S / 580T, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 696, or relative to the reference sequence corresponding to SEQ ID NO: 696.

58. The engineered pyruvate oxidase of Claim 1, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 6, 14-636, and 692-876, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 6, 14-636, and 692-876.

59. The engineered pyruvate oxidase of Claim 1, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12-604 of SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696, or to a reference sequence corresponding to SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696.

60. The engineered pyruvate oxidase of Claim 1, wherein the amino acid sequence of the engineered pyruvate oxidase comprises amino acid residues 12-604 of an even-numbered SEQ ID NO. of SEQ ID NOs: 14-636 and 692-876, or a sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOs: 14-636 and 692-876.

61. The engineered pyruvate oxidase of Claim 1, wherein the amino acid sequence of the engineered pyruvate oxidase comprises amino acid residues 12-604 of SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696, or a sequence comprising SEQ ID NO: 16, 56, 184, 264, 290, 400, or 696.

62. The engineered pyruvate oxidase of any one of Claims 1-61, wherein the engineered pyruvate oxidase has activity in the conversion of pyruvate, oxygen, and phosphate to acetyl phosphate, carbon dioxide and hydrogen peroxide.

63. The engineered pyruvate oxidase of any one of Claims 1-62, comprising at least one improved property as compared to a wild-type or reference pyruvate oxidase.

64. The engineered pyruvate oxidase of Claim 63, wherein the improved property comprises increased activity, increased thermostability, increased activity at high temperature, or increased soluble expression, as compared to a wild-type or reference pyruvate oxidase.

65. The engineered pyruvate oxidase of Claim 63 or 64, wherein the reference pyruvate oxidase has an amino acid sequence corresponding to SEQ ID NO: 6, 16, 56, 184, 264, 290, 400, or 69666. The engineered pyruvate oxidase of any one of Claims 1-65, wherein the pyruvate oxidase is purified.

67. A recombinant polynucleotide comprising a polynucleotide sequence encoding at least one engineered pyruvate oxidase of any one of Claims 1-65.

68. The recombinant polynucleotide of Claim 67, comprising a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34-1812 of SEQ ID NO: 5, 15, 55, 183, 263, 289, 399, or 695, or to a reference polynucleotide sequence of SEQ ID NO: 5, 15, 55, 183, 263, 289, 399, or 695, wherein the recombinant polynucleotide encodes a polypeptide having pyruvate oxidase activity.

69. The recombinant polynucleotide of Claim 67, comprising a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34-1812 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 13-635 and 691-875, or to a reference polynucleotide sequence of SEQ ID NOs: 13-635 and 691-875, wherein the recombinant polynucleotide encodes a polypeptide having pyruvate oxidase activity.

70. The recombinant polynucleotide of any one of Claims 67-69, wherein the polynucleotide sequence is codon-optimized.

71. The recombinant polynucleotide of Claim 67, wherein the polynucleotide sequence comprises nucleotide residues 34-1812 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 13-635 and 691-875, or comprises an odd-numbered SEQ ID NO. of SEQ ID NOs: 13-635 and 691-875.

72. The recombinant polynucleotide of Claim 67, wherein the polynucleotide sequence comprises nucleotide residues 34-1812 of SEQ ID NO: 5, 15, 55, 183, 263, 289, 399, or 695, or comprises SEQ ID NO: 5, 15, 55, 183, 263, 289, 399, or 695.

73. The recombinant polynucleotide of any one of Claims 67-72, wherein the polynucleotide sequence is operably linked to a control sequence.

74. An expression vector comprising at least one recombinant polynucleotide of any one of Claims 67-73.

75. A host cell comprising at least one expression vector of Claim 74.

76. A method of producing an engineered pyruvate oxidase polypeptide in a host cell comprising culturing a host cell of Claim 75, under suitable culture conditions, such that at least one engineered pyruvate oxidase is produced.

77. The method of Claim 76, further comprising recovering at least one pyruvate oxidase from the culture and / or host cells.

78. The method of Claim 76 or 77, further comprising the step of purifying the at least one engineered pyruvate oxidase.

79. A composition comprising an engineered pyruvate oxidase of any one of Claims 1- 66.

80. The composition of Claim 79, further comprising a cofactor.

81. The composition of Claim 80, wherein the cofactor comprises FAD, TPP, thiamine, and / or a divalent metal ion.

82. The composition of any one of Claims 79-81, further comprising a second enzyme.

83. The composition of Claim 82, wherein the second enzyme comprises an acetate kinase and / or catalase.

84. The composition of Claim 82 or 83, wherein the composition further comprises one or more additional enzymes.

85. The composition of Claim 84, wherein the one or more additional enzymes comprises an adenylate kinase, adenosine kinase, terminal nucleotidyl transferase, 3’0-kinase, or inorganic pyrophosphatase.

86. A method of producing acetyl phosphate, comprising contacting an engineered pyruvate oxidase of any one of Claims 1-66 with pyruvate under suitable reaction conditions for the production of acetyl phosphate.

87. The method of Claim 86, wherein the suitable reaction conditions comprise oxygen and phosphate.

88. The method of Claim 86 or 87, wherein the suitable reaction conditions comprise FAD, TPP, thiamine, and / or a divalent metal ion.

89. The method of any one of Claims 86-88, wherein the method further comprises providing a second enzyme in a paired reaction, wherein the second enzyme uses acetylphosphate as a substrate or degrades hydrogen peroxide.

90. The method of Claim 89, wherein the second enzyme is acetate kinase.

91. The method of Claim 89, wherein the second enzyme is catalase.

Images