Engineered acetate kinase variant enzymes

Abstract

The present invention provides engineered acetate kinase (AcK) enzymes, polypeptides having AcK activity, and polynucleotides encoding these enzymes, as well as vectors and host cells containing these polynucleotides and polypeptides. Methods for producing AcK enzymes are also provided. The present invention further provides compositions containing AcK enzymes and methods for using engineered AcK enzymes. The present invention is particularly useful in the production of pharmaceutical compounds.

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 869,667, filed July 2, 2019, which is incorporated by reference in its entirety for all purposes.

[0002] The present invention provides engineered acetate kinase (AcK) enzymes, polypeptides having AcK activity, and polynucleotides encoding these enzymes, as well as vectors and host cells containing these polynucleotides and polypeptides. Methods for producing AcK enzymes are also provided. The present invention further provides compositions containing AcK enzymes and methods for using engineered AcK enzymes. The present invention is particularly useful in the production of pharmaceutical compounds.

[0003] Reference to a sequence listing, table, or computer program An official copy of the Sequence Listing has been submitted herewith via EFS-Web as an ASCII formatted text file with the filename "CX2-191WO2_ST25.txt", created on June 29, 2020, and 1.74 MB in size. The Sequence Listing filed via EFS-Web is a part of the present specification and is incorporated herein by reference in its entirety. [Background technology]

[0004] A retrovirus called human immunodeficiency virus (HIV) is the causative agent of acquired immune deficiency syndrome (AIDS), a complex disease that involves the progressive destruction of the immune system of affected individuals and degeneration of the central and peripheral nervous systems. A common feature of retroviral replication is the reverse transcription of the viral RNA genome by the virus-encoded reverse transcriptase enzyme, which generates a DNA copy of the HIV sequence necessary for viral replication. Some compounds, such as MK-8591, are known reverse transcriptase inhibitors and are useful in the treatment of AIDS and similar diseases. Although several compounds are known to inhibit HIV reverse transcriptase, there is still a need in the art for additional compounds that are more effective in inhibiting this enzyme and thereby ameliorating the effects of AIDS.

[0005] Nucleoside analogs, such as MK-8591 (Merck), are effective inhibitors of HIV reverse transcriptase due to their similarity to natural nucleosides used in DNA synthesis. When reverse transcriptase binds to these analogs, they halt DNA synthesis by inhibiting the processive nature of reverse transcriptase. Enzyme halts lead to premature termination of the DNA molecule, rendering it ineffective. However, producing nucleoside analogs by standard chemical synthesis techniques can be challenging due to their chemical complexity. Summary of the Invention [Means for solving the problem]

[0006] The present invention provides engineered acetate kinase (AcK) enzymes, polypeptides having AcK activity, and polynucleotides encoding these enzymes, as well as vectors and host cells containing these polynucleotides and polypeptides. Methods for producing AcK enzymes are also provided. The present invention further provides compositions containing AcK enzymes and methods for using engineered AcK enzymes. The present invention is particularly useful in the production of pharmaceutical compounds.

[0007] In some embodiments, the present invention provides an engineered acetate kinase comprising a polypeptide sequence or functional fragment thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:2, SEQ ID NO:12, and / or SEQ ID NO:600, wherein the engineered acetate kinase comprises at least one substitution or set of substitutions in the polypeptide sequence, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:2, SEQ ID NO:12, and / or SEQ ID NO:600.

[0008] In some embodiments, the present invention provides an engineered acetate kinase comprising a polypeptide sequence or functional fragment thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:2, wherein the engineered acetate kinase comprises at least one substitution or set of substitutions in the polypeptide sequence, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:2. In some embodiments, the engineered acetate kinase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO:2 or a functional fragment thereof, and the engineered acetate kinase is selected from the group consisting of 12 / 259, 15, 16, 18, 23, 25, 26, 31, 32, 39, 40, 41, 44, 47, 47 / 411, 50, 51, 55, 58 / 135, 59, 64, 70, 76, 76 / 232 / 262, 76 / 232 / 364 / 386, 76 / 273, 85, 92, 96 / 119, 101, 102, 104, 107, 108, 116, 122, 135, 135 / 392, 136, 137, 140, 141, 143, 144, 145 / 400, 152, 154, 159, 161, 164, 183, 191, 19 4, 197, 215, 216, 222, 224, 229, 232, 236, 251, 256, 258, 259 / 284, 260, 261, 263 / 391, 268, 273, 274, 276, 283, 284, 286, 287, 288, 289, 290, 292, 297, 298, 298 / 405, 299, 300, 301, 302, 303, 304, 305, 308, 310, 312, 314, 316, 317, 32 2, 323, 337, 346, 347, 348, 349, 351, 352, 352 / 405, 354, 355, 356, 362, 366, 368, 371, 372, 373, 374, 375, 376, 377, 378, 380, 386, 390, 391, 392, 398, 399, and 407, wherein the amino acid positions of the polypeptide sequence are:The numbering is with reference to SEQ ID NO: 2. In some additional embodiments, the engineered acetate kinase is 12K / 259V, 15S, 16K, 16R, 16Y, 18C, 23A, 23F, 23G, 23H, 23L, 23N, 25L, 25V, 26G, 31D, 32T, 39A, 39V, 40L, 41C, 44L, 47D, 47S, 47S / 411F, 50K, 50N, 50Q, 51F, 55P, 58S / 135T, 59A, 59G, 59V, 64K, 70K, 70T, 76V, 76V / 232S / 262L, 76V / 232S / 364I / 386K, 76V / 273V, 85H, 92D, 9 6T / 119I, 101G, 101I, 101M, 101T, 102A, 104A, 104L, 107K, 108L, 108R, 108 W, 116D, 122C, 122E, 122G, 122T, 122V, 122W, 135L, 135N, 135T, 135T / 392W , 136A, 136E, 136G, 136T, 136V, 137K, 137R, 140F, 140P, 140R, 140S, 140W, 140Y, 141R, 143C, 144K, 144Q, 145E / 400V, 152Q, 154M, 154Q, 154T, 154V, 1 59F, 161M, 161S, 161V, 164G, 164H, 164L, 183A, 183G, 183S, 191I, 194K, 19 7P, 197Q, 197R, 215E, 215F, 215L, 215M, 216A, 216S, 222I, 224I, 229G, 229 N, 232S, 236G, 251G, 256L, 256M, 256Q, 258V, 259V / 284L, 260E, 260T, 261A , 263T / 391G, 268L, 273V, 274W, 276V, 283K, 284L, 286I, 287A, 287D, 287G, 287L, 287T, 288G, 289A, 289S, 290L, 292P, 297L, 297M, 297Q, 298F, 298K, 2 98L, 298L / 405E, 298Q, 298T, 298V, 298W, 299Q, 299R, 299S, 299T, 300G, 30 0S, 300T, 301E, 301K, 301Q, 301R, 302A, 302F, 302G, 302R, 302S, 303E, 304 A, 304C, 304D, 304E, 304Q, 304R, 304V, 305N, 308R, 310A, 310G, 310M, 312K,312P, 312R, 314V, 316A, 316G, 316M, 316Q, 316R, 316V, 317A, 317G, 317I, 317L, 317S, 322G, 32 2N, 322S, 323P, 323S, 337L, 346L, 347Q, 347R, 347T, 347V, 348E, 349V, 351L, 351R, 351S, 351V, 352A, 352F, 352G, 352K, 352P, 352T, 352V, 352V / 405Q, 354G, 354L, 354P, 354S, 354V, 354W, 355 D, 355E, 355K, 355M, 356G, 362I, 366E, 366G, 366I, 366L, 366P, 366R, 366S, 366W, 368E, 368G, 3 68L, 368N, 368Q, 368R, 368S, 368V, 371N, 371V, 372D, 372F, 372N, 372V, 373P, 373V, 374D, 374E , 374H, 374L, 374M, 374V, 375L, 376E, 376L, 376M, 376R, 376S, 376T, 376W, 377M, 377V, 378A, 38 and 407L, and at least one substitution or set of substitutions selected from: 0G, 386K, 390E, 390G, 390S, 390T, 390V, 391G, 391R, 392G, 392W, 398H, 398W, 398Y, 399A, 399P, 399S, and 407L, and the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:2. In some additional embodiments, the engineered acetate kinase is R12K / P259V, V15S, I16K, I16R, I16Y, S18C, I23A, I23F, I23G, I23H, I23L, I23N, Y25L, Y25V, Q26G, E31D, G32T, G39A, G39V, I40L, A41C, I44L, E47D, E47S, E47S / I411F, R50K, R50N, R50Q, L51F, V55P, E 58S / A135T, K59A, K59G, K59V, R64K, E70K, E70T, I76V, I76V / C232S / I262L, I76V / C232S / L364I / R386K, I76V / M273 V, L85H, K92D, A96T / L119I, V101G, V101I, V101M, V101T, V102A, G104A, G104L, R107K, F108L, F108R, F108W, E116D,I122C、I122E、I122G、I122T、I122V、I122W、A135L、A135N、A135T、A135T / V392W、N136A、N136E、N136G、N136T、N136V、L137K、L137R、I140F、I140P、I140R、I140S、I140W、I140Y、K141R、A143C、M144K、M144Q、K145E / I400V、N152Q、A154M、A154Q、A154T、A154V、A159F、H161M、H161S、H161V、I164G、I164H、I164L、I183A、I183G、I183S、T191I、R194K、S197P、S197Q、S197R、T215E、T215F、T215L、T215M、C216A、C216S、A222I、V224I、Y229G、Y229N、C232S、S236G、S251G、P256L、P256M、P256Q、I258V、P259V / Y284L、F260E、F260T、F261A、M263T / V391G、I268L、M273V、Y274W、I276V、V283K、Y284L、L286I、S287A、S287D、S287G、S287L、S287T、K288G、G289A、G289S、F290L、S292P、I297L、I297M、I297Q、E298F、E298K、E298L、E298L / K405E、E298Q、E298T、E298V、E298W、E299Q、E299R、E299S、E299T、A300G、A300S、A300T、A301E、A301K、A301Q、A301R、L302A、L302F、L302G、L302R、L302S、K303E、G304A、G304C、G304D、G304E、G304Q、G304R、G304V、D305N、C308R、L310A、L310G、L310M、L312K、L312P、L312R、I314V、D316A、D316G、D316M、D316Q、D316R、D316V、Y317A、Y317G、Y317I、Y317L、Y317S、Y322G、Y322N、Y322S、I323P、I323S、V337L、S346L、P347Q、P347R、P347T、P347V、I348E、T349V、E351L、E351R、E351S、E351V、D352A、D352F、D352G, D352K, D352P, D352T, D352V, D352V / K405Q, C354G, C354L, C354P, C354S, C 354V, C354W, S355D, S355E, S355K, S355M, Y356G, V362I, K366E, K366G, K366I, K3 66L, K366P, K366R, K366S, K366W, K368E, K368G, K368L, K368N, K368Q, K368R, K36 8S, K368V, E371N, E371V, T372D, T372F, T372N, T372V, I373P, I373V, R374D, R374 E, R374H, R374L, R374M, R374V, G375L, K376E, K376L, K376M, K376R, K376S, K376T, K376W, E377M, E377V, G378A, I380G, R386K, L390E, L390G, L390S, L390T, L390V, V391G, V391R, V392G, V392W, L398H, L398W, L398Y, M399A, M399P, M399S, and I407L, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:2. In some embodiments, the engineered acetate kinase comprises a polypeptide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence of at least one engineered acetate kinase variant listed in Table 2-1.

[0009] In some further embodiments, the engineered acetate kinase comprises a polypeptide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:2.

[0010] In some embodiments, the present invention provides an engineered acetate kinase comprising a polypeptide sequence or functional fragment thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:12, wherein the engineered acetate kinase comprises at least one substitution or set of substitutions in the polypeptide sequence, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:12. In some embodiments, the engineered acetate kinase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 12 or a functional fragment thereof, and the engineered acetate kinase is selected from the group consisting of 15 / 70 / 154 / 297 / 352 / 355, 15 / 154 / 191 / 297 / 2 98 / 301 / 348 / 352 / 391, 15 / 154 / 191 / 297 / 355, 15 / 154 / 297 / 298 / 348 / 352 / 355 / 391, 15 / 191 / 297 / 298 / 301 / 348 / 352 / 355 / 391, 15 / 191 / 298 / 352 / 355, 15 / 297 / 298 / 355, 23 / 101 / 102 / 122 / 140 / 143 / 316 / 372, 23 / 101 / 102 / 140, 23 / 101 / 374, 23 / 122 / 316 / 372 / 374, 23 / 122 / 316 / 374 / 395, 23 / 140 / 316 / 374, 29 / 154 / 191 / 298 / 348 / 355, 51 / 101 / 102 / 135 / 242 / 316 / 374, 51 / 101 / 316, 70 / 154 / 162 / 191 / 297 / 301 / 355 / 391, 70 / 154 / 191 / 297 / 352, 70 / 154 / 191 / 298 / 348 / 352 / 355 / 391, 70 / 154 / 191 / 298 / 352, 70 / 154 / 297 / 298 / 348 / 355, 70 / 154 / 297 / 352 / 355, 70 / 191 / 297 / 298 / 352 / 391, 101 / 102 / 122 / 140 / 142 / 316 / 372 / 374, 101 / 136 / 242 / 372, 102 / 135 / 140 / 316, 102 / 136 / 140 / 142 / 143 / 316,102 / 142 / 316, 122 / 140 / 142 / 164 / 242, 122 / 142 / 316, 122 / 143 / 242 / 374, 135 / 136 / 140 / 142 / 143 / 242 / 316 / 372 / 374, 135 / 140 / 143 / 242 / 374 / 395, 135 / 140 / 316, 136 / 242, 142 / 316, 142 / 316 / 372 / 374, 142 / 316 / 374, 143 / 316, 154 / 191 / 2 and 154 / 297 / 298, 154 / 191 / 297 / 298 / 301 / 352 / 355, 154 / 191 / 297 / 298 / 352, 154 / 191 / 298 / 301 / 348 / 352 / 391, 154 / 191 / 298 / 348 / 352, and 154 / 297 / 298, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:12. In some additional embodiments, the engineered acetate kinase is 15N / 70K / 154Q / 297N / 352T / 355K, 15N / 154Q / 191I / 297L / 298F / 301R / 348E / 352T / 391R, 15N / 154Q / 191I / 297N / 355K, 15N / 154Q / 297N / 298F / 348E / 352G / 35 5K / 391R, 15N / 191I / 297L / 298F / 301R / 348E / 352T / 355K / 391R, 15N / 191I / 298F / 352T / 355K, 1 5N / 297L / 298F / 355K, 23H / 101I / 102A / 122G / 140P / 143C / 316R / 372F, 23H / 101I / 102A / 140P, 2 3H / 101I / 374L, 23H / 122G / 316R / 372F / 374L, 23H / 122G / 316R / 374E / 395K, 23H / 140P / 316R / 3 74E, 29D / 154Q / 191I / 298F / 348E / 355K, 51F / 101I / 102A / 135L / 242T / 316R / 374L, 51F / 101I / 3 16R, 70K / 154Q / 162H / 191I / 297N / 301R / 355K / 391R, 70K / 154Q / 191I / 297L / 352T, 70K / 154Q / 1 91I / 298F / 348E / 352T / 355K / 391R, 70K / 154Q / 191I / 298F / 352T, 70K / 154Q / 297L / 352G / 355K,70K / 154Q / 297N / 298F / 348E / 355K, 70K / 191I / 297N / 298F / 352T / 391R, 101I / 102A / 122G / 140P / 14 2V / 316R / 372F / 374L, 101I / 136V / 242T / 372F, 102A / 135L / 140P / 316R, 102A / 136A / 140P / 142V / 14 3C / 316R, 102A / 142V / 316R, 122G / 140P / 142V / 164H / 242T, 122G / 142V / 316R, 122G / 143C / 242T / 37 4E, 135L / 136A / 140P / 142V / 143C / 242T / 316R / 372F / 374L, 135L / 140P / 143C / 242T / 374L / 395K, 135 L / 140P / 316R, 136V / 242T, 142V / 316R, 142V / 316R / 372F / 374E, 142V / 316R / 374L, 143C / 316R, 154 Q / 191I / 297L / 298F, 154Q / 191I / 297L / 298F / 301R / 352T / 355K, 154Q / 191I / 297N / 298F, 154Q / 191 and at least one substitution or set of substitutions selected from I / 297N / 298F / 352G, 154Q / 191I / 298F / 301R / 348E / 352T / 391R, 154Q / 191I / 298F / 348E / 352T, and 154Q / 297L / 298F, and the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:12. In some further embodiments, the engineered acetate kinase is V15N / E70K / A154Q / I297N / D352T / S355K, V15N / A154Q / T191I / I297L / L298F / A301R / I348E / D352T / V391R, V15N / A154Q / T191I / I297N / S355K, V15N / A154Q / I297N / L298F / I348E / D352G / S355K / V391R, V15N / T191I / I297L / L298F / A301R / I348E / D352T / S355K / V391R, V15N / T191I / L2 98F / D352T / S355K, V15N / I297L / L298F / S355K, I23H / V101I / V102A / I122G / I140P / A143C / D316R / T372F,I23H / V101I / V102A / I140P、I23H / V101I / R374L、I23H / I122G / D316R / T372F / R374L、I23H / I122G / D316R / R374E / N395K、I23H / I140P / D316R / R374E、E29D / A154Q / T191I / L298F / I348E / S355K、L51F / V101I / V102A / A135L / L242T / D316R / R374L、L51F / V101I / D316R、E70K / A154Q / Q162H / T191I / I297N / A301R / S355K / V391R、E70K / A154Q / T191I / I297L / D352T、E70K / A154Q / T191I / L298F / I348E / D352T / S355K / V391R、E70K / A154Q / T191I / L298F / D352T、E70K / A154Q / I297L / D352G / S355K、E70K / A154Q / I297N / L298F / I348E / S355K、E70K / T191I / I297N / L298F / D352T / V391R、V101I / V102A / I122G / I140P / A142V / D316R / T372F / R374L、V101I / N136V / L242T / T372F、V102A / A135L / I140P / D316R、V102A / N136A / I140P / A142V / A143C / D316R、V102A / A142V / D316R、I122G / I140P / A142V / I164H / L242T、I122G / A142V / D316R、I122G / A143C / L242T / R374E、A135L / N136A / I140P / A142V / A143C / L242T / D316R / T372F / R374L、A135L / I140P / A143C / L242T / R374L / N395K、A135L / I140P / D316R、N136V / L242T、A142V / D316R、A142V / D316R / T372F / R374E、A142V / D316R / R374L、A143C / D316R、A154Q / T191I / I297L / L298F、A154Q / T191I / I297L / L298F / A301R / D352T / S355K、A154Q / T191I / I297N / L298F、A154Q / T191I / I297N / L298F / D352G、and A154Q / I297L / L298F, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 12. In some embodiments, the engineered acetate kinase comprises a polypeptide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence of at least one engineered acetate kinase variant described in Table 3-1. In some further embodiments, the engineered acetate kinase comprises a polypeptide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 12. In some additional embodiments, the engineered acetate kinase is a variant engineered polypeptide set forth in SEQ ID NO: 12.

[0011] In some embodiments, the present invention provides an engineered acetate kinase comprising a polypeptide sequence or functional fragment thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 600, wherein the engineered acetate kinase comprises at least one substitution or set of substitutions in the polypeptide sequence, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 600. In some embodiments, the engineered acetate kinase comprises a polypeptide sequence or functional fragment thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 600, wherein the engineered acetate kinase comprises at least one substitution or set of substitutions at one or more positions selected from 23 / 242 / 374, 23 / 374, 70 / 374, 242 / 298, 242 / 374, 298 / 374, and 374, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 600. In some additional embodiments, the engineered acetate kinase comprises at least one substitution or set of substitutions selected from 23H / 242T / 374L, 23H / 374L, 70K / 374L, 242T / 298F, 242T / 374L, 298F / 374L, and 374L, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 600. In some additional embodiments, the engineered acetate kinase comprises at least one substitution or set of substitutions selected from I23H / L242T / R374L, I23H / R374L, E70K / R374L, L242T / L298F, L242T / R374L, L298F / R374L, and R374L, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 600.In some embodiments, the engineered acetate kinase comprises a polypeptide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence of at least one engineered acetate kinase variant listed in Table 4-1.

[0012] In some further embodiments, the engineered acetate kinase comprises a polypeptide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence of at least one engineered acetate kinase variant set forth in an even-numbered sequence of SEQ ID NOs: 2-696. In some additional embodiments, the engineered acetate kinase comprises a polypeptide sequence set forth in an even-numbered sequence of SEQ ID NOs: 2-696. In some further embodiments, the engineered acetate kinase comprises at least one improved property relative to wild-type Thermotoga maritima acetate kinase. In yet some additional embodiments, the improved property comprises improved activity toward a substrate relative to wild-type acetate kinase. In some embodiments, the substrate comprises a nucleoside triphosphate and acetate and / or an acetyl phosphate and a nucleoside diphosphate. In some further embodiments, the substrates comprise adenosine diphosphate (ADP) and acetyl phosphate and / or adenosine triphosphate (ATP) and acetate. In some additional embodiments, the improved property comprises improved production of adenosine triphosphate (ATP) compared to wild-type acetate kinase. In some further embodiments, the engineered acetate kinase is purified. The present invention also provides compositions comprising at least one engineered acetate kinase provided herein. In some embodiments, the present invention provides compositions comprising one engineered acetate kinase provided herein.

[0013] The present invention also provides polynucleotide sequences encoding at least one engineered acetate kinase provided herein. In some embodiments, the polynucleotide sequence encoding at least one engineered acetate kinase comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO:1, SEQ ID NO:11, and / or SEQ ID NO:599. In some embodiments, the polynucleotide sequence of the engineered acetate kinase comprises at least one substitution at one or more positions. In some further embodiments, the polynucleotide sequence encoding at least one engineered acetate kinase or functional fragment thereof comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO:1. In some further embodiments, the polynucleotide sequence encoding at least one engineered acetate kinase or functional fragment thereof comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 11. In some further embodiments, the polynucleotide sequence encoding at least one engineered acetate kinase or functional fragment thereof comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 599. In some additional embodiments, the polynucleotide sequence is operably linked to a regulatory sequence. In some embodiments, the polynucleotide sequence is codon-optimized. In some further embodiments, the polynucleotide comprises the odd-numbered sequences of SEQ ID NOs: 1-695. The present invention also provides expression vectors comprising at least one polynucleotide sequence encoding an acetate kinase provided herein. In some embodiments, the expression vector comprises one polynucleotide sequence encoding an acetate kinase provided herein.The present invention also provides a host cell comprising at least one expression vector provided herein.The present invention also provides a host cell comprising at least one polynucleotide sequence encoding acetate kinase provided herein.

[0014] The present invention also provides methods for producing an engineered acetate kinase in a host cell, the methods comprising culturing the host cells provided herein under suitable conditions such that at least one engineered acetate kinase is produced. In some embodiments, the methods further comprise recovering the at least one engineered acetate kinase from the culture and / or host cells. In some additional embodiments, the methods further comprise purifying the at least one engineered acetate kinase. DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention provides engineered acetate kinase (AcK) enzymes, polypeptides having AcK activity, and polynucleotides encoding these enzymes, as well as vectors and host cells containing these polynucleotides and polypeptides. Methods for producing AcK enzymes are also provided. The present invention further provides compositions containing AcK enzymes and methods for using engineered AcK enzymes. The present invention is particularly useful in the production of pharmaceutical compounds.

[0016] Unless otherwise defined, all scientific and technical terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. 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 materials well known to those skilled in the art. Standard techniques, or modifications thereof, are used for chemical synthesis and chemical analysis. All patents, patent applications, articles, and publications mentioned herein, both above and below, are hereby expressly incorporated by reference.

[0017] Although any suitable method and material similar or equivalent to the method and material described herein can be used in the practice of the present invention, some methods and materials are described herein.It should be understood that the present invention is not limited to the specific methodology, protocols, and reagents described, since they may vary depending on the context in which they are used by those skilled in the art.Therefore, the terms defined immediately below will be more fully explained with reference to the present invention as a whole.

[0018] It should 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 should not be construed as limiting the subject matter described. Numerical ranges include the numbers defining that 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. Similarly, every maximum (or minimum) numerical limitation disclosed herein is also intended to include every lower (or higher) numerical limitation, as if such lower (or higher) numerical limitations were expressly written herein.

[0019] Abbreviation Abbreviations used for genetically encoded amino acids are conventional and are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartate (Asp or D), cysteine (Cys or C), glutamate (Glu or E), glutamine (Gln or Q), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0020] When a three-letter abbreviation is used, an amino acid is referred to as an amino acid having an α-carbon (C α ) can be in either the L- or D-configuration about the α-carbon. For example, "Ala" specifically denotes alanine with no configuration about the α-carbon, while "D-Ala" and "L-Ala" specifically denote D-alanine and L-alanine, respectively. When single-letter abbreviations are used, an uppercase letter specifically denotes an amino acid in the L-configuration about the α-carbon, and a lowercase letter specifically denotes an amino acid in the D-configuration about the α-carbon. For example, "A" specifically denotes L-alanine, and "a" specifically denotes D-alanine. When a polypeptide sequence is presented as a series of single-letter or three-letter abbreviations (or mixtures thereof), the sequence is represented in the amino (N) to carboxy (C) direction according to common convention.

[0021] The abbreviations used for genetically coded nucleosides are conventional and are as follows: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). Unless specifically depicted, the abbreviated nucleoside may be either a ribonucleoside or a 2'-deoxyribonucleoside. Nucleosides may be designated as either ribonucleosides or 2'-deoxyribonucleosides, either individually or in aggregate. When a nucleic acid sequence is represented as a series of single-letter abbreviations, the sequence is represented in the 5' to 3' direction, according to common convention, and the phosphate is not indicated.

[0022] definition In reference to the present invention, the technical and scientific terms used in the description herein have the meanings that are commonly understood by those of ordinary skill in the art, unless specifically defined otherwise. Accordingly, the following terms are intended to have the following meanings:

[0023] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "one polypeptide" includes more than one polypeptide.

[0024] Similarly, "comprise," "comprises," "comprising," "include," "includes," and "including" are intended to be interchangeable and not limiting. Thus, as used herein, the term "comprising" and its cognates are used in their inclusive sense (i.e., equivalent to the term "including" and its corresponding cognates).

[0025] It should be further understood that where the descriptions of various embodiments use the term "comprising," those skilled in the art will understand that in some specific instances, an embodiment can alternatively be described using the language "consisting essentially of" or "consisting of."

[0026] As used herein, the term "about" refers to an acceptable error for a particular value. In some instances, "about" refers to within 0.05%, 0.5%, 1.0%, or 2.0% of a given value range. In some instances, "about" refers to within 1, 2, 3, or 4 standard deviations of a given value.

[0027] As used herein, the "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.

[0028] As used herein, "ATCC" refers to the American Type Culture Collection, whose biorepository collection includes genes and strains.

[0029] As used herein, "NCBI" refers to the National Center for Biological Information and the sequence databases provided therein.

[0030] As used herein, "acetate kinase" and "AcK" refer to an enzyme (EC 2.7.2.1) that mediates the reversible interconversion of nucleoside triphosphates (e.g., ATP) and acetate to acetyl phosphate and nucleoside diphosphates (e.g., ADP), or a variant enzyme derived from such an AcK enzyme, whether or not such variant enzyme retains the same functionality as the source (i.e., "parent") enzyme.

[0031] "Protein," "polypeptide," and "peptide" are used interchangeably herein to refer to a polymer of at least two amino acids covalently joined by an amide bond, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). This definition includes D- and L-amino acids, mixtures of D- and L-amino acids, and polymers comprising D- and L-amino acids and mixtures of D- and L-amino acids.

[0032] "Amino acids" are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter symbols.

[0033] As used herein, "hydrophilic amino acid or residue" refers to an amino acid or residue having a side chain that exhibits a hydrophobicity of less than zero according to the normalized consensus hydrophobicity scale of Eisenberg et al. (Eisenberg et al., J. Mol. Biol., 179:125-142

[1984] ). Genetically encoded hydrophilic amino acids include L-Thr (T), L-Ser (S), L-His (H), L-Glu (E), L-Asn (N), L-Gln (Q), L-Asp (D), L-Lys (K), and L-Arg (R).

[0034] As used herein, "acidic amino acid or residue" refers to a hydrophilic amino acid or residue having a side chain that exhibits a pKa value of less than about 6 when the amino acid is included in a peptide or polypeptide. Acidic amino acids typically have a side chain that is negatively charged at physiological pH due to loss of a hydrogen ion. Genetically encoded acidic amino acids include L-Glu (E) and L-Asp (D).

[0035] As used herein, "basic amino acid or residue" refers to a hydrophilic amino acid or residue having a side chain that exhibits a pKa value greater than about 6 when the amino acid is included in a peptide or polypeptide. Basic amino acids typically have a side chain that is positively charged at physiological pH due to association with a hydronium ion. Genetically encoded basic amino acids include L-Arg (R) and L-Lys (K).

[0036] As used herein, "polar amino acid or residue" refers to a hydrophilic amino acid or residue having a side chain that is uncharged at physiological pH but has at least one bond in which an electron pair commonly shared by two atoms is held more tightly by one of the atoms. Genetically encoded polar amino acids include L-Asn (N), L-Gln (Q), L-Ser (S), and L-Thr (T).

[0037] As used herein, "hydrophobic amino acid or residue" refers to an amino acid or residue having a side chain that exhibits a hydrophobicity greater than zero according to the normalized consensus hydrophobicity scale of Eisenberg et al. (Eisenberg et al., J. Mol. Biol., 179:125-142

[1984] ). Genetically encoded hydrophobic amino acids include L-Pro (P), L-Ile (I), L-Phe (F), L-Val (V), L-Leu (L), L-Trp (W), L-Met (M), L-Ala (A), and L-Tyr (Y).

[0038] As used herein, "aromatic amino acid or residue" refers to a hydrophilic or hydrophobic amino acid or residue having a side chain containing at least one aromatic or heteroaromatic ring. Genetically encoded aromatic amino acids include L-Phe (F), L-Tyr (Y), and L-Trp (W). Depending on the pKa of its heteroaromatic nitrogen atom, L-His (H) may be classified as a basic or aromatic residue because its side chain contains a heteroaromatic ring, whereas histidine is classified herein as a hydrophobic residue or "constrained residue" (see below).

[0039] As used herein, a "constrained amino acid or residue" refers to an amino acid or residue that has a constrained geometry. As used herein, constrained residues include L-Pro (P) and L-His (H). Histidine has a constrained geometry because it has a relatively small imidazole ring. Proline also has a constrained geometry because it has a five-membered ring.

[0040] As used herein, "nonpolar amino acid or residue" refers to a hydrophobic amino acid or residue having a side chain that is uncharged at physiological pH and has a bond in which the electron pair commonly shared by two atoms is generally held equally by each of the two atoms (i.e., the side chain is nonpolar). Genetically encoded nonpolar amino acids include L-Gly (G), L-Leu (L), L-Val (V), L-Ile (I), L-Met (M), and L-Ala (A).

[0041] As used herein, "aliphatic amino acid or residue" refers to a hydrophobic amino acid or residue having an aliphatic hydrocarbon side chain. Genetically encoded aliphatic amino acids include L-Ala (A), L-Val (V), L-Leu (L), and L-Ile (I). Note that cysteine (or "L-Cys" or "[C]") is unusual in that it can form disulfide bridges with other L-Cys (C) amino acids or other sulfanyl- or sulfhydryl-containing amino acids. "Cysteine-like residues" include cysteine and other amino acids that contain sulfhydryl moieties available for disulfide bridge formation. The ability of L-Cys (C) (and other amino acids with SH-containing side chains) to exist in a peptide in either the reduced, free -SH form or the oxidized, disulfide-bridged form affects whether L-Cys (C) confers a net hydrophobic or hydrophilic character to the peptide. L-Cys(C) exhibits a hydrophobicity of 0.29 according to the normalized consensus scale of Eisenberg (Eisenberg et al., 1984, supra), but for purposes of this disclosure, L-Cys(C) should be understood to be categorized in its own unique group.

[0042] As used herein, "small amino acid or residue" refers to an amino acid or residue having a side chain composed of a total of three or fewer carbon and / or heteroatoms (excluding the α-carbon and hydrogen). Small amino acids or residues may be further categorized as aliphatic, nonpolar, polar, or acidic small amino acids or residues according to the above definitions. Genetically encoded small amino acids include L-Ala (A), L-Val (V), L-Cys (C), L-Asn (N), L-Ser (S), L-Thr (T), and L-Asp (D).

[0043] As used herein, "hydroxyl-containing amino acid or residue" refers to an amino acid that contains a hydroxyl (-OH) moiety. Genetically encoded hydroxyl-containing amino acids include L-Ser (S), L-Thr (T), and L-Tyr (Y).

[0044] As used herein, "polynucleotide" and "nucleic acid" refer to two or more nucleotides covalently linked together. A polynucleotide can be composed entirely of ribonucleotides (i.e., RNA), entirely of 2'-deoxyribonucleotides (i.e., DNA), or a mixture of ribonucleotides and 2'-deoxyribonucleotides. Nucleosides are typically linked together via standard phosphodiester linkages, although a polynucleotide can contain one or more non-standard linkages. A polynucleotide can be single-stranded or double-stranded, or can contain both single-stranded and double-stranded regions. Moreover, while a polynucleotide is typically composed of naturally occurring coding nucleobases (i.e., adenine, guanine, uracil, thymine, and cytosine), it can contain 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 that encode an amino acid sequence.

[0045] As used herein, "nucleoside" refers to a glycosylamine comprising a nucleobase (i.e., a nitrogenous base) and a five-carbon sugar (e.g., ribose or deoxyribose). Non-limiting examples of nucleosides include cytidine, uridine, adenosine, guanosine, thymidine, and inosine. In contrast, the term "nucleotide" refers to a glycosylamine comprising a nucleobase, a five-carbon sugar, and one or more phosphate groups. In some embodiments, a nucleoside can be phosphorylated by a kinase to produce a nucleotide.

[0046] As used herein, "nucleoside diphosphate" refers to a glycosylamine containing a nucleobase (i.e., a nitrogenous base), a five-carbon sugar (e.g., ribose or deoxyribose), and a diphosphate (i.e., pyrophosphate) moiety. In some embodiments herein, "nucleoside diphosphate" is abbreviated as "NDP." Non-limiting examples of nucleoside diphosphates include cytidine diphosphate (CDP), uridine diphosphate (UDP), adenosine diphosphate (ADP), guanosine diphosphate (GDP), thymidine diphosphate (TDP), and inosine diphosphate (IDP). The terms "nucleoside" and "nucleotide" may be used interchangeably in some contexts.

[0047] As used herein, "coding sequence" refers to a portion of a nucleic acid (eg, a gene) that encodes the amino acid sequence of a protein.

[0048] As used herein, the terms "biocatalyst," "biocatalytic," "biotransformation," and "biosynthesis" refer to the use of enzymes to carry out chemical reactions on organic compounds.

[0049] As used herein, "wild-type" and "naturally occurring" refer to forms found in nature. For example, a wild-type polypeptide or polynucleotide sequence is a sequence that can be isolated from a natural source and is present in an organism that has not been intentionally modified by human manipulation.

[0050] As used herein, "recombinant," "engineered," "variant," and "non-naturally occurring," when used in reference to a cell, nucleic acid, or polypeptide, refer to a material that has been altered in a manner that does not otherwise occur in nature, or a material that corresponds to the natural or native form of the material. In some embodiments, the cell, nucleic acid, or polypeptide is identical to a naturally occurring cell, nucleic acid, or polypeptide, but is produced or derived by synthetic materials and / or manipulation using recombinant technology. Non-limiting examples include recombinant cells that express genes not found in the native (non-recombinant) form of the cell, or that express native genes that are otherwise expressed at different levels, among others.

[0051] The term "percent (%) sequence identity" as used herein refers to a comparison of polynucleotides or polypeptides, and is determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence due to the optimal alignment of the two sequences. The percentage can be calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in both sequences to generate the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to generate the percentage of sequence identity. Alternatively, the percentage can be calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in both sequences, or where the nucleic acid base or amino acid residue is aligned with a gap to generate the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to generate the percentage of sequence identity. Those skilled in the art will recognize that there are many established algorithms available for aligning two sequences. Optimal alignment of sequences for comparison can be performed by any suitable method, including, but not limited to, the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 2:482

[1981] ), the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 48:443

[1970] ), by the similarity search method of Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444

[1988] ), 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 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 in Altschul et al. (see Altschul et al., J. Mol. Biol., 215: 403-410

[1990] ; and Altschul et al., Nucl. Acids Res., 3389-3402

[1977] , respectively). Software for performing BLAST analyses is publicly available through the website of the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in a query sequence that, when aligned with words of the same length in a database sequence, match or meet some positive threshold score T. T is referred to as the neighborhood word score threshold (see 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 possible to increase the cumulative alignment score. For nucleotide sequences, the cumulative score is calculated using 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 is terminated if the cumulative alignment score falls by an amount X from its maximum achieved value; if the accumulation of one or more negative-scoring residue alignments causes the cumulative score to be zero or less; or if 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 word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915

[1989] ). Exemplary sequence alignments and determinations of percent sequence identity can be employed using the BESTFIT or GAP programs in the GCG Wisconsin software package (Accelrys, Madison WI) using the default parameters provided.

[0052] As used herein, a "reference sequence" refers to a defined sequence used as the basis for sequence and / or activity comparison. A reference sequence can be a subset of a larger sequence, such as a segment of a full-length gene or polypeptide sequence. Generally, a reference sequence is at least 20 nucleotides or amino acid residues in length, at least 25 residues in length, at least 50 residues in length, at least 100 residues in length, or the entire length of the nucleic acid or polypeptide. Because two polynucleotides or polypeptides can each contain (1) sequences that are similar between the two sequences (i.e., a portion of the complete sequence), and (2) additional sequences that differ between the two sequences, sequence comparison between two (or more) polynucleotides or polypeptides is typically performed by comparing the 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, although the reference sequence may have one or more changes in the primary sequence.

[0053] As used herein, a "comparison window" refers to a conceptual segment of at least about 20 contiguous nucleotide positions or amino acid residues within which a sequence may be compared to a reference sequence of at least 20 contiguous nucleotides or amino acids, and within which the portion of the sequence in the comparison window may contain 20 percent or less additions or deletions (i.e., gaps) relative to the reference sequence (no additions or deletions) for optimal alignment of the two sequences. The comparison window may be longer than 20 contiguous residues, including windows of 30, 40, 50, 100, or longer, as appropriate.

[0054] As used herein, "corresponding," "with reference to," and "compared to," when used in the context of numbering a given amino acid or polynucleotide sequence, refer to the numbering of residues in a specified reference sequence when comparing the given amino acid or polynucleotide sequence to the reference sequence. In other words, the residue numbers or residue positions in a given polymer are explicitly stated with respect to the reference sequence, rather than the actual numerical positions of the residues in the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as the amino acid sequence of an engineered acetate kinase, can be aligned with a reference sequence by introducing gaps to optimize residue matching between the two sequences. In these cases, although gaps exist, the numbering of residues in a given amino acid or polynucleotide sequence is done with respect to the reference sequence to which it is aligned.

[0055] As used herein, "substantial identity" refers to a polynucleotide or polypeptide sequence that shares at least 80 percent sequence identity, at least 85 percent identity, at least 89-95 percent sequence identity, or more usually at least 99 percent sequence identity with a reference sequence over a comparison window of at least 20 residue positions, often over a window of at least 30-50 residues, where the percentage of sequence identity is calculated by comparing the reference sequence to a sequence that contains deletions or additions totaling 20 percent or less of the reference sequence over the comparison window. In some specific embodiments, as applied to polypeptides, the term "substantial identity" means that two polypeptide sequences share at least 80 percent sequence identity, preferably at least 89 percent sequence identity, at least 95 percent sequence identity, or even higher identity (e.g., 99 percent sequence identity) when optimally aligned, e.g., by the programs GAP or BESTFIT using default gap weighting. In some embodiments, residue positions that are not identical in the compared sequences differ by conservative amino acid substitutions.

[0056] As used herein, "amino acid difference" and "residue difference" refer to the difference in amino acid residue at a position of a polypeptide sequence compared to the amino acid residue at the corresponding position in a reference sequence. In some examples, the reference sequence has a histidine tag, but the numbering is maintained compared to an equivalent reference sequence without the histidine tag. The position of the amino acid difference is generally referred to herein as "Xn," where n refers to the corresponding position in the reference sequence for which the residue difference is based. For example, "a residue difference at position X93 compared to SEQ ID NO:4" refers to the difference in the amino acid residue at the polypeptide position corresponding to position 93 of SEQ ID NO:4. Thus, if the reference polypeptide of SEQ ID NO:4 has a serine at position 93, then "a residue difference at position X93 compared to SEQ ID NO:4" is an amino acid substitution of any residue other than serine at the polypeptide position corresponding to position 93 of SEQ ID NO:4. In most examples herein, a specific amino acid residue difference at a position is referred to as "XnY," where "Xn" designates the corresponding position above and "Y" is the single-letter identifier of the amino acid found in the engineered polypeptide (i.e., the residue that differs from the reference polypeptide). In some examples (e.g., tables presented in the Examples), the present invention also provides specific amino acid differences, designated 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. In some examples, the polypeptides of the present invention may contain one or more amino acid residue differences compared to the reference sequence, as indicated by a list of designated positions where the residue difference occurs compared to the reference sequence. In some embodiments, when more than one amino acid can be used at a specific residue position in the polypeptide, the various amino acid residues that can be used are separated by a " / " (e.g., X307H / X307P or X307H / P). A slash can also be used to indicate multiple substitutions within a given variant (i.e., more than one substitution is present in a given sequence, such as in a combinatorial variant).In some embodiments, the invention includes engineered polypeptide sequences that contain one or more amino acid differences, including conservative or non-conservative amino acid substitutions. In some additional embodiments, the invention provides engineered polypeptide sequences that contain both conservative and non-conservative amino acid substitutions.

[0057] As used herein, "conservative amino acid substitution" refers to the replacement of a residue with a different residue having a similar side chain, and thus typically involves the replacement of an amino acid in a polypeptide with an amino acid within the same or similar defined class of amino acids. By way of non-limiting example, in some embodiments, an amino acid having an aliphatic side chain is replaced with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine), an amino acid having a hydroxyl side chain is replaced with another amino acid having a hydroxyl side chain (e.g., serine and threonine), an amino acid having an aromatic side chain is replaced with another amino acid having an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine), an amino acid having a basic side chain is replaced with another amino acid having a basic side chain (e.g., lysine and arginine), an amino acid having an acidic side chain is replaced with another amino acid having 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.

[0058] As used herein, a "non-conservative substitution" refers to the replacement of an amino acid in a polypeptide with an amino acid having significantly different side chain properties. Non-conservative substitutions may use amino acids between groups rather than within a defined group and affect (a) the structure of the peptide backbone in the area of substitution (e.g., proline for glycine), (b) the charge or hydrophobicity, or (c) the bulk of the side chain. By way of non-limiting example, exemplary non-conservative substitutions may 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.

[0059] As used herein, "deletion" refers to a modification to a polypeptide by removing one or more amino acids from a reference polypeptide. Deletions can include removing one or more amino acids, two or more amino acids, five or more amino acids, ten or more amino acids, fifteen or more amino acids, or twenty or more amino acids, up to 10% of the total number of amino acids comprising the reference enzyme, or up to 20% of the total number of amino acids, while retaining enzymatic activity and / or improving the properties of the engineered acetate kinase enzyme. Deletions can be directed to internal and / or terminal portions of the polypeptide. In various embodiments, deletions can include continuous segments or can be discontinuous. Deletions are typically indicated by "-" in the amino acid sequence.

[0060] As used herein, "insertion" refers to a modification of a polypeptide by adding one or more amino acids from a reference polypeptide. The insertion may be an insertion in the internal portion of the polypeptide, or may be an insertion at the carboxy or amino terminus. As used herein, an insertion includes fusion proteins known in the art. The insertion may be a continuous segment of amino acids, or may be separated by one or more amino acids in a naturally occurring polypeptide.

[0061] The term "amino acid substitution set" or "substitution set" refers to a group of amino acid substitutions in a polypeptide sequence relative to a reference sequence. A substitution set can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions. In some embodiments, a substitution set refers to the set of amino acid substitutions present in any of the variant acetate kinases described in the tables provided in the Examples.

[0062] "Functional fragment" and "biologically active fragment" are used interchangeably herein and refer to a polypeptide that has amino- and / or carboxy-terminal deletions and / or internal deletions, but where the remaining amino acid sequence is identical to the corresponding positions in the sequence to which it is compared (e.g., a full-length engineered acetate kinase of the invention), and which retains substantially all of the activity of the full-length polypeptide.

[0063] As used herein, an "isolated polypeptide" refers to a polypeptide that has been substantially separated from other contaminants (e.g., proteins, lipids, and polynucleotides) with which it is naturally associated. The term encompasses polypeptides that have been removed or purified from their naturally occurring environment or expression system (e.g., within a host cell or via in vitro synthesis). Recombinant acetate kinase polypeptides may be present intracellularly, in cell culture medium, or prepared in various forms, such as as a lysate or isolated preparation. Thus, in some embodiments, a recombinant acetate kinase polypeptide may be an isolated polypeptide.

[0064] As used herein, "substantially pure polypeptide" or "purified protein" refers to a composition in which the polypeptide species is the predominant species present (i.e., more abundant than any other individual macromolecular species in the composition, on a molar or weight basis); generally, a composition is substantially purified when the species of interest constitutes at least about 50 percent, by molar or percent weight, of the macromolecular species present. However, in some embodiments, a composition comprising acetate kinase comprises acetate kinase that is less than 50% pure (e.g., about 10%, about 20%, about 30%, about 40%, or about 50%). Generally, a substantially pure acetate kinase composition will constitute 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 percent weight, present in the composition. In some embodiments, the species of interest is purified to essential homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods), and 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 recombinant acetate kinase polypeptide is a substantially pure polypeptide composition.

[0065] As used herein, "improved enzymatic property" refers to at least one improved property of an enzyme. In some embodiments, the present invention provides engineered acetate kinase polypeptides that exhibit any improved enzymatic property relative to a reference acetate kinase polypeptide, and / or a wild-type acetate kinase polypeptide, and / or another engineered acetate kinase polypeptide. In this manner, the level of "improvement" can be determined and compared among various acetate kinase polypeptides, including wild-type and engineered acetate kinases. Improved properties include, but are not limited to, properties such as increased protein expression, increased thermoactivity, increased thermostability, increased pH activity, increased stability, increased enzymatic activity, increased substrate specificity or affinity, increased specific activity, increased resistance to substrate or end-product inhibition, increased chemical stability, improved chemical selectivity, improved solvent stability, increased tolerance to acidic pH, increased resistance to proteolytic activity (i.e., reduced susceptibility to proteolysis), reduced aggregation, increased solubility, and an altered temperature profile. In additional embodiments, the term is used in reference to at least one improved property of an acetate kinase enzyme. In some embodiments, the present invention provides engineered acetate kinase polypeptides that exhibit any improved enzymatic property relative to a reference acetate kinase polypeptide and / or a wild-type acetate kinase polypeptide and / or another engineered acetate kinase polypeptide, such that the level of "improvement" can be determined and compared among various acetate kinase polypeptides, including wild-type and engineered acetate kinases.

[0066] As used herein, "increased enzymatic activity" and "enhanced catalytic activity" refer to improved properties of an engineered polypeptide, which may be expressed by an increase in specific activity (e.g., product produced / time / weight protein) or an increase in percent conversion of substrate to product (e.g., percent conversion of a starting amount of substrate to product in a specified period of time using a specified amount of enzyme) compared to a reference enzyme. In some embodiments, the terms refer to improved properties of an engineered acetate kinase polypeptide provided herein, which may be expressed by an increase in specific activity (e.g., product produced / time / weight protein) or an increase in percent conversion of substrate to product (e.g., percent conversion of a starting amount of substrate to product in a specified period of time using a specified amount of acetate kinase) compared to a reference acetate kinase enzyme. In some embodiments, the terms are used in reference to improved acetate kinase enzymes provided herein. Exemplary methods for determining the enzymatic activity of an engineered acetate kinase of the invention are provided in the Examples. Any property related to enzymatic activity, a change of which can lead to increased enzymatic activity, can be affected, including the classical enzymatic properties of Km, Vmax, or kcat. For example, the improvement in enzymatic activity can be from about 1.1-fold the enzymatic activity of the corresponding wild-type enzyme to as much as 2-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold, or more enzymatic activity of a naturally occurring acetate kinase or another engineered acetate kinase from which the acetate kinase polypeptide is derived.

[0067] As used herein, "conversion" refers to the enzymatic conversion (or biotransformation) of a substrate to a corresponding product. "Percent conversion" refers to the percent of a substrate that is converted to a product within a given period of time under specified conditions. Thus, the "enzyme activity" or "activity" of an acetate kinase polypeptide can be expressed as the "percent conversion" of substrate to product within a specified period of time.

[0068] An enzyme with "generalist properties" (or "generalist enzyme") refers to an enzyme that exhibits improved activity with a broader range of substrates compared to the parent sequence. A generalist enzyme need not necessarily demonstrate improved activity with every possible substrate. In some embodiments, the present invention provides acetate kinase variants with generalist properties in that they demonstrate similar or improved activity with a broad range of sterically and electronically diverse substrates compared to the parent gene. Additionally, the generalist enzymes provided herein have been engineered to be improved across a broad range of diverse molecules to increase production of metabolites / products.

[0069] The term "stringent hybridization conditions" as used herein refers to the conditions under which nucleic acid hybrids are stable.As known to those skilled in the art, hybrid stability is reflected in the melting temperature (Tm) of hybrids.Generally, hybrid stability is a function of ionic strength, temperature, G / C content and the presence of chaotropic agents. Tm values for polynucleotides can be calculated using known methods for predicting melting temperatures (e.g., Baldino et al., Meth. Enzymol., 168:761-777

[1989] ; Bolton et al., Proc. Natl. Acad. Sci. USA 48:1390

[1962] ; Bresslauer et al., Proc. Natl. Acad. Sci. USA 83:8893-8897

[1986] ; Freier et al., Proc. Natl. Acad. Sci. USA 83:9373-9377

[1986] ; Kierzek et al., Biochem., 25:7840-7846

[1986] ; Rychlik et al., Nucl. Acids Res., 18:6409-6412

[1990] ). (See, e.g., Nucl. Acids Res., 19:698

[1991] ; Sambrook et al., supra); 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. 26:227-259

[1991] .) In some embodiments, the polynucleotide encodes a polypeptide disclosed herein and hybridizes under defined conditions, e.g., moderately stringent or highly stringent conditions, to the complement of a sequence encoding an engineered acetate kinase enzyme of the invention.

[0070] As used herein, "stringency of hybridization" refers to hybridization conditions, such as washing conditions, in nucleic acid hybridization. Generally, hybridization reactions are carried out under low stringency conditions, followed by washing at various but higher stringencies. The term "moderately stringent hybridization" refers to conditions that allow target DNA to bind to complementary nucleic acids that have about 60% identity with the target DNA, preferably about 75% identity, about 85% identity, or about 90% or more identity with the target polynucleotide. Exemplary moderately stringent conditions are equivalent to hybridization in 50% formamide, 5x Denhart's solution, 5x SSPE, 0.2% SDS at 42°C, followed by washing in 0.2x SSPE, 0.2% SDS at 42°C. "High stringency hybridization" generally refers to conditions that are about 10°C or lower than the thermal melting temperature, Tm, determined under solution conditions for a defined polynucleotide sequence. In some embodiments, high stringency conditions refer to conditions that allow hybridization of only those nucleic acid sequences that form stable hybrids at 65°C in 0.018M NaCl (i.e., if a hybrid is not stable at 65°C in 0.018M NaCl, it is not stable under high stringency conditions as contemplated herein). High stringency conditions can be provided, for example, by hybridization under conditions equivalent to 42°C in 50% formamide, 5x Denhart's solution, 5x SSPE, 0.2% SDS, followed by a wash in 0.1x SSPE and 0.1% SDS at 65°C. Another high stringency condition is hybridization under conditions equivalent to hybridization in 5×SSC containing 0.1% (weight / volume) SDS at 65° C., and washing in 0.1×SSC containing 0.1% SDS at 65° C. Other high stringency conditions, as well as moderately stringent conditions, are described in the references cited above.

[0071] As used herein, "codon optimization" refers to changing the codons of a polynucleotide encoding a protein to those preferentially used in a particular organism so that the encoded protein can be efficiently expressed in the target organism. Although the genetic code is degenerate in that most amino acids are represented by several codons called "synonymous" or "synonymous" codons, it is well known that the codon usage by a particular organism is non-random and biased toward certain codon triplets. This codon usage bias can be higher for certain genes, genes with common functions or ancestral origins, highly expressed proteins compared to proteins with low copy numbers, and the total protein-coding region of an organism's genome. In some embodiments, a polynucleotide encoding an acetate kinase enzyme can be codon-optimized for optimal production in the host organism selected for expression.

[0072] As used herein, "preferred," "optimal," and "high codon usage bias" codons, when used alone or in combination, interchangeably refer to codons that are used in protein-coding regions at a higher frequency than other codons that encode the same amino acid. Preferred codons can be determined in relation to codon usage in a single gene, a set of genes of common function or origin, highly expressed genes, codon frequency in the total protein-coding region of an entire organism, codon frequency in the total protein-coding region of closely related organisms, or a combination thereof. Codons whose frequency increases with gene expression level are typically optimal codons for expression. A variety of methods are known for determining codon frequency (e.g., codon usage, relative synonymous codon usage), and codon preference in a particular organism, and the effective number of codons used in a gene, including multivariate analysis using, for example, cluster analysis or correspondence analysis (see, e.g., GCG CodonPreference, Genetics Computer Group Wisconsin Package; CodonW, Peden, University of Nottingham; McInerney, Bioinform., 14:372-73

[1998] ; Stenico et al., Nucl. Acids Res., 222437-46

[1994] ; and Wright, Gene 87:23-29

[1990] ). Codon usage tables are available for many different organisms (see, e.g., Wada et al., Nucl. Acids Res., 20:2111-2118

[1992] ; Nakamura et al., Nucl. Acids Res., 28:292

[2000] ; Duret et al., supra; Henaut and Danchin, in Escherichia coli and Salmonella, Neidhardt et al. (eds.), ASM Press, Washington DC, pp. 2047-2066

[1996] ). The data source for obtaining codon usage can rely on any available nucleotide sequence capable of encoding a protein.These datasets include nucleic acid sequences that are 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, NY

[2001] ; Uberbacher, Meth. Enzymol., 266:259-281

[1996] ; and Tiwari et al., Comput. Appl. Biosci., 13:263-270

[1997] ).

[0073] As used herein, the term "control sequences" includes all components necessary or advantageous for the expression of polynucleotides and / or polypeptides 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 sequence, signal peptide sequence, initiation sequence, and transcription terminator. At a minimum, control sequences include a promoter, and transcription and translation stop signals. Control sequences may be provided with linkers for the purpose of introducing specific restriction sites to facilitate ligation of the control sequences with the coding region of the nucleic acid sequence encoding the polypeptide.

[0074] "Operably linked" is defined herein as a configuration in which a control sequence is suitably positioned relative to a polynucleotide of interest (i.e., in a functional relationship) such that the control sequence directs or regulates expression of the polynucleotide and / or polypeptide of interest.

[0075] A "promoter sequence" refers to a nucleic acid sequence recognized by a host cell for expression of a polynucleotide of interest, such as a coding sequence. The promoter sequence contains transcriptional control sequences that mediate expression of the polynucleotide of interest. The promoter may be any nucleic acid sequence, including mutant, truncated, and hybrid promoters, that exhibits transcriptional activity in the host cell of choice and that can be derived from a gene encoding an extracellular or intracellular polypeptide that is either homologous or heterologous to the host cell.

[0076] The phrase "suitable reaction conditions" refers to conditions in an enzyme conversion reaction solution (e.g., enzyme loading range, substrate loading, temperature, pH, buffer, co-solvent, etc.) that allow an acetate kinase polypeptide of the invention to convert a substrate into a desired product compound. Some exemplary "suitable reaction conditions" are provided herein.

[0077] As used herein, "load," e.g., "compound load" or "enzyme load," refers to the concentration or amount of a component in a reaction mixture at the start of the reaction.

[0078] As used herein, "substrate" in the context of an enzymatic conversion reaction process refers to a compound or molecule that is acted upon by an engineered enzyme (e.g., an engineered acetate kinase polypeptide) provided herein.

[0079] As used herein, an "increase" in the yield of a product (e.g., a deoxyribose phosphate analog) from a reaction occurs when a particular component (e.g., an acetate kinase enzyme) present during the reaction produces more product compared to a reaction carried out under the same conditions with the same substrate and other substituents, but not in the absence of the component of interest.

[0080] A reaction is said to be "substantially free" of a particular enzyme if the amount of that enzyme relative to other enzymes involved in catalyzing the reaction is less than about 2%, less than about 1%, or less than about 0.1% (weight / weight).

[0081] As used herein, "fractionating" a liquid (e.g., a culture broth) means applying a separation process (e.g., salting out, column chromatography, size exclusion, and filtration), or a combination of such processes, to provide a solution in which the desired protein comprises a higher percentage of total protein in solution than in the original liquid product.

[0082] As used herein, "starting composition" refers to any composition that includes at least one substrate. In some embodiments, the starting composition includes any suitable substrate.

[0083] As used herein, "product" in the context of an enzymatic conversion process refers to a compound or molecule that results from the action of an enzyme polypeptide on a substrate.

[0084] As used herein, "equilibration," as used herein, refers to the process of resulting in steady-state concentrations of chemical species in a chemical or enzymatic reaction (e.g., the interconversion of two species A and B), including the interconversion of stereoisomers, as determined by the forward and reverse rate constants of the chemical or enzymatic reaction.

[0085] As used herein, "alkyl" refers to a saturated hydrocarbon group of 1 to 18 (inclusive) carbon atoms, more preferably 1 to 8 (inclusive), and most preferably 1 to 6 (inclusive) carbon atoms, either straight or branched. Alkyl groups having a designated number of carbon atoms are indicated in parentheses (e.g., (C1-C4) alkyl refers to alkyl of 1 to 4 carbon atoms).

[0086] As used herein, "alkenyl" refers to a group of 2 to 12 carbon atoms (inclusive), either straight or branched, containing at least one double bond, but optionally containing more than one double bond.

[0087] As used herein, "alkynyl" refers to a group of 2 to 12 carbon atoms (inclusive), either straight or branched, containing at least one triple bond, and optionally containing more than one triple bond, plus optionally one or more double bond moieties.

[0088] As used herein, "heteroalkyl," "heteroalkenyl," and "heteroalkynyl" refer to alkyl, alkenyl, and alkynyl, as defined herein, in which one or more carbon atoms are each independently replaced with the same or different heteroatom or heteroatom group. Heteroatoms and / or heteroatom groups with which carbon atoms can be replaced include, but are not limited to, -O-, -S-, -SO-, -NRα-, -PH-, -S(O)-, -S(O)2, -S(O)NRα-, -S(O)2NRα-, and the like, including combinations thereof, where each Rα is independently selected from hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl.

[0089] As used herein, "alkoxy" refers to the group -ORβ, where Rβ is an alkyl group as defined above, including optionally substituted alkyl groups also as defined herein.

[0090] As used herein, "aryl" refers to an unsaturated aromatic carbocyclic group of 6 to 12 carbon atoms (inclusive) having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl). Exemplary aryls include phenyl, pyridyl, naphthyl, and the like.

[0091] As used herein, "amino" refers to the -NH group. Substituted amino refers to the -NHR, NR, and NR groups, where each R is independently selected from substituted or unsubstituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy, aryl, heteroaryl, heteroarylalkyl, acyl, alkoxycarbonyl, sulfanyl, sulfinyl, sulfonyl, and the like. Exemplary amino groups include, but are not limited to, dimethylamino, diethylamino, trimethylammonium, triethylammonium, methylsulfonylamino, furanyl-oxy-sulfamino, and the like.

[0092] As used herein, "oxo" refers to =O.

[0093] As used herein, "oxy" refers to the divalent group --O--, which can have a variety of substituents to form different oxy groups, including ethers and esters.

[0094] As used herein, "carboxy" refers to --COOH.

[0095] As used herein, "carbonyl" refers to -C(O)-, which can have a variety of substituents to form different carbonyl groups including acids, acid halides, aldehydes, amides, esters, and ketones.

[0096] As used herein, "alkyloxycarbonyl" refers to --C(O)OR.epsilon., where R.epsilon. is an alkyl group, as defined herein, which can be optionally substituted.

[0097] As used herein, "aminocarbonyl" refers to -C(O)NH. Substituted aminocarbonyl refers to -C(O)NRδRδ, where the amino group NRδRδ is as defined herein.

[0098] As used herein, "halogen" and "halo" refer to fluoro, chloro, bromo, and iodo.

[0099] As used herein, "hydroxy" refers to --OH.

[0100] As used herein, "cyano" refers to --CN.

[0101] As used herein, "heteroaryl" refers to an aromatic heterocyclic group of 1 to 10 (inclusive) carbon atoms and 1 to 4 (inclusive) heteroatoms selected from oxygen, nitrogen, and sulfur within the ring. Such heteroaryl groups can have a single ring (e.g., pyridyl or furyl) or multiple condensed rings (e.g., indolizinyl or benzothienyl).

[0102] As used herein, "heteroarylalkyl" refers to an alkyl substituted by a heteroaryl (i.e., a heteroaryl-alkyl group) preferably having from 1 to 6 (inclusive) carbon atoms in the alkyl portion and from 5 to 12 (inclusive) ring atoms in the heteroaryl portion. Such heteroarylalkyl groups are exemplified by pyridylmethyl, and the like.

[0103] As used herein, "heteroarylalkenyl" refers to an alkenyl substituted by a heteroaryl (i.e., a heteroaryl-alkenyl group) preferably having from 2 to 6 (inclusive) carbon atoms in the alkenyl moiety and from 5 to 12 (inclusive) ring atoms in the heteroaryl moiety.

[0104] As used herein, "heteroarylalkynyl" refers to an alkynyl substituted by a heteroaryl (i.e., a heteroaryl-alkynyl group) preferably having from 2 to 6 (inclusive) carbon atoms inclusively in the alkynyl moiety and from 5 to 12 (inclusive) ring atoms inclusively in the heteroaryl moiety.

[0105] As used herein, "heterocycle," "heterocyclic," and interchangeably, "heterocycloalkyl" refer to saturated or unsaturated groups having a single ring or multiple condensed rings of 2 to 10 (inclusive) carbon ring atoms and 1 to 4 (inclusive) heteroatoms selected from nitrogen, sulfur, or oxygen within the ring. Such heterocyclic groups can have a single ring (e.g., piperidinyl or tetrahydrofuryl) or multiple condensed rings (e.g., indolinyl, dihydrobenzofuran, or quinuclidinyl). Examples of heterocycles include, but are not limited to, furan, thiophene, thiazole, oxazole, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, pyrrolidine, indoline, and the like.

[0106] As used herein, the term "membered ring" is intended to encompass any cyclic structure. The number preceding the term "membered" indicates the number of skeletal atoms that make up the ring. Thus, for example, cyclohexyl, pyridine, pyran, and thiopyran are six-membered rings, while cyclopentyl, pyrrole, furan, and thiophene are five-membered rings.

[0107] Unless otherwise specified, any hydrogen position in the aforementioned groups may be replaced by any of the following radicals: 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, amido and (heterocycle)oxy, and (heterocycle)alkyl; preferred heteroatoms are oxygen, nitrogen, and sulfur. It is understood that when open valences exist for these substituents, they can be further substituted with alkyl, cycloalkyl, aryl, heteroaryl, and / or heterocyclic groups, and when these open valences exist at carbon, they can be further substituted with halogen and oxygen-, nitrogen-, or sulfur-linked substituents, and when multiple such open valences exist, these groups can be joined to form a ring, either by direct bond formation or by forming a bond to a new heteroatom, preferably oxygen, nitrogen, or sulfur.It is further understood that the above substitutions can be made provided that replacing a hydrogen with a substituent does not introduce unacceptable instability into the molecules of the invention and is otherwise chemically reasonable.

[0108] As used herein, the term "culturing" refers to growing a population of microbial cells under any suitable conditions (e.g., using liquid, gel, or solid media).

[0109] Recombinant polypeptides can be produced using any suitable method known in the art. A gene encoding a wild-type polypeptide of interest can be cloned into a vector, such as a plasmid, and expressed in a desired host, such as E. coli. Variants of recombinant polypeptides can be generated by various methods known in the art. Indeed, there are a wide variety of different mutagenesis techniques well known to those skilled in the art. In addition, mutagenesis kits are also available from many commercial molecular biology suppliers. Methods are available for performing specific substitutions at defined amino acids (site-directed), specific or random mutations in local regions of a gene (region-directed), or random mutagenesis of the entire gene (e.g., saturation mutagenesis). Many suitable methods for generating enzyme variants are known to those skilled in the art, including, but not limited to, site-directed mutagenesis of single- or double-stranded DNA using PCR, cassette mutagenesis, gene synthesis, error-prone PCR, shuffling, and chemical saturation mutagenesis, or any other suitable method known in the art. Mutagenesis and directed evolution methods can be readily applied to enzyme-encoding polynucleotides to generate libraries of variants, which can be expressed, screened, and assayed. Any suitable mutagenesis and directed evolution method is useful in the present invention and is well known in the art (e.g., U.S. 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, 6,166,794, 6,167,795, 6,168,796, 6,169,797, 6,170,798, 6,171,799, 6,172,799, 6,173,799, 6,174,799, 6,175,799, 6,176,799, 6,177,799, 6,178,799, 6,179 ... , 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,No. 344,356, same as No. 6,352,859, same as No. 6,355,484, same as No. 6,358,740, same as No. 6,358,742, same as No. 6,365,377, same as No. 6,365,408, same as No. 6,368,861, same as No. 6,372,497, same as No. 6,337,186 , same as No. 6,376,246, same as No. 6,379,964, same as No. 6,387,702, same as No. 6,391,552, same as No. 6,391,640, same as No. 6,395,547, same as No. 6,406,855, same as No. 6,406,910, same as No. 6,413,745, same as No. 6,413 ,774, same as No. 6,420,175, same as No. 6,423,542, same as No. 6,426,224, same as No. 6,436,675, same as No. 6,444,468, same as No. 6,455,253, same as No. 6,479,652, same as No. 6,482,647, same as No. 6,483,011, same as No. No. 6,484,105, same as No. 6,489,146, same as No. 6,500,617, same as No. 6,500,639, same as No. 6,506,602, same as No. 6,506,603, same as No. 6,518,065, same as No. 6,519,065, same as No. 6,521,453, same as No. 6,528,31 No. 1, same as No. 6,537,746, same as No. 6,573,098, same as No. 6,576,467, same as No. 6,579,678, same as No. 6,586,182, same as No. 6,602,986, same as No. 6,605,430, same as No. 6,613,514, same as No. 6,653,072, same as No. 6,6 No. 86,515, same as No. 6,703,240, same as No. 6,716,631, same as No. 6,825,001, same as No. 6,902,922, same as No. 6,917,882, same as No. 6,946,296, same as No. 6,961,664, same as No. 6,995,017, same as No. 7,024,312, Same as No. 7,058,515, Same as No. 7,105,297, Same as No. 7,148,054, Same as No. 7,220,566, Same as No. 7,288,375, Same as No. 7,384,387, Same as No. 7,421,347, Same as No. 7,430,477, Same as No. 7,462,469, Same as No. 7,534, No. 564, same as No. 7,620,500, same as No. 7,620,502, same as No. 7,629,170, same as No. 7,702,464, same as No. 7,747,391, same as No. 7,747,393, same as No. 7,751,986, same as No. 7,776,598, same as No. 7,783,428, same as No. 7,No. 795,030, same as No. 7,853,410, same as No. 7,868,138, same as No. 7,783,428, same as No. 7,873,477, same as No. 7,873,499, same as No. 7,904,249, same as No. 7,957,912, same as No. 7,981,614, same as No. 8,014,961, same as No. 8,029,988, same as No. 8,048,674, same as No. 8,05 No. 8,001, same as No. 8,076,138, same as No. 8,108,150, same as No. 8,170,806, same as No. 8,224,580, same as No. 8,377,681, same as No. 8,383,346, same as No. 8,457,903, same as No. 8,504,498, same as No. 8,589,085, same as No. 8,762,066, same as No. 8,768,871, same as No. 9,593,326, and all relevant U.S., and PCT and non-U.S. counterparts; Ling et al., Anal. Biochem., 254(2):157-78

[1997] ; Dale et al., Meth. Mol. Biol., 57:369-74

[1996] ; Smith, Ann. Rev. Genet., 19:423-462

[1985] ; Botstein et al., Science, 229:1193-1201

[1985] ; Carter, Biochem. J., 237:1-7

[1986] ; Kramer et al., Cell, 38:879-887

[1984] ; Wells et al., Gene, 34:315-323

[1985] ; Minshull et al., Curr. Op. Chem. Biol., 3:284-290

[1999] ; Christians et al., Nat. Biotechnol., 17:259-264

[1999] ; Crameri et al., Nature, 391:288-291

[1998] ; Crameri, et al., Nat. Biotechnol., 15:436-438

[1997] ; Zhang et al., Proc. Nat. Acad. Sci. USA, 94:4504-4509

[1997] ; Crameri et al., Nat. Biotechnol., 14:315-319

[1996] ; Stemmer, Nature, 370:389-391

[1994] ; Stemmer, Proc. Nat. Acad. Sci. USA, 91:10747-10751

[1994] ; WO95 / 22625; WO97 / 0078; WO97 / 35966; WO98 / 27230; WO00 / 42651; WO01 / 75767; and WO2009 / 152336).

[0110] In some embodiments, the enzyme clones obtained after mutagenesis treatment are screened by subjecting the enzyme preparation to a defined temperature (or other assay conditions) and measuring the amount of enzyme activity remaining after heat treatment or other suitable assay conditions. Clones containing polynucleotides encoding polypeptides are then isolated from the gene, sequenced to identify nucleotide sequence changes (if any), and used to express the enzyme in host cells. Measuring enzyme activity from an expression library can be performed using any suitable method known in the art (e.g., standard biochemical techniques, such as HPLC analysis).

[0111] After the variants are produced, they can be screened for any desired properties (e.g., high or increased activity, or low or reduced activity, increased thermal activity, increased thermostability, and / or acidic pH stability, etc.). In some embodiments, "recombinant acetate kinase polypeptides" (also referred to herein as "engineered acetate kinase polypeptides," "variant acetate kinase enzymes," "acetate kinase variants," and "acetate kinase combinatorial variants") are useful. In some embodiments, "recombinant acetate kinase polypeptides" (also referred to herein as "engineered acetate kinase polypeptides," "variant acetate kinase enzymes," "acetate kinase variants," and "acetate kinase combinatorial variants") are useful.

[0112] As used herein, a "vector" is a DNA construct for introducing a DNA sequence into a cell. In some embodiments, the vector is an expression vector operably linked to a suitable control sequence capable of causing expression of a polypeptide encoded by the DNA sequence in a suitable host. In some embodiments, an "expression vector" has a promoter sequence operably linked to a DNA sequence (e.g., a transgene) to drive expression in a host cell, and in some embodiments, also includes a transcription terminator sequence.

[0113] As used herein, the term "expression" includes any step involved in producing a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from the cell.

[0114] As used herein, the term "produce" refers to the production of a protein and / or other compound by a cell. The term encompasses any step involved in producing a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses the secretion of a polypeptide from the cell.

[0115] As used herein, an amino acid or nucleotide sequence (e.g., a promoter sequence, signal peptide, terminator sequence, etc.) is "heterologous" to another sequence to which it is operably linked if the two sequences are not associated in nature. For example, a "heterologous polynucleotide" is any polypeptide introduced into a host cell by laboratory techniques, including polynucleotides that have been removed from the host cell, subjected to laboratory manipulation, and then reintroduced into the host cell.

[0116] As used herein, the terms "host cell" and "host strain" refer to a suitable host for an expression vector containing the DNA provided herein (e.g., a polynucleotide encoding an acetate kinase variant). In some embodiments, the host cell is a prokaryotic or eukaryotic cell that has been transformed or transfected with a vector constructed using recombinant DNA techniques known in the art.

[0117] The term "analog" refers to a polypeptide having greater than 70% sequence identity but less than 100% sequence identity (e.g., greater than 75%, greater than 78%, greater than 80%, greater than 83%, greater than 85%, greater than 88%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99% sequence identity) to a reference polypeptide. In some embodiments, an analog refers to a polypeptide containing one or more non-naturally occurring amino acid residues and naturally occurring amino acids, including, but not limited to, homoarginine, ornithine, and norvaline. In some embodiments, an analog also includes one or more D-amino acid residues and non-peptide linkages between two or more amino acid residues.

[0118] The term "effective amount" means an amount sufficient to produce a desired result. One of ordinary skill in the art can determine what an effective amount is by using routine experimentation.

[0119] The terms "isolated" and "purified" are used to refer to a molecule (e.g., an isolated nucleic acid, polypeptide, etc.) or other component that has been removed from at least one other component with which it is naturally associated. The term "purified" does not require absolute purity, but rather is intended as a relative definition.

[0120] As used herein, "stereoselectivity" refers to the preferential formation of one stereoisomer over another in a chemical or enzymatic reaction. Stereoselectivity can be partial, where the formation of one stereoisomer is favored over the other, or complete, where only one stereoisomer is formed. When the stereoisomers are enantiomers, the stereoselectivity is referred to as enantioselectivity, which is the proportion (typically reported as a percentage) of one enantiomer over the sum of the two. This is commonly alternatively reported in the art (typically as a percentage) as enantiomeric excess ("ee"), calculated according to the formula [major enantiomer - minor enantiomer] / [major enantiomer + minor enantiomer]. When the stereoisomers are diastereoisomers, the stereoselectivity is called diastereoselectivity, which is the proportion (typically reported as a percentage) of one diastereomer in a mixture of two diastereomers, and is commonly reported alternatively as diastereomeric excess ("de"). Enantiomeric excess and diastereomeric excess are types of stereomeric excess.

[0121] As used herein, "regioselectivity," and "regioselective reaction" refer to a reaction in which one direction of making or breaking a bond occurs preferentially over all other possible directions. A reaction can be completely (100%) regioselective, where discrimination is complete; substantially regioselective (at least 75%), where the reaction product at one site is greater than the reaction product at the other site; or partially regioselective (x%, where the percentage is set depending on the reaction of interest).

[0122] As used herein, "chemoselectivity" refers to the preferential formation of one product over another in a chemical or enzymatic reaction.

[0123] As used herein, "pH-stable" refers to an acetate kinase polypeptide that maintains similar activity (e.g., greater than 60% to 80%) compared to the untreated enzyme after exposure to high or low pH (e.g., 4.5-6 or 8-12) for a period of time (e.g., 0.5-24 hours).

[0124] As used herein, "thermostable" refers to an acetate kinase polypeptide that, after exposure to elevated temperatures (e.g., 40-80°C) for a period of time (e.g., 0.5-24 hours), maintains similar activity (e.g., greater than 60% to 80%) compared to the wild-type enzyme exposed to the same elevated temperature.

[0125] As used herein, "solvent-stable" refers to an acetate kinase polypeptide that, after exposure to various concentrations (e.g., 5-99%) of a solvent (e.g., ethanol, isopropyl alcohol, dimethyl sulfoxide [DMSO], tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene, butyl acetate, methyl tert-butyl ether, etc.) for a period of time (e.g., 0.5-24 hours), maintains similar activity (e.g., greater than 60% to 80%) compared to the wild-type enzyme exposed to the same solvent at the same concentration.

[0126] As used herein, "heat and solvent stable" refers to an acetate kinase polypeptide that is both heat stable and solvent stable.

[0127] As used herein, "optional" and "optionally" mean that the subsequently described event or circumstance may or may not occur, and that the description includes cases where the event or circumstance occurs and cases where it does not. Those of skill in the art will understand that with respect to any molecule described as containing one or more optional substituents, only sterically realistic and / or synthetically feasible compounds are meant to be included.

[0128] As used herein, "optionally substituted" refers to all subsequent modifiers in a term or series of chemical groups. For example, in the term "optionally substituted arylalkyl," the "alkyl" and "aryl" portions of the molecule can be substituted or unsubstituted, and in the series "optionally substituted alkyl, cycloalkyl, aryl, and heteroaryl," the alkyl, cycloalkyl, aryl, and heteroaryl groups can be substituted or unsubstituted independently of each other.

[0129] Detailed Description of the Invention The present invention provides engineered acetate kinase (AcK) enzymes, polypeptides having AcK activity, and polynucleotides encoding these enzymes, as well as vectors and host cells containing these polynucleotides and polypeptides. Methods for producing AcK enzymes are also provided. The present invention further provides compositions containing AcK enzymes and methods for using engineered AcK enzymes. The present invention is particularly useful in the production of pharmaceutical compounds.

[0130] In some embodiments, the present invention provides enzymes suitable for recycling ATP from ADP to produce phosphorylated glycerol and glyceraldehyde derivatives bearing bulky substituents at the C2 carbon of glycerol, particularly phosphorylated ethynyl-glycerol and ethynyl-glyceraldehyde, which are intermediates for the in vitro enzymatic synthesis of the depicted unnatural nucleoside analog of compound (1). [ka]

[0131] Producing phosphorylated glyceraldehyde derivatives, such as compound 5, can be difficult. However, the corresponding non-phosphorylated glyceraldehyde derivative 6 can be generated by oxidizing the glycerol derivative 7 with alcohol oxidase. Once formed, glycerol aldehyde can be phosphorylated to the desired intermediate 5 by pantothenate kinase (PanK), as shown in Scheme I. [ka]

[0132] The production of the phosphorylated intermediate (5) utilizes ATP as the phosphate donor. The resulting ADP is then recycled to ATP by AcK using acetyl phosphate, as depicted in Scheme II. The improved AcK enzymes of the present invention have improved activity in recycling ATP from ADP using acetyl phosphate. [ka]

[0133] In some embodiments, an engineered AcK polypeptide of the present disclosure is part of a multi-enzyme system for producing a compound, such as a nucleoside analog of compound (1). In some embodiments, the engineered AcK polypeptide is part of a multi-enzyme system that includes one or more of the following enzymes: pantothenate kinase, phosphopentomutase, purine nucleoside phosphorylase, alcohol oxidase, aldolase, and / or sucrose phosphorylase.

[0134] Engineered AcK polypeptide The present invention provides engineered AcK polypeptides, polynucleotides encoding the polypeptides, methods for preparing the polypeptides, and methods for using the polypeptides. Where a description refers to a polypeptide, it should be understood that this also describes the polynucleotide encoding the polypeptide. In some embodiments, the present invention provides engineered, non-naturally occurring AcK enzymes with improved properties compared to wild-type AcK enzymes. Any suitable reaction conditions are useful in the present invention. In some embodiments, methods are used to analyze the improved properties of engineered polypeptides for performing phosphorylation reactions. In some embodiments, the reaction conditions are modified with respect to conditions including the concentration or amount of engineered AcK, substrate, buffer, solvent, cofactor, pH, temperature, and reaction time, and / or conditions for immobilizing the engineered AcK polypeptide to a solid support, as described in detail below and in the Examples.

[0135] In some embodiments, additional reaction components or techniques are utilized to supplement the reaction conditions, including taking measures to stabilize or prevent inactivation of enzymes, reduce product inhibition, or shift the equilibrium of the reaction toward the formation of the desired product.

[0136] In some further embodiments, any of the above processes for converting substrate compounds to product compounds may further include one or more steps selected from extraction, isolation, purification, crystallization, filtration, and / or lyophilization of the product compound. Methods, techniques, and protocols for extracting, isolating, purifying, and / or crystallizing products from biocatalytic reaction mixtures produced by the processes provided herein are known to those skilled in the art and / or obtainable through routine experimentation. Additionally, illustrative methods are provided in the following examples.

[0137] Engineered AcK polynucleotides encoding engineered polypeptides, expression vectors, and host cells The present invention provides polynucleotides encoding the engineered enzyme polypeptides described herein. In some embodiments, the polynucleotides are operably linked to one or more heterologous regulatory sequences that control gene expression to create recombinant polynucleotides capable of expressing the polypeptides. In some embodiments, an expression construct containing at least one heterologous polynucleotide encoding an engineered enzyme polypeptide is introduced into a suitable host cell to express the corresponding enzyme polypeptide.

[0138] As will be apparent to those skilled in the art, the availability of protein sequences and knowledge of the codons corresponding to various amino acids provides a description of all polynucleotides capable of encoding the polypeptides of the invention. The degeneracy of the genetic code, in which the same amino acid is coded for by alternative or synonymous codons, allows for the creation of an extremely large number of nucleic acids, all of which encode engineered enzyme (e.g., AcK) polypeptides. Thus, the present invention provides methods and compositions for producing any and all possible variations of enzyme polynucleotides that can be made by selecting combinations based on potential codon choices and that encode the enzyme polypeptides described herein; all such variations should be considered specifically disclosed with respect to any polypeptide described herein, including the amino acid sequences presented in the Examples (e.g., various Tables).

[0139] In some embodiments, codons are preferably optimized for utilization by the host cell chosen for protein production. For example, preferred codons used in bacteria are typically used for expression in bacteria. Thus, a codon-optimized polynucleotide encoding an engineered enzyme polypeptide contains preferred codons at about 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% of the codon positions in the full-length coding region.

[0140] In some embodiments, the enzyme polynucleotide encodes an engineered polypeptide having an enzymatic activity with the properties disclosed herein, wherein the polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a reference sequence selected from the SEQ ID NOs provided herein, or the amino acid sequence of any variant (e.g., a variant provided in the Examples), and one or more residue differences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid residue positions) compared to the amino acid sequence of the reference polynucleotide or any variant disclosed in the Examples. In some embodiments, the reference polypeptide sequence comprises SEQ ID NO:2, while in some other embodiments, the reference polypeptide sequence comprises SEQ ID NO:12, and in yet some other embodiments, the reference polypeptide sequence comprises SEQ ID NO:600.

[0141] In some embodiments, a polynucleotide is capable of hybridizing under high stringency conditions to a reference polynucleotide sequence selected from any of the polynucleotide sequences provided herein or their complementary strands, or a polynucleotide sequence encoding any of the variant enzyme polypeptides provided herein. In some embodiments, a polynucleotide capable of hybridizing under high stringency conditions encodes an enzyme polypeptide comprising an amino acid sequence that differs by one or more residues compared to the reference sequence.

[0142] In some embodiments, an isolated polynucleotide encoding any of the engineered enzyme polypeptides herein is engineered in various ways to facilitate expression of the enzyme polypeptide. In some embodiments, the polynucleotide encoding the enzyme polypeptide comprises an expression vector in which one or more regulatory sequences are present to regulate expression of the enzyme polynucleotide and / or polypeptide. Manipulation of the isolated polynucleotide prior to insertion into the vector may be desirable or necessary depending on the expression vector used. Techniques for modifying polynucleotides and nucleic acid sequences using recombinant DNA methods are well known in the art. In some embodiments, regulatory sequences include promoters, leader sequences, polyadenylation sequences, propeptide sequences, signal peptide sequences, and transcription terminators, among others. In some embodiments, a suitable promoter is 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 those 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 genes (e.g., Villa-Kamaroff et al., Proc. Natl. Acad. Sci. USA 75: 3727-3731). Examples of promoters that can be used include, but are not limited to, promoters derived from the tac promoter (see, e.g., DeBoer et al., Proc. Natl. Acad. Sci. USA 80: 21-25

[1983] ), as well as promoters derived from the tac promoter (see, e.g., DeBoer et al., Proc. Natl. Acad. Sci. USA 80: 21-25

[1983] ).Exemplary promoters for filamentous fungal host cells include promoters obtained from the genes for Aspergillus 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 triosephosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (see, e.g., WO 96 / 00787), as well as the NA2-tpi promoter (Aspergillus niger neutral alpha-amylase and Aspergillus Examples of promoters that can be used include, but are not limited to, promoters from the Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase genes. Other useful promoters for yeast host cells are known in the art (see, e.g., Romanos et al., Yeast 8:423-488

[1992] ).

[0143] In some embodiments, the control sequence is also 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' end of the nucleic acid sequence encoding the enzyme polypeptide. Any suitable terminator that is functional in the host cell of choice is useful in the present invention. 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., supra).

[0144] In some embodiments, the control sequence is also a suitable leader sequence (i.e., a nontranslated region of an mRNA important for translation by the host cell). In some embodiments, the leader sequence is operably linked to the 5' end of the nucleic acid sequence encoding the enzyme polypeptide. Any suitable leader sequence that is functional in the host cell of choice is useful in the present invention. Exemplary leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triosephosphate 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 alpha factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0145] In some embodiments, the control sequence is also a polyadenylation sequence (i.e., a sequence operably linked to the 3' end of a nucleic acid sequence that, upon transcription, is recognized by a host cell as a signal for the addition of polyadenosine residues to the transcribed mRNA). Any suitable polyadenylation sequence that is functional in the host cell of choice is useful in the present invention. Exemplary polyadenylation 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 polyadenylation sequences for yeast host cells are known (see, e.g., Guo and Sherman, Mol. Cell. Biol., 15:5983-5990

[1995] ).

[0146] In some embodiments, the control sequence is also a signal peptide (i.e., a coding region encoding an amino acid sequence linked to the amino terminus of a polypeptide which directs the encoded polypeptide into the secretory pathway of a cell). In some embodiments, the 5' end of the coding sequence of the nucleic acid sequence naturally contains a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region which 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 is useful for expressing an engineered polypeptide. Useful signal peptide coding regions for bacterial host cells include, but are not limited to, those obtained from the genes encoding Bacillus NC1B 11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM), and Bacillus subtilis prsA. Additional signal peptides are known in the art (see, e.g., Simonen and Palva, Microbiol. Rev., 57:109-137

[1993] ). In some embodiments, effective signal peptide coding regions for filamentous fungal host cells include, but are not limited to, signal peptide coding regions 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, the signal peptides from the genes for Saccharomyces cerevisiae alpha factor and Saccharomyces cerevisiae invertase.

[0147] In some embodiments, the control sequence is also a propeptide-coding region that encodes an amino acid sequence located at the amino terminus of the polypeptide. The resulting polypeptide is referred to as a "proenzyme," "propolypeptide," or "zymogen." 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 can be obtained from any suitable source, including, but 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). When both a signal peptide and a propeptide region are present at the amino terminus of a polypeptide, the propeptide region is located next to the amino terminus of the polypeptide, and the signal peptide region is located next to the amino terminus of the propeptide region.

[0148] In some embodiments, regulatory sequences are also utilized. These sequences facilitate the regulation of polypeptide expression relative to the growth of the host cell. Examples of regulatory systems are systems that turn gene expression on or off in response to chemical or physical stimuli, including the presence of regulatory compounds. 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 the GAL1 system. In filamentous fungi, suitable regulatory sequences include, but are not limited to, the TAKA alpha-amylase promoter, the Aspergillus niger glucoamylase promoter, and the Aspergillus oryzae glucoamylase promoter.

[0149] In another aspect, the present invention is directed to recombinant expression vectors comprising a polynucleotide encoding an engineered enzyme polypeptide and one or more expression control regions, such as a promoter and terminator, an origin of replication, etc., depending on the type of host into which they will be introduced. In some embodiments, the various nucleic acids and control sequences described herein are joined together to produce a recombinant expression vector that contains one or more convenient restriction sites to allow for insertion or substitution of a nucleic acid sequence encoding an enzyme polypeptide at such site. Alternatively, in some embodiments, the nucleic acid sequences of the present invention are expressed by inserting the nucleic acid sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression. In some embodiments involving the creation of an expression vector, the coding sequence is placed in the vector such that the coding sequence is operably linked to appropriate control sequences for expression.

[0150] The recombinant expression vector can be any suitable vector (e.g., a plasmid or a virus) that can be easily subjected to recombinant DNA procedures and can cause the expression of the enzyme polynucleotide sequence. The selection of the vector typically depends on the compatibility of the vector with the host cell into which the vector will be introduced. The vector can be a linear or closed circular plasmid.

[0151] In some embodiments, the expression vector is an autonomously replicating vector (i.e., a vector that exists as an extrachromosomal entity whose replication is independent of chromosomal replication, such as a plasmid, extrachromosomal element, minichromosome, or artificial chromosome). The vector may contain any means for ensuring self-replication. In some alternative embodiments, the vector is one that, when introduced into a host cell, integrates into the genome and replicates together with the chromosome into which it is integrated. Furthermore, in some embodiments, a single vector or plasmid, or two or more vectors or plasmids and / or transposons that together contain the total DNA to be introduced into the genome of the host cell are utilized.

[0152] In some embodiments, the expression vector contains one or more selectable markers that allow for easy selection of transformed cells. A "selectable marker" is a gene whose product provides biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, etc. Examples of bacterial selectable markers include, but are not limited to, the dal genes of Bacillus subtilis or Bacillus licheniformis, or markers that confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol, or tetracycline resistance. Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamidase; e.g., from A. nidulans or A. orzyae), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase; e.g., from S. hygroscopicus), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase; e.g., from A. nidulans or A. orzyae), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), and equivalents thereof.

[0153] In another aspect, the present invention provides a host cell comprising at least one polynucleotide encoding at least one engineered enzyme polypeptide of the present invention, wherein the polynucleotide is operably linked to one or more control sequences for expression of the engineered enzyme in the host cell. Suitable host cells for use in expressing polypeptides encoded by the expression vectors of the present invention are well known in the art and include, but are not limited to, bacterial cells, such as E. coli, 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(ΔfhuA) and BL21). Examples of bacterial selectable markers include, but are not limited to, the dal genes of Bacillus subtilis or Bacillus licheniformis, or markers that confer antibiotic resistance, such as ampicillin, kanamycin, chloramphenicol, and / or tetracycline resistance.

[0154] In some embodiments, the expression vectors of the invention contain elements that allow for integration of the vector into the host cell genome or autonomous replication of the vector in the cell independent of the genome. In some embodiments involving integration into the host cell genome, the vector relies on the nucleic acid sequence encoding the polypeptide, or any other element of the vector, to integrate the vector into the genome by homologous or non-homologous recombination.

[0155] In some alternative embodiments, the expression vector contains an additional nucleic acid sequence that directs integration into the host cell genome by homologous recombination. The additional nucleic acid sequence allows the vector to integrate into the host cell genome at a precise location in the chromosome. To increase the likelihood of integration at a precise location, the integration element preferably contains a sufficient number of nucleotides, for example, 100 to 10,000 base pairs, preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000 base pairs, that are highly homologous to the corresponding target sequence to enhance the probability of homologous recombination. The integration element can be any sequence that is homologous to the target sequence in the host cell genome. Furthermore, the integration element can be a non-coding or coding nucleic acid sequence. Alternatively, the vector can be integrated into the host cell genome by non-homologous recombination.

[0156] In the case of autonomous replication, the vector may further comprise an origin of replication that enables the vector to replicate autonomously in the host cell. Examples of bacterial origins of replication include the P15A ori, or the origins of replication of plasmids pBR322, pUC19, pACYC177 (which have a P15A ori), or pACYC184, which enable replication in E. coli, and pUB110, pE194, or pTA1060, which enable replication in Bacillus. Examples of origins of replication for use in yeast host cells are the 2 micron origin of replication, ARS1, ARS4, a combination of ARS1 and CEN3, and a combination of ARS4 and CEN6. The origin of replication may be one that has a mutation that makes its function temperature-sensitive in the host cell (see, e.g., Ehrlich, Proc. Natl. Acad. Sci. USA 75:1433

[1978] ).

[0157] In some embodiments, more than one copy of the nucleic acid sequence of the present invention is inserted into a host cell to increase production of the gene product. The increased copy number of the nucleic acid sequence can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene together with the nucleic acid sequence, and cells containing the amplified copy of the selectable marker gene and thereby the additional copy of the nucleic acid sequence can be selected by culturing the cells in the presence of an appropriate selectable agent.

[0158] Many expression vectors for use in the present invention are commercially available. Suitable commercially available expression vectors include, but are not limited to, the p3xFLAG™ expression vector (Sigma-Aldrich Chemicals), which contains a CMV promoter and hGH polyadenylation site for expression in mammalian host cells, and a pBR322 origin of replication and an ampicillin resistance marker for amplification in E. coli. Other suitable expression vectors include, but are not limited to, pBluescriptII SK(-) and pBK-CMV (Stratagene), as well as plasmids derived from pBR322 (Gibco BRL), pUC (Gibco BRL), pREP4, pCEP4 (Invitrogen), or pPoly (see, e.g., Lathe et al., Gene 57:193-201

[1987] ).

[0159] Thus, in some embodiments, a vector containing a sequence encoding at least one variant acetate kinase is transformed into a host cell to allow propagation of the vector and expression of the variant acetate kinase. In some embodiments, the variant acetate kinase may be post-translationally modified to remove the signal peptide, and in some instances, cleaved after secretion. In some embodiments, the transformed host cell is cultured in a suitable nutrient medium under conditions that allow expression of the variant acetate kinase. Any suitable medium useful for culturing host cells is useful in the present invention, including, but not limited to, minimal or complex media containing appropriate supplements. In some embodiments, the host cells are grown in HTP medium. Suitable media are available from various commercial sources or may be prepared according to published recipes (e.g., catalogs of the American Type Culture Collection).

[0160] In another aspect, the present invention provides a host cell comprising a polynucleotide encoding an improved acetate kinase polypeptide provided herein, the polynucleotide being operably linked to one or more regulatory sequences for expressing the acetate kinase enzyme in the host cell. Host cells for use in expressing the acetate kinase polypeptide encoded by the expression vector of the present invention are well known in the art and include, but are not limited to, bacterial cells such as E. coli, Bacillus megaterium, Lactobacillus kefir, 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. Appropriate culture media and growth conditions for the above host cells are well known in the art.

[0161] Polynucleotides for expressing acetate kinase can be introduced into cells by various methods known in the art. Techniques include electroporation, biolistic particle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion, among others. Various methods for introducing polynucleotides into cells are known to those skilled in the art.

[0162] In some embodiments, the host cell is a eukaryotic cell. Suitable eukaryotic host cells include, but are not limited to, fungal cells, algae cells, insect cells, and plant cells. Suitable fungal host cells include, but are not limited to, Ascomycota, Basidiomycota, Deuteromycota, Zygomycota, and Fungi Deuteromycota. In some embodiments, the fungal host cell is a yeast cell or a filamentous fungal cell. Filamentous fungal host cells of the present invention include all filamentous fungal forms of the subdivisions Eumycotina and Oomycota. Filamentous fungi are characterized by vegetative mycelium with cell walls composed of chitin, cellulose, and other complex polysaccharides. The filamentous fungal host cells of the present invention are morphologically distinct from yeast.

[0163] In some embodiments of the invention, the filamentous fungal host cell is selected from the group consisting of Achlya, Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Cephalosporium, Chrysosporium, Cochliobolus, Corynascus, Cryphonectria, Cryptococcus, Coprinus, Coriolus, Diplodia, Endothis, Fusarium, Gibberella, Gliocladium, Humicola, Hypocrea, Myceliophthora, Mucor, Neurospora, Penicilli The present invention is directed to cells of any suitable genus and species, including, but not limited to, um, Podospora, Phlebia, Piromyces, Pyricularia, Rhizomucor, Rhizopus, Schizophyllum, Scytalidium, Sporotrichum, Talaromyces, Thermoascus, Thielavia, Trametes, Tolypocladium, Trichoderma, Verticillium, and / or Volvariella, and / or sexual or asexual forms thereof, and synonyms, basionyms, or taxonomic equivalents thereof.

[0164] In some embodiments of the invention, the host cell is a yeast cell, including, but not limited to, a cell of a Candida, Hansenula, Saccharomyces, Schizosaccharomyces, Pichia, Kluyveromyces, or Yarrowia species. In some embodiments of the invention, the yeast cells are Hansenula polymorpha, Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Saccharomyces diastaticus, Saccharomyces norbensis, Saccharomyces kluyveri, Schizosaccharomyces pombe, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia kodamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia quercuum, Pichia pijperi, Pichia stipitis, Pichia methanolica, Pichia angusta, Kluyveromyces lactis, Candida albicans, or Yarrowia lipolytica.

[0165] In some embodiments of the invention, the host cells are algal cells, such as Chlamydomonas (e.g., C. reinhardtii) and Phormidium (P. sp. ATCC29409).

[0166] In some other embodiments, the host cell is a prokaryotic cell. Suitable prokaryotic cells include, but are not limited to, gram-positive, gram-negative, and gram-variant bacterial cells.Any suitable bacterial organism is useful in the present invention, including but not limited to Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Acinetobacter, Acidothermus, Arthrobacter, Azobacter, Bacillus, Bifidobacterium, Brevibacterium, Butyrivibrio, Buchnera, Campestris, Camplyobacter, Clostridium, Corynebacterium, Chromatium, Coprococcus, Escherichia, Enterococcus, Enterobacter, Erwinia, Fusobacterium, Faecalibacterium, Francisella, Flavobacterium, Geobacillus, Haemophilus, Helicobacter, Klebsiella, Lactobacillus, Lactococcus, Ilyobacter, Micrococcus, Microbacterium, Mesorhizobium, Methylobacterium, Methylobacterium, Mycobacterium, Neisseria, Pantoea, Pseudomonas, Prochlorococcus, Rhodobacter, Rhodopseudomonas, Rhodopseudomonas, Roseburia, Rhodospirillum, Rhodococcus, Scenedesmus, Streptomyces, Streptococcus, Synecoccus, Saccharomonospora, Staphylococcus, Serratia, Salmonella, Shigella, Thermoanaerobacterium, Tropheryma, Tularensis, Temecula, Thermosynechococcus, Thermococcus, Ureaplasma, Xanthomonas, Xylella, Yersinia, and Zymomonas.In some embodiments, the host cell is a species of Agrobacterium, Acinetobacter, Azobacter, Bacillus, Bifidobacterium, Buchnera, Geobacillus, Campylobacter, Clostridium, Corynebacterium, Escherichia, Enterococcus, Erwinia, Flavobacterium, Lactobacillus, Lactococcus, Pantoea, Pseudomonas, Staphylococcus, Salmonella, Streptococcus, Streptomyces, or Zymomonas. In some embodiments, the bacterial host strain is non-pathogenic to humans. In some embodiments, the bacterial host strain is an industrial strain. Many industrial bacterial strains are known and suitable for the present invention. In some embodiments of the present invention, the bacterial host strain is an Agrobacterium species (e.g., A. radiobacter, A. rhizogenes, and A. rubi). In some embodiments of the invention, the bacterial host cell is an Arthrobacter species (e.g., A. aurescens, A. citreus, A. globiformis, A. hydrocarboglutamicus, A. mysorens, A. nicotianae, A. paraffineus, A. protophonniae, A. roseoparaffinus, A. sulfureus, and A. ureafaciens). In some embodiments of the invention, the bacterial host cell is a Bacillus species (e.g., B. thuringensis, B. anthracis, B. megaterium, B. subtilis, B. lentus, B. circulans, B. pumilus, B. lautus, B. coagulans, B. brevis, B. firmus, B. alkaophius, B. licheniformis, B. clausii, B. stearothermophilus, B. halodurans, and B. amyloliquefaciens).In some embodiments, the host cell is an industrial Bacillus strain, including, but not limited to, B. subtilis, B. pumilus, B. licheniformis, B. megaterium, B. clausii, B. stearothermophilus, or B. amyloliquefaciens. In some embodiments, the Bacillus host cell is B. subtilis, B. licheniformis, B. megaterium, B. stearothermophilus, and / or B. amyloliquefaciens. In some embodiments, the bacterial host cell is a Clostridium species (e.g., C. acetobutylicum, C. tetani E88, C. lituseburense, C. saccharobutylicum, C. perfringens, and C. beijerinckii). In some embodiments, the bacterial host cell is a Corynebacterium species (e.g., C. glutamicum and C. acetoacidophilum). In some embodiments, the bacterial host cell is an Escherichia species (e.g., E. coli). In some embodiments, the host cell is Escherichia coli W3110. In some embodiments, the bacterial host cell is an Erwinia species (e.g., E. uredovora, E. carotovora, E. ananas, E. herbicola, E. punctata, and E. terreus). In some embodiments, the bacterial host cell is a Pantoea species (e.g., P. citrea and P. agglomerans). In some embodiments, the bacterial host cell is a Pseudomonas species (e.g., P. putida, P. aeruginosa, P. mevalonii, and P. sp. D-01 10). In some embodiments, the bacterial host cell is a Streptococcus species (e.g., S. equisimiles, S. pyogenes, and S. uberis).In some embodiments, the bacterial host cell is a Streptomyces species (e.g., S. ambofaciens, S. achromogenes, S. avermitilis, S. coelicolor, S. aureofaciens, S. aureus, S. fungidicus, S. griseus, and S. lividans). In some embodiments, the bacterial host cell is a Zymomonas species (e.g., Z. mobilis and Z. lipolytica).

[0167] Many prokaryotic and eukaryotic strains useful in the present invention are readily publicly available from several culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and the Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

[0168] In some embodiments, host cells are genetically modified to possess characteristics that improve protein secretion, protein stability, and / or other properties desirable for protein expression and / or secretion. Genetic modifications can be achieved through genetic engineering techniques and / or classical microbiology techniques (e.g., chemical or UV mutagenesis followed by selection). Indeed, in some embodiments, host cells are produced using a combination of recombinant modification and classical selection techniques. Recombinant techniques can be used to introduce, delete, inhibit, or modify nucleic acid molecules to increase the yield of acetate kinase variants in host cells and / or culture media. For example, knocking out Alp1 function results in cells that are protease-deficient, and knocking out pyr5 function results in cells with a pyrimidine-deficient phenotype. One genetic engineering approach uses homologous recombination to specifically target genes in vivo, thereby inducing targeted gene modifications and suppressing expression of the encoded protein. In an alternative approach, siRNA, antisense, and / or ribozyme technologies are useful for inhibiting gene expression. A variety of methods are known in the art for reducing protein expression in cells, including, but not limited to, deletion of all or part of the gene encoding the protein and site-directed mutagenesis to disrupt the expression or activity of the gene product (see, e.g., Chaveroche et al., Nucl. Acids Res., 28:22 e97

[2000] ; Cho et al., Molec. Plant Microbe Interact., 19:7-15

[2006] ; Maruyama and Kitamoto, Biotechnol Lett., 30:1811-1817

[2008] ; Takahashi et al., Mol. Gen. Genom., 272: 344-352

[2004] ; and You et al., Arch. Microbiol., 191:615-622

[2009] , all of which are incorporated herein by reference).Random mutagenesis followed by screening for desired mutations is also useful (see, e.g., Combier et al., FEMS Microbiol. Lett., 220:141-8

[2003] ; and Firon et al., Eukary. Cell 2:247-55

[2003] , both of which are incorporated by reference).

[0169] Introduction of the vector or DNA construct into the host cell can be accomplished using any suitable method known in the art, including, but not limited to, calcium phosphate transfection, DEAE-dextran mediated transfection, PEG-mediated transformation, electroporation, or other common techniques known in the art. In some embodiments, the Escherichia coli expression vector pCK100900i (see U.S. Pat. No. 9,714,437, hereby incorporated by reference) is useful.

[0170] In some embodiments, engineered host cells of the invention (i.e., "recombinant host cells") are cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants, or amplifying acetate kinase polynucleotides. Culture conditions, e.g., temperature, pH, etc., are those previously used for the host cell selected for expression and are well known to those of skill in the art. As noted, many standard references and textbooks are available for the culture and production of many cells, including cells of bacterial, plant, animal (especially mammalian), and archebacterial origin.

[0171] In some embodiments, cells expressing a variant acetate kinase polypeptide of the invention are grown under batch or continuous fermentation conditions. Classical "batch fermentation" is a closed system in which the composition of the medium is set at the beginning of the fermentation and is not subject to artificial alterations during fermentation. A variation of the batch system is "fed-batch fermentation," which is useful in the present invention. In this variation, substrate is added gradually as the fermentation progresses. Fed-batch systems are useful when catabolite repression may inhibit cellular metabolism and when it is desirable to limit the amount of substrate in the medium. Batch and fed-batch fermentation are common and well known in the art. "Continuous fermentation" is an open system in which a defined fermentation medium is continuously added to a bioreactor and an equal amount of conditioned medium is simultaneously removed for processing. Continuous fermentation generally maintains the culture at a constant high density when the cells are primarily in logarithmic growth phase. Continuous fermentation systems strive to maintain steady-state growth conditions. Methods for modulating nutrients and growth factors for continuous fermentation processes, as well as techniques for maximizing the rate of product formation, are well known in the field of industrial microbiology.

[0172] In some embodiments of the invention, cell-free transcription / translation systems are useful for producing variant acetate kinases. Several systems are commercially available and the methods are well known to those skilled in the art.

[0173] The present invention provides methods for producing a variant acetate kinase polypeptide or a biologically active fragment thereof. In some embodiments, the method includes providing a host cell transformed with a polynucleotide encoding an amino acid sequence comprising at least about 70% (or at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%) sequence identity to SEQ ID NO:2, SEQ ID NO:12, and / or SEQ ID NO:600, and comprising at least one mutation provided herein; culturing the transformed host cell in a culture medium under conditions in which the host cell expresses the encoded variant acetate kinase polypeptide; and optionally recovering or isolating the expressed variant acetate kinase polypeptide and / or recovering or isolating the culture medium containing the expressed variant acetate kinase polypeptide. In some embodiments, the method further provides, optionally, lysing the transformed host cell after expressing the encoded acetate kinase polypeptide, and optionally recovering and / or isolating the expressed variant acetate kinase polypeptide from the cell lysate. The present invention further provides a method for producing a variant acetate kinase polypeptide, comprising culturing a host cell transformed with the variant acetate kinase polypeptide under conditions suitable for production of the variant acetate kinase polypeptide and recovering the variant acetate kinase polypeptide. Typically, the acetate kinase polypeptide is recovered or isolated from the host cell culture medium, from the host cells, or both, using protein recovery techniques well known in the art, including those described herein. In some embodiments, the host cells are harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract is retained for further purification. Microbial cells employed in protein expression can be disrupted by any convenient method, including, but not limited to, freeze-thaw cycling, sonication, mechanical disruption, and / or the use of cell lysing agents, as well as many other suitable methods well known to those of skill in the art.

[0174] The engineered acetate kinase enzyme expressed in the host cells can be recovered from the cells and / or culture medium using any one or more of the techniques known in the art for protein purification, including lysozyme treatment, sonication, filtration, salting out, ultracentrifugation, and chromatography, among others. A suitable solution for lysing and highly efficient extraction of proteins from bacteria, such as E. coli, is commercially available under the trade name CelLytic B™ (Sigma-Aldrich). Thus, in some embodiments, the resulting polypeptide is recovered / isolated and, if necessary, purified by any of a number of methods known in the art. For example, in some embodiments, the polypeptide is isolated from the nutrient medium by conventional procedures, including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, chromatography (e.g., ion exchange, affinity, hydrophobic interaction, chromatofocusing, and size exclusion), or precipitation. In some embodiments, a protein refolding step is used to complete configuration of the mature protein, if desired. Additionally, in some embodiments, high-performance liquid chromatography (HPLC) is employed in a final purification step. For example, in some embodiments, methods known in the art are useful in the present invention (see, e.g., Parry et al., Biochem. J., 353:117

[2001] ; and Hong et al., Appl. Microbiol. Biotechnol., 73:1331

[2007] , both of which are incorporated herein by reference). Indeed, any suitable purification method known in the art is useful in the present invention.

[0175] Chromatographic techniques for isolating acetate kinase polypeptides include, but are not limited to, reverse-phase chromatography, high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, and affinity chromatography. Conditions for purifying a particular enzyme depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc., and are known to those skilled in the art.

[0176] In some embodiments, affinity techniques are useful for isolating improved acetate kinase enzymes. For affinity chromatography purification, any antibody that specifically binds to an acetate kinase polypeptide can be used. To produce antibodies, various host animals, including but not limited to rabbits, mice, and rats, can be immunized by injection with acetate kinase. The acetate kinase polypeptide can be attached to a suitable carrier, such as BSA, via a side-chain functional group or a linker attached to a side-chain functional group. Various adjuvants, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, Pluronic® polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacillus Calmette-Guerin) and Corynebacterium parvum, can be used to increase the immune response depending on the host species.

[0177] In some embodiments, acetate kinase variants are prepared and used in the form of crude extracts or isolated or purified preparations of cells expressing the enzyme. In some embodiments, acetate kinase variants are prepared as lyophilisates, powder forms (e.g., acetone powders), or enzyme solutions. In some embodiments, acetate kinase variants are in the form of substantially pure preparations.

[0178] In some embodiments, the acetate kinase polypeptide is attached to any suitable solid substrate. Solid substrates include, but are not limited to, solid phases, surfaces, and / or membranes. Solid supports include, but are not limited to, organic polymers such as polystyrene, polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy, and polyacrylamide, as well as copolymers and grafts thereof. Solid supports can also be inorganic, such as glass, silica, controlled pore glass (CPG), reverse-phase silica, or metals such as gold or platinum. The substrate configuration can be in the form of beads, spheres, particles, granules, gels, membranes, or surfaces. Surfaces can be planar, substantially planar, or non-planar. Solid supports can be porous or non-porous and can have expanded or non-expanded features. Solid supports can be configured in the form of wells, depressions, or other containers, vessels, features, or locations. Multiple supports can be configured in an array with various locations addressable for robotic delivery of reagents or by detection methods and / or instruments.

[0179] In some embodiments, acetate kinase variants are purified using immunological methods. In one approach, antibodies raised against variant acetate kinase polypeptides (e.g., against polypeptides comprising SEQ ID NO: 2, SEQ ID NO: 12, and / or SEQ ID NO: 600, and / or immunogenic fragments thereof) using conventional methods are immobilized on beads, mixed with cell culture medium under conditions in which the variant acetate kinase binds, and then precipitated. In a related approach, immunochromatography is useful.

[0180] In some embodiments, the variant acetate kinase is expressed as a fusion protein comprising a non-enzymatic moiety. In some embodiments, the variant acetate kinase sequence is fused to a purification-facilitating domain. As used herein, the term "purification-facilitating domain" refers to a domain that mediates purification of the polypeptide to which it is fused. Suitable purification domains include, but are not limited to, metal-chelating peptides, histidine-tryptophan modules that enable purification on immobilized metals, glutathione-binding sequences (e.g., GST), hemagglutinin (HA) tags (corresponding to an epitope derived from the influenza hemagglutinin protein; see, e.g., Wilson et al., Cell 37:767

[1984] ), maltose-binding protein sequences, the FLAG epitope utilized in the FLAGS extension / affinity purification system (e.g., systems available from Immunex Corp.), and the like. One expression vector contemplated for use in the compositions and methods described herein provides for expression of a fusion protein comprising a polypeptide of the invention fused to a polyhistidine tract separated by an enterokinase cleavage site. The histidine residues facilitate purification on IMIAC (immobilized metal ion affinity chromatography; see, e.g., Porath et al., Prot. Exp. Purif., 3:263-281

[1992] ), while the enterokinase cleavage site provides a means for separating the variant acetate kinase polypeptide from the fusion protein. pGEX vectors (Promega) can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). Generally, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption to ligand-agarose beads (e.g., glutathione-agarose in the case of GST-fusions) followed by elution in the presence of free ligand.

[0181] Thus, in another aspect, the invention provides methods for producing an engineered enzyme polypeptide, the method comprising culturing a host cell capable of expressing a polynucleotide encoding the engineered enzyme polypeptide under conditions suitable for expression of the polypeptide, hi some embodiments, the method further comprises isolating and / or purifying the enzyme polypeptide described herein.

[0182] Suitable culture medium and growth conditions for host cells are well known in the art.Any suitable method for introducing polynucleotides into cells for the expression of enzyme polypeptides is contemplated to be useful in the present invention.Suitable techniques include, but are not limited to, electroporation, particle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion.

[0183] Various features and embodiments of the present invention are illustrated in the following representative examples, which are intended to be illustrative and not limiting. [Example]

[0184] The following examples, including experiments and results achieved, are provided for illustrative purposes only and should not be construed as limiting the invention. Indeed, a variety of suitable sources exist for many of the reagents and equipment described below. It is not intended that the invention be limited to any particular source for any reagent or equipment item.

[0185] In the experimental disclosure that follows, the following abbreviations apply: M (molar); mM (millimolar), uM and μM (micromolar); nM (nanomolar); mol (mole); gm and g (grams); mg (milligrams); ug and μg (micrograms); L and l (liters); ml and mL (milliliters); cm (centimeters); mm (millimeters); um and μm (micrometers); sec. (seconds); min (minutes); h and hr (hours); U (unit); MW (molecular weight); rpm (revolutions per minute); ps i and PSI (pounds per square inch); °C (degrees Celsius); RT and rt (room temperature); RH (relative humidity); CV (coefficient of variation); CAM and cam (chloramphenicol); PMBS (polymyxin B sulfate); IPTG (isopropyl β-Dl-thiogalactopyranoside); LB (Luria 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 (a commonly used laboratory E. coli strain, available from the Coli Genetic Stock Center [CGSC], New Haven, CT); HTP (high-throughput); HPLC (high-performance liquid chromatography); HPLC-UV (HPLC-ultraviolet-visible detector); 1H NMR (proton nuclear magnetic resonance spectroscopy); FIOPC (fold improvement 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, 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); Infors (Infors USA Inc., Annapolis Junction, MD); and Thermotron (Thermotron, Inc., Holland, MI). .

[0186] Example 1 Production of engineered polypeptides in pCK110900 A polynucleotide (SEQ ID NO: 1) encoding a naturally occurring wild-type polypeptide (SEQ ID NO: 2) from Thermotoga maritima having acetate kinase activity was cloned into the pCK110900 vector system (see, e.g., U.S. Pat. No. 9,714,437, hereby incorporated by reference in its entirety), and then expressed in E. coli W3110fhuA under the control of the lac promoter.

[0187] In a 96-well format, single colonies were picked and grown in 180 μL of LB containing 1% glucose and 30 μg / mL CAM at 30°C, 200 rpm, and 85% humidity. After overnight growth, 20 μL of the grown culture was transferred to a deep-well plate containing 380 μL of TB with 30 μg / mL CAM. The culture was grown at 30°C, 250 rpm, and 85% humidity. The optical density (OD) of the culture was measured. 600When the chromatin (K) reached 0.6-0.8, expression of the acetate kinase gene was induced by adding IPTG to a final concentration of 1 mM. Growth was continued for 18-20 hours after induction. Cells were harvested by centrifugation at 4000 rpm at 4°C for 10 minutes, and the medium was discarded. The cell pellet was stored at -80°C until ready for use. Before performing the assay, the cell pellet was resuspended in 400 μL of lysis buffer containing 1 g / L lysozyme and 50 mM potassium phosphate at pH 7.5 with 0.5 g / L PMBS. The plate was agitated at moderate shaking speed on a microtiter plate shaker at room temperature for 2 hours. The plate was then centrifuged at 4000 rpm at 4°C for 20 minutes, and the clarified supernatant was used in the HTP assay reaction described below.

[0188] The shake flask procedure can be used to generate shake flask powders (SFP) of engineered acetate kinase polypeptides, which are useful for secondary screening assays and / or use in the biocatalytic processes described herein. Shake flask powder (SFP) preparations of enzymes provide more purified preparations of engineered enzymes (e.g., up to 30% total protein) compared to the cell lysates used in HTP assays and also allow for the use of more concentrated enzyme solutions. To initiate cultures, a single colony of E. coli containing a plasmid encoding the engineered polypeptide of interest was inoculated into 5 mL of LB medium with 30 μg / mL CAM and 1% glucose. The culture was grown overnight (at least 16 hours) in a 30°C incubator with shaking at 250 rpm. The grown culture was cultured in 250 mL of TB medium with 30 μg / mL CAM in a 1 L shake flask until a final OD 600 The 250 mL culture was diluted to an OD of 0.05 at 30°C and 250 rpm. 600The cells were grown until a pH of 0.6-0.8 was reached. Expression of the acetate kinase gene was induced by adding IPTG to a final concentration of 1 mM, and growth was continued for an additional 18-20 hours. The culture was transferred to pre-weighed centrifuge bottles and then harvested by centrifugation at 7,000 rpm at 4°C for 10 minutes. The supernatant was discarded, and the remaining cell pellet was weighed. In some embodiments, the cells were stored at -80°C until ready for use. For lysis, the cell pellet was resuspended in 6 mL of cold 50 mM potassium phosphate, pH 7.5, per gram of cell pellet. The resuspended cells were lysed using a 110 L MICROFLUIDIZER® Processor System (Microfluidics). Cell debris was removed by centrifugation at 10,000 rpm at 4°C for 60 minutes. The clarified lysate was collected, frozen at -80°C, and then lyophilized using standard methods known in the art. Lyophilization of the frozen clarified lysate provides a dry shake-flask powder containing the crude engineered polypeptide.

[0189] Example 2 Evolution and screening of engineered polypeptides derived from SEQ ID NO:2 for improved acetate kinase (AcK) activity A polynucleotide (SEQ ID NO: 1) encoding a polypeptide having acetate kinase activity of SEQ ID NO: 2 was used to generate the engineered polypeptides in Table 2-1. These polypeptides exhibited improved acetate kinase activity under desired conditions compared to the starting polypeptide (e.g., the ability to promote ATP recycling via ADP and acetyl phosphate, as measured by the percent conversion of ethynylglyceraldehyde to ethynylglyceraldehyde phosphate product under ATP-limited conditions). Engineered polypeptides having amino acid sequences with even-numbered sequence identifiers were generated from the "backbone" amino acid sequence of SEQ ID NO: 2 described below, along with the HTP assay and analytical methods described in Table 2-2.

[0190] Directed evolution began with the polynucleotide set forth in SEQ ID NO: 1. Libraries of engineered polypeptides were generated using a variety of well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using HTP assays, an analytical method that measures the ability of polypeptides to promote ATP recycling via ADP and acetyl phosphate (measured via the conversion of an ethynylglyceraldehyde substrate to an ethynylglyceraldehyde phosphate product under ATP-limiting conditions).

[0191] Enzyme assays were performed in a 96-well format in a total volume of 50 μL / well containing HTP enzyme lysate, final concentrations of 20 g / L ethynylglyceraldehyde, 2 equivalents of acetyl phosphate, 0.3 g / L ATP, 0.5 g / L engineered pantothenate kinase (SEQ ID NO: 698), 10 mM MgCl, 100 mM potassium phosphate, pH 7.5. Reactions were performed by adding to each well: (i) 25 μL of a solution containing 40 g / L ethynylglyceraldehyde, 300 mM acetyl phosphate, 0.6 g / L ATP, 1 g / L engineered pantothenate kinase (SEQ ID NO: 698), 20 mM MgCl, 200 mM potassium phosphate (the pH of the mixture was adjusted to 7.8); (ii) 25 μL of 0.3% (vol / vol) diluted AcK HTP lysate. The reaction plate was heat sealed and shaken at 30°C and 600 rpm overnight.

[0192] After overnight incubation (approximately 16 hours), 100 μL of 50 mM potassium phosphate, pH 7.5, was mixed with the sample. To a separate plate, 20 μL of sample was transferred and mixed with 180 μL of a 10 g / L solution of O-benzylhydroxylamine in methanol. The plate was sealed and shaken at 25°C and 400 rpm for 20-30 minutes. The sample was further diluted 4-fold in methanol before UPLC analysis using the method described in Table 2-2 below. The data shown in Table 2-1 reflect activity relative to SEQ ID NO:2, calculated as the peak area of compound 5 formed by the variant enzyme compared to the peak area of compound 5 formed by SEQ ID NO:2 under the specified reaction conditions.

[0193] Selected hit variants were grown in 250 mL shake flasks to generate enzyme powders, whose activity was assessed in 0.0002-0.2 g / L SF powder, 3 g / L ethynylglyceraldehyde, 2 equivalents of acetyl phosphate, 0.3 g / L ATP, 0.5 g / L engineered pantothenate kinase (SEQ ID NO: 698), 10 mM MgCl, 100 mM potassium phosphate, pH 6.8 at 30°C, 600 rpm for 3 hours using an assay similar to that described above. [Table 2-1-1] [Table 2-1-2] [Table 2-1-3] [Table 2-1-4] [Table 2-1-5] [Table 2-1-6] [Table 2-1-7] [Table 2-1-8] [Table 2-1-9] [Table 2-1-10] [Table 2-2-1] [Table 2-2-2]

[0194] Example 3 Evolution and screening of engineered polypeptides derived from SEQ ID NO: 12 for improved acetate kinase (AcK) activity An engineered polynucleotide (SEQ ID NO:11) encoding a polypeptide having acetate kinase activity of SEQ ID NO:12 was used to generate the engineered polypeptides in Table 3-1. These polypeptides exhibited improved acetate kinase activity under desired conditions (e.g., the ability to promote ATP recycling via ADP and acetyl phosphate as measured by the percent conversion of ethynylglyceraldehyde to ethynylglyceraldehyde phosphate product under ATP-limited conditions) compared to the starting polypeptide. Engineered polypeptides having amino acid sequences with even-numbered sequence identifiers were generated from the "backbone" amino acid sequence of SEQ ID NO:12 described below, along with the HTP assay and analytical methods described in Table 2-2.

[0195] Directed evolution began with the polynucleotide set forth in SEQ ID NO: 11. Libraries of engineered polypeptides were generated using a variety of well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using HTP assays, an analytical method that measures the ability of polypeptides to promote ATP recycling via ADP and acetyl phosphate (measured via the conversion of an ethynylglyceraldehyde substrate to an ethynylglyceraldehyde phosphate product under ATP-limiting conditions).

[0196] Enzyme assays were performed in a 96-well format in a total volume of 50 μL / well containing HTP enzyme lysate, final concentrations of 20 g / L ethynylglyceraldehyde, 2 equivalents of acetyl phosphate, 0.3 g / L ATP, 0.5 g / L engineered pantothenate kinase (SEQ ID NO: 700), 10 mM MgCl, 100 mM potassium phosphate, pH 7.5. Reactions were performed by adding to each well: (i) 25 μL of a solution containing 40 g / L ethynylglyceraldehyde, 300 mM acetyl phosphate, 0.6 g / L ATP, 1 g / L engineered pantothenate kinase (SEQ ID NO: 700), 20 mM MgCl, 200 mM potassium phosphate (the pH of the mixture was adjusted to 7.8); (ii) 25 μL of 0.3% (vol / vol) diluted AcK HTP lysate. The reaction plate was heat sealed and shaken at 30°C and 600 rpm overnight.

[0197] After overnight incubation (approximately 16 hours), 100 μL of 50 mM potassium phosphate, pH 7.5, was mixed with the sample. In a separate plate, 20 μL of sample was transferred and mixed with 180 μL of a 10 g / L solution of O-benzylhydroxylamine in methanol. The plate was sealed and shaken at 25°C and 400 rpm for 20-30 minutes. The sample was further diluted 4-fold in methanol before UPLC analysis using the method described in Table 2-2. The data shown in Table 3-1 reflect the activity relative to SEQ ID NO: 12, calculated as the peak area of compound 5 formed by the variant enzyme compared to the peak area of compound 5 formed by SEQ ID NO: 12 under the specified reaction conditions.

[0198] Hit variants were grown in 250 mL shake flasks to generate enzyme powders, whose activity was assessed in 0.0002-0.2 g / L SF powder, 3 g / L ethynylglyceraldehyde, 2 equivalents of acetyl phosphate, 0.3 g / L ATP, 0.5 g / L engineered pantothenate kinase (SEQ ID NO: 700), 10 mM MgCl, 100 mM potassium phosphate, pH 6.8 at 30°C, 600 rpm for 3 hours using an assay similar to that described above. [Table 3-1-1] [Table 3-1-2]

[0199] Example 4 Evolution and screening of engineered polypeptides derived from SEQ ID NO:600 for improved acetate kinase (AcK) activity A polynucleotide encoding a polypeptide having acetate kinase activity of SEQ ID NO:600 (SEQ ID NO:599) was used to generate the engineered polypeptides in Table 4-1. These polypeptides exhibited improved acetate kinase activity under desired conditions (e.g., the ability to promote ATP recycling via ADP and acetyl phosphate as measured by the percent conversion of ethynylglycerol to ethynylglycerol phosphate product under ATP-limited conditions) compared to the starting polypeptide. Engineered polypeptides having amino acid sequences of even-numbered sequence identifiers were generated from the "backbone" amino acid sequence of SEQ ID NO:600 described below, along with the assays and analytical methods described in Table 4-2.

[0200] Directed evolution began with the polynucleotide set forth in SEQ ID NO: 599. Libraries of engineered polypeptides were generated using a variety of well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using HTP assays, an analytical method that measures the ability of polypeptides to promote ATP recycling via ADP and acetyl phosphate (measured via the conversion of an ethynylglycerol substrate to an ethynylglycerol phosphate product under ATP-limiting conditions).

[0201] Enzyme assays were performed in a 96-well format with a total volume of 50 μL per well containing HTP enzyme lysate, final concentrations of 50 g / L ethynylglycerol, 1.25 equivalents of acetyl phosphate, 0.1 mol% ATP, 0.5 g / L engineered pantothenate kinase (SEQ ID NO: 702), 10 mM MgCl, 50 mM Bis-Tris buffer pH 7.0. Reactions were performed by adding to each well: (i) 40 μL of a solution containing 62.5 g / L ethynylglycerol, 1.5625 equivalents of mM acetyl phosphate, 0.125 mol% ATP, 0.625 g / L engineered pantothenate kinase (SEQ ID NO: 702), 12.5 mM MgCl, 50 mM Bis-Tris buffer pH 7.0. The pH of the final master mix was pH = 6.8. (ii) 10 μL of 50-fold diluted AcK HTP lysate. The reaction plate was heat sealed and shaken at 30°C and 600 rpm overnight.

[0202] After overnight incubation (approximately 18 hours), the reaction samples were derivatized with 5,5',5''-[2,2',2''-nitrilotris(methylene-tris(1H-benzimidazole-2,1-diyl))]tripentanoic acid tripotassium hydrate ((BimC4A)3) to generate chromophore-containing species and allow simple reaction monitoring. The reaction mixture was then incubated at 45 °C for 1 hour with 5 equivalents of benzyl azide, 5 mol% copper sulfate, 7.5 mol% (BimC4A)3, and 20 mol% sodium ascorbate in 9:1 water:DMSO to achieve click chemistry. After the reaction, 5 μL of the reaction mixture was combined with 220 μL of (BimC4A)3 derivatization solution in a new 96-well plate. The click chemistry reaction was incubated at 45 °C for 1 hour. Samples were then filtered by centrifugation using a 0.22 micron 96-well filter plate in preparation for analysis by UPLC-UV (Table 4-2).

[0203] Hit variants were grown in 250 mL shake flasks to generate enzyme powders. The activity of the enzyme powders was evaluated using an assay similar to that described above, containing 0.0125-0.05 g / L SF powder, 50 g / L ethynylglycerol, 1.25 equivalents of acetyl phosphate, 0.1 mol% ATP, 0.25-1.5 g / L engineered pantothenate kinase (SEQ ID NO: 702), 10 mM MgCl2, 50 mM Bis-Tris buffer pH 7.0 (final master mix pH = 6.8), at 30 °C, 600 rpm for 18 hours. Data from the shake flask powder study are shown in Table 4-1. [Table 4-1] [Table 4-2]

[0204] For example, certain embodiments provide the following: (Item 1) 1. An engineered acetate kinase comprising a polypeptide sequence or functional fragment thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:2, SEQ ID NO:12, and / or SEQ ID NO:600, wherein said engineered acetate kinase comprises at least one substitution or set of substitutions in said polypeptide sequence, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:2, SEQ ID NO:12, and / or SEQ ID NO:600. (Item 2) 2. The engineered acetate kinase of item 1, comprising a polypeptide sequence or functional fragment thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to SEQ ID NO:2, wherein the engineered acetate kinase comprises at least one substitution or set of substitutions in the polypeptide sequence, and the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:2. (Item 3) The engineered acetate kinase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:2 or a functional fragment thereof, and the engineered acetate kinase is 1, 50, 51, 55, 58 / 135, 59, 64, 70, 76, 76 / 232 / 262, 76 / 232 / 364 / 386, 76 / 273, 85, 92, 96 / 119, 101, 102, 104, 107, 108, 116, 122, 135, 135 / 392, 136, 137, 140, 141, 143, 144, 145 / 400, 152, 154, 159, 161, 164, 183, 191, 194, 197, 215, 216, 222, 2 24, 229, 232, 236, 251, 256, 258, 259 / 284, 260, 261, 263 / 391, 268, 273, 274, 276, 283, 284, 286, 287, 288, 289, 290, 292, 297, 298 / 405, 299, 300, 301, 302, 303, 304, 305, 308, 310, 312, 314, 316, 317, 322, 323, 337, 346, 347, 348, 349, 351, 35 3. The engineered acetate kinase of item 2, comprising at least one substitution or set of substitutions at one or more positions selected from: 2, 352 / 405, 354, 355, 356, 362, 366, 368, 371, 372, 373, 374, 375, 376, 377, 378, 380, 386, 390, 391, 392, 398, 399, and 407, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:2. (Item 4) 2. The engineered acetate kinase of item 1, comprising a polypeptide sequence or functional fragment thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to SEQ ID NO: 12, wherein the engineered acetate kinase comprises at least one substitution or set of substitutions in the polypeptide sequence, and the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 12. (Item 5) the engineered acetate kinase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12 or a functional fragment thereof, and the engineered acetate kinase is selected from the group consisting of 15 / 70 / 154 / 297 / 352 / 355, 15 / 154 / 191 / 297 / 298 / 301 / 348 / 352 / 391, 15 / 154 / 191 / 297 / 355, 15 / 154 / 297 / 298 / 348 / 352 / 355 / 3 91, 15 / 191 / 297 / 298 / 301 / 348 / 352 / 355 / 391, 15 / 191 / 298 / 352 / 355, 15 / 297 / 298 / 355, 23 / 101 / 102 / 122 / 140 / 143 / 316 / 372, 23 / 101 / 102 / 140, 23 / 101 / 374, 23 / 122 / 316 / 372 / 374, 23 / 122 / 316 / 374 / 395, 23 / 140 / 316 / 374, 29 / 154 / 191 / 298 / 348 / 355, 51 / 101 / 102 / 135 / 242 / 316 / 374, 51 / 101 / 316, 70 / 15 4 / 162 / 191 / 297 / 301 / 355 / 391, 70 / 154 / 191 / 297 / 352, 70 / 154 / 191 / 298 / 348 / 352 / 355 / 391, 70 / 154 / 191 / 298 / 352, 70 / 154 / 297 / 298 / 348 / 355, 70 / 154 / 297 / 352 / 355, 70 / 191 / 297 / 298 / 352 / 391, 101 / 102 / 122 / 140 / 142 / 316 / 372 / 374, 101 / 136 / 242 / 372, 102 / 135 / 140 / 316, 102 / 136 / 140 / 142 / 143 / 316, 102 / 142 / 316, 122 / 140 / 142 / 164 / 242, 122 / 142 / 316, 122 / 143 / 242 / 374, 135 / 136 / 140 / 142 / 143 / 242 / 316 / 372 / 374, 135 / 140 / 143 / 242 / 374 / 395, 135 / 140 / 316, 136 / 242, 142 / 316, 142 / 316 / 372 / 374, 142 / 316 / 374, 143 / 316, 154 / 191 / 297 / 298, 154 / 191 / 297 / 298 / 301 / 352 / 355, 154 / 191 / 297 / 298 / 352,3. The engineered acetate kinase of item 2, comprising at least one substitution or set of substitutions at one or more positions selected from 154 / 191 / 298 / 301 / 348 / 352 / 391, 154 / 191 / 298 / 348 / 352, and 154 / 297 / 298, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 12. (Item 6) 2. The engineered acetate kinase of item 1, comprising a polypeptide sequence or functional fragment thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 600, wherein the engineered acetate kinase comprises at least one substitution or set of substitutions in the polypeptide sequence, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 600. (Item 7) 3. The engineered acetate kinase of item 2, wherein the engineered acetate kinase comprises a polypeptide sequence or functional fragment thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 600, wherein the engineered acetate kinase comprises at least one substitution or set of substitutions at one or more positions selected from 23 / 242 / 374, 23 / 374, 70 / 374, 242 / 298, 242 / 374, 298 / 374, and 374, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 600. (Item 8) 2. The engineered acetate kinase of item 1, comprising a polypeptide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence of at least one engineered acetate kinase variant listed in Table 2-1, Table 3-1, and / or Table 4-1. (Item 9) 2. The engineered acetate kinase of item 1, comprising a polypeptide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:2. (Item 10) 2. The engineered acetate kinase of item 1, comprising a polypeptide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:12. (Item 11) 2. The engineered acetate kinase of item 1, comprising a variant engineered polypeptide as set forth in SEQ ID NO: 12. (Item 12) 2. The engineered acetate kinase of item 1, comprising a polypeptide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 600. (Item 13) 2. The engineered acetate kinase of item 1, comprising a variant engineered polypeptide as set forth in SEQ ID NO: 600. (Item 14) 2. The engineered acetate kinase of item 1, comprising a polypeptide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence of at least one engineered acetate kinase variant set forth in an even-numbered sequence of SEQ ID NOS: 2-696. (Item 15) 2. The engineered acetate kinase according to item 1, comprising a polypeptide sequence set forth in the even-numbered sequences of SEQ ID NOs: 2 to 696. (Item 16) 16. The engineered acetate kinase of any of items 1 to 15, comprising at least one improved property compared to wild-type M. thermoga acetate kinase. (Item 17) 17. The engineered acetate kinase of paragraph 16, wherein the improved properties comprise improved activity towards the substrate compared to wild-type acetate kinase. (Item 18) 18. The engineered acetate kinase of item 17, wherein the substrate comprises adenosine diphosphate and acetyl phosphate. (Item 19) 17. The engineered acetate kinase of claim 16, wherein the improved properties include improved production of adenosine triphosphate compared to wild-type acetate kinase. (Item 20) 20. The engineered acetate kinase of any of items 1 to 19, which is purified. (Item 21) 21. The engineered acetate kinase of any of items 1 to 20, which is part of a multi-enzyme system for producing nucleoside analogues. (Item 22) 22. A composition comprising at least one engineered acetate kinase according to any of items 1 to 21. (Item 23) 22. A polynucleotide sequence encoding at least one engineered acetate kinase according to any of items 1 to 21. (Item 24) 1. A polynucleotide sequence encoding at least one engineered acetate kinase, wherein said polynucleotide sequence comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:1, SEQ ID NO:11, and / or SEQ ID NO:599, and wherein said polynucleotide sequence of said engineered acetate kinase comprises at least one substitution at one or more positions. (Item 25) 24. The polynucleotide sequence of item 23, encoding at least one engineered acetate kinase or functional fragment thereof, comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to SEQ ID NO:1. (Item 26) 24. The polynucleotide sequence of item 23, encoding at least one engineered acetate kinase or functional fragment thereof, comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to SEQ ID NO: 11. (Item 27) 24. The polynucleotide sequence of Item 23, encoding at least one engineered acetate kinase or functional fragment thereof, comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to SEQ ID NO: 599. (Item 28) 28. The polynucleotide sequence according to any one of items 23 to 27, operably linked to a regulatory sequence. (Item 29) 29. The polynucleotide sequence according to any one of items 23 to 28, which is codon-optimized. (Item 30) 30. The polynucleotide sequence according to any one of Items 23 to 29, comprising odd-numbered sequences of SEQ ID NOs: 1 to 695. (Item 31) 31. An expression vector comprising at least one polynucleotide sequence according to any one of items 23 to 30. (Item 32) A host cell comprising at least one expression vector according to item 31. (Item 33) A host cell comprising at least one polynucleotide sequence according to any one of items 23 to 30. (Item 34) 34. A method for producing an engineered acetate kinase in a host cell, comprising culturing the host cell of items 32 and / or 33 under suitable conditions such that at least one engineered acetate kinase is produced. (Item 35) 35. The method of claim 34, further comprising recovering the at least one engineered acetate kinase from the culture and / or host cell. (Item 36) 36. The method of items 34 and / or 35, further comprising purifying the at least one engineered acetate kinase. All publications, patents, patent applications, and other documents cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated by reference herein for all purposes.

[0205] While various specific embodiments have been illustrated and described, it will be recognized that various changes can be made without departing from the spirit and scope of the invention.

Claims

1. 1. An engineered acetate kinase comprising a polypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:2 or a functional fragment thereof, wherein said engineered acetate kinase comprises at least one substitution at position 298 in said polypeptide sequence selected from the group consisting of 298F, 298L, 298K, 298Q, 298T, 298V and 298W, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:2, and wherein said engineered acetate kinase comprises improved production of adenosine triphosphate compared to the wild-type acetate kinase of SEQ ID NO:

2.

2. the engineered acetate kinase comprises a polypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:2 or a functional fragment thereof, and the engineered acetate kinase is 59, 64, 70, 76, 76 / 232 / 262, 76 / 232 / 364 / 386, 76 / 273, 85, 92, 96 / 119, 101, 102, 104, 107, 108, 116, 122, 135, 135 / 392, 136, 137, 140, 141, 143, 144, 145 / 400, 152, 154, 159, 161, 164, 183, 191, 194, 197, 215, 216, 222, 224, 229, 232, 2 36, 251, 256, 258, 259 / 284, 260, 261, 263 / 391, 268, 273, 274, 276, 283, 284, 286, 287, 288, 289, 290, 292, 297, 405, 299, 300, 301, 302, 303, 304, 305, 308, 310, 312, 314, 316, 317, 322, 323, 337, 346, 347, 348, 349, 351, 352, 352 / 405, 2. The engineered acetate kinase of claim 1, further comprising at least one substitution or set of substitutions at one or more positions selected from 354, 355, 356, 362, 366, 368, 371, 372, 373, 374, 375, 376, 377, 378, 380, 386, 390, 391, 392, 398, 399, and 407, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:

2.

3. 2. The engineered acetate kinase of claim 1, comprising a polypeptide sequence or functional fragment thereof having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:12, wherein the engineered acetate kinase comprises at least one substitution or set of substitutions in the polypeptide sequence, and the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:

12.

4. the engineered acetate kinase comprises a polypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12 or a functional fragment thereof, and the engineered acetate kinase is selected from the group consisting of: 15 / 70 / 154 / 297 / 352 / 355, 15 / 154 / 191 / 297 / 355, 15 / 154 / 297 / 348 / 352 / 391, 15 / 154 / 191 / 297 / 355, 15 / 154 / 297 / 348 / 352 / 355 / 391, 15 / 191 / 297 / 301 / 34 8 / 352 / 355 / 391, 15 / 191 / 352 / 355, 15 / 297 / 355, 23 / 101 / 102 / 122 / 140 / 143 / 316 / 372, 23 / 101 / 102 / 140, 23 / 101 / 374, 23 / 122 / 316 / 372 / 374, 23 / 122 / 316 / 374 / 395, 23 / 140 / 316 / 374, 29 / 154 / 191 / 348 / 355, 51 / 101 / 102 / 135 / 242 / 316 / 374, 51 / 101 / 316, 70 / 154 / 162 / 191 / 297 / 301 / 355 / 391, 70 / 154 / 191 / 297 / 352, 70 / 154 / 191 / 348 / 352 / 355 / 391, 70 / 154 / 191 / 352, 70 / 154 / 297 / 348 / 355, 70 / 154 / 297 / 352 / 355, 70 / 191 / 297 / 352 / 391, 101 / 102 / 122 / 140 / 142 / 316 / 372 / 374, 101 / 136 / 242 / 372, 102 / 135 / 140 / 316, 102 / 136 / 140 / 142 / 143 / 316, 102 / 142 / 316, 122 / 140 / 142 / 164 / 242, 122 / 142 / 316, 122 / 143 / 242 / 374, 135 / 136 / 140 / 142 / 143 / 242 / 316 / 372 / 374, 135 / 140 / 143 / 242 / 374 / 395, 135 / 140 / 316, 136 / 242, 142 / 316, 142 / 316 / 372 / 374, 142 / 316 / 374, 143 / 316, 154 / 191 / 297, 154 / 191 / 297 / 301 / 352 / 355, 154 / 191 / 297 / 352, 154 / 191 / 301 / 348 / 352 / 391, 154 / 191 / 348 / 352,and 154 / 297, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:

12.

5. 2. The engineered acetate kinase of claim 1, comprising a polypeptide sequence or functional fragment thereof having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:600, wherein said engineered acetate kinase comprises at least one substitution or set of substitutions in said polypeptide sequence, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:

600.

6. 2. The engineered acetate kinase of claim 1, wherein the engineered acetate kinase comprises a polypeptide sequence or functional fragment thereof having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to SEQ ID NO:600, and wherein the engineered acetate kinase further comprises at least one substitution or set of substitutions at one or more positions selected from 23 / 242 / 374, 23 / 374, 70 / 374, 242, 242 / 374, and 374, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:

600.

7. 2. The engineered acetate kinase of claim 1, comprising a polypeptide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:

2.

8. 2. The engineered acetate kinase of claim 1, comprising a polypeptide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:

12.

9. 10. The engineered acetate kinase of claim 1, comprising a variant engineered polypeptide as set forth in SEQ ID NO:

12.

10. 2. The engineered acetate kinase of claim 1, comprising a polypeptide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:

600.

11. 10. The engineered acetate kinase of claim 1, comprising a variant engineered polypeptide as set forth in SEQ ID NO:

600.

12. 10. The engineered acetate kinase of claim 1, comprising a polypeptide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence of at least one engineered acetate kinase variant set forth in the even-numbered sequences of SEQ ID NOS: 4-14, 22, 64 and 592-696.

13. 10. The engineered acetate kinase of claim 1, comprising a polypeptide sequence set forth in the even-numbered sequences of SEQ ID NOs: 4-14, 22, 64 and 592-696.

14. 14. The engineered acetate kinase of any of claims 1-13, further comprising at least one improved property compared to wild-type Thermotoga maritima acetate kinase.

15. 15. The engineered acetate kinase of claim 14, wherein the improved properties comprise improved activity towards a substrate compared to wild-type acetate kinase.

16. 16. The engineered acetate kinase of claim 15, wherein the substrate comprises adenosine diphosphate and acetyl phosphate.

17. 17. The engineered acetate kinase of any one of claims 1 to 16, which is purified.

18. 18. The engineered acetate kinase of any one of claims 1 to 17, which is part of a multi-enzyme system for producing nucleoside analogues.

19. A composition comprising at least one engineered acetate kinase according to any of claims 1 to 18.

20. A polynucleotide encoding at least one engineered acetate kinase according to any one of claims 1 to 18.

21. 1. A polynucleotide encoding at least one engineered acetate kinase, said polynucleotide comprising a polynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:1, wherein said polynucleotide sequence of said engineered acetate kinase comprises at least one substitution at one or more positions, wherein said engineered acetate kinase comprises at least one substitution at position 298 of the polypeptide sequence selected from the group consisting of 298F, 298L, 298K, 298Q, 298T, 298V and 298W, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:2, and wherein said engineered acetate kinase comprises improved production of adenosine triphosphate compared to the wild-type acetate kinase of SEQ ID NO:

2.

22. 21. The polynucleotide of claim 20, encoding at least one engineered acetate kinase or functional fragment thereof, comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:

11.

23. 21. The polynucleotide of claim 20, encoding at least one engineered acetate kinase or functional fragment thereof comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:

599.

24. The polynucleotide according to any one of claims 20 to 23, which is operably linked to a control sequence.

25. The polynucleotide of any one of claims 20 to 24, which is codon-optimized.

26. A polynucleotide according to any one of claims 20 to 25, comprising the odd-numbered sequences of SEQ ID NOs: 3 to 13, 21, 63 and 591 to 695.

27. An expression vector comprising at least one polynucleotide according to any one of claims 20 to 26.

28. 28. A host cell comprising at least one expression vector according to claim 27.

29. A host cell comprising at least one polynucleotide according to any one of claims 20 to 26.

30. 30. A method for producing an engineered acetate kinase in a host cell, comprising culturing the host cell of claims 28 and / or 29 under suitable conditions such that at least one engineered acetate kinase is produced.

31. 31. The method of claim 30, further comprising recovering the at least one engineered acetate kinase from the culture and / or host cell.

32. 32. The method of claims 30 and / or 31, further comprising purifying the at least one engineered acetate kinase.

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