Engineered acetate kinase variant enzymes

Abstract

The present invention provides engineered acetate kinase (AcK), polypeptides with AcK activity and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. Also provided is a method for producing the AcK enzyme. The present invention also provides a composition comprising the AcK enzyme and a method for using the engineered AcK enzyme. The present invention is particularly useful for the production of pharmaceutical compounds.

Description

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

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

[0003] Reference to a sequence listing, table or computer program

[0004] An official copy of the sequence listing is submitted with the specification via EFS-Web as an ASCII formatted text file, with the file name "CX2-191WO2_ST25.txt", creation date June 29, 2020, and size of 1.74 megabytes. The sequence listing submitted via EFS-Web is part of the specification and is incorporated herein by reference in its entirety. Background of the Invention

[0006] The retrovirus known as human immunodeficiency virus (HIV) is the pathogen of acquired immunodeficiency syndrome (AIDS), which is a complex disease involving the gradual destruction of the immune system of the affected individual and the degeneration of the central and peripheral nervous systems. A common feature of retroviral replication is that the viral RNA genome is reversely transcribed by a virally encoded reverse transcriptase to produce a DNA copy of the HIV sequence required for viral replication. Some compounds such as MK-8591 are known reverse transcriptase inhibitors and can be used to treat AIDS and similar diseases. Although there are some compounds known to inhibit HIV reverse transcriptase, there is still a need in the art for other compounds that can more effectively inhibit this enzyme and thereby improve the effect on AIDS.

[0007] Due to its similarity to the natural nucleosides for synthesizing DNA, nucleoside analogs such as MK-8591 (Merck) are effective inhibitors of the reverse transcriptase of HIV. The combination of reverse transcriptase and these analogs stagnates the synthesis of DNA by inhibiting the progressive nature of reverse transcriptase. The stagnation of enzyme causes the premature termination of DNA molecules, making it invalid. However, the production of nucleoside analogs by standard chemical synthesis techniques may be challenging due to its chemical complexity. SUMMARY OF THE INVENTION

[0009] The present invention provides engineered acetate kinase (AcK), polypeptides with AcK activity and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. Also provided are methods for producing AcK enzymes. The present invention also provides compositions comprising AcK enzymes, and methods for using engineered AcK enzymes. The present invention is particularly useful for the production of pharmaceutical compounds.

[0010] In some embodiments, the present invention provides an engineered acetate kinase, comprising 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, SEQ ID NO:12 and / or SEQ ID NO:600, or a functional fragment thereof, 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.

[0011] In some embodiments, the present invention provides an engineered acetate kinase comprising 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, 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 NO:2 A polypeptide sequence or a 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, and wherein the engineered acetate kinase comprises at least one substitution or set of substitutions at one or more positions 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, 194, 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, 306 8, 310, 312, 314, 316, 317, 322, 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, and wherein 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 comprises at least one substitution or set of substitutions selected from the group consisting of 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, 70 K、70T、76V、76V / 232S / 262L、76V / 232S / 364I / 386K、76V / 273V、85H、92D、96T / 119I、101G、101I、101M、101T、102A、104A、104L、107K、108L、108R、108W、1 16D、122C、122E、122G、122T、122V、122W、135L、135N、135T、135T / 392W、136 A、136E、136G、136T、136V、137K、137R、140F、140P、140R、140S、140W、140Y、1 41R、143C、144K、144Q、145E / 400V、152Q、154M、154Q、154T、154V、159F、161 M、161S、161V、164G、164H、164L、183A、183G、183S、191I、194K、197P、197Q、1 97R、215E、215F、215L、215M、216A、216S、222I、224I、229G、229N、232S、236 G、251G、256L、256M、256Q、258V、259V / 284L、260E、260T、261A、263T / 391G、 268L、273V、274W、276V、283K、284L、286I、287A、287D、287G、287L、287T、28 8G、289A、289S、290L、292P、297L、297M、297Q、298F、298K、298L、298L / 405E、 298Q、298T、298V、298W、299Q、299R、299S、299T、300G、300S、300T、301E、30 1K、301Q、301R、302A、302F、302G、302R、302S、303E、304A、304C、304D、304E、 304Q、304R、304V、305N、308R、310A、310G、310M、312K、312P、312R、314V、31 6A、316G、316M、316Q、316R、316V、317A、317G、317I、317L、317S、322G、322N、322S, 323P, 323S, 337L, 346L, 347Q, 347R, 347T, 347V, 348E, 349V, 351L, 3 51R, 351S, 351V, 352A, 352F, 352G, 352K, 352P, 352T, 352V, 352V / 405Q, 354 G, 354L, 354P, 354S, 354V, 354W, 355D, 355E, 355K, 355M, 356G, 362I, 366E, 366G, 366I, 366L, 366P, 366R, 366S, 366W, 368E, 368G, 368L, 368N, 368Q, 36 392G, 392W, 398H, 398W, 398Y, 399A, 399P, 399S and 407L, wherein 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 comprises at least one substitution or set of substitutions selected from the group consisting of 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, E58S / A135T, K59A, K59G, K59V, R64K, E70K, E70T, I76V, I76 V / C232S / I262L, I76V / C232S / L364I / R386K, I76V / M273V, L85H, K92D, A96T / L119I, V101G, V101I, V101M, V101T, V102A, G104A, G104L, R107K, F108L, F 108R, F108W, E116D, I122C, I122E, I122G, I122T, I122V, I122W, A135L, A13 5N, 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、C354V、C354W、S355D、S355E、S355K、S355M、Y356G、V362I, K366E, K366G, K366I, K366L, K366P, K366R, K366S, K366W, K368E, K368G, K368L, K368N, K368Q, K368R, K368 S, K368V, E371N, E371V, T372D, T372F, T372N, T372V, I373P, I373V, R374D, R374E, R374H, R374L, R374M, R374V, G37 76M, 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 sequences 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.

[0012] In some additional 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.

[0013] In some embodiments, the present invention provides an engineered acetate kinase, comprising 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, 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 NO:12 A polypeptide sequence or a functional fragment thereof having a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, and wherein the engineered acetate kinase comprises at least one substitution or set of substitutions at one or more positions selected from the group consisting of: 15 / 70 / 154 / 297 / 352 / 355, 15 / 154 / 191 / 297 / 298 / 301 / 348 / 352 / 391, 15 / 154 / 1 91 / 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 / 1 54 / 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、1 54 / 191 / 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 comprises at least one substitution or set of substitutions selected from the group consisting of: 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 / 355K / 391 R、15N / 191I / 297L / 298F / 301R / 348E / 352T / 355K / 391R、15N / 191I / 298F / 352T / 355K、15N / 297L / 298F / 3 55K、23H / 101I / 102A / 122G / 140P / 143C / 316R / 372F、23H / 101I / 102A / 140P、23H / 101I / 374L、23H / 122G / 3 16R / 372F / 374L, 23H / 122G / 316R / 374E / 395K, 23H / 140P / 316R / 374E, 29D / 154Q / 191I / 298F / 348E / 355K , 51F / 101I / 102A / 135L / 242T / 316R / 374L, 51F / 101I / 316R, 70K / 154Q / 162H / 191I / 297N / 301R / 355K / 391 R、70K / 154Q / 191I / 297L / 352T、70K / 154Q / 191I / 298F / 348E / 352T / 355K / 391R、70K / 154Q / 191I / 298F / 3 52T, 70K / 154Q / 297L / 352G / 355K, 70K / 154Q / 297N / 298F / 348E / 355K, 70K / 191I / 297N / 298F / 352T / 391R,101I / 102A / 122G / 140P / 142V / 316R / 372F / 374L, 101I / 136V / 242T / 372F, 102A / 1 35L / 140P / 316R, 102A / 136A / 140P / 142V / 143C / 316R, 102A / 142V / 316R, 122G / 140 P / 142V / 164H / 242T, 122G / 142V / 316R, 122G / 143C / 242T / 374E, 135L / 136A / 140P / 142V / 143C / 242T / 316R / 372F / 374L, 135L / 140P / 143C / 242T / 374L / 395K, 135L / 1 40P / 316R, 136V / 242T, 142V / 316R, 142V / 316R / 372F / 374E, 142V / 316R / 374L, 14 3C / 316R, 154Q / 191I / 297L / 298F, 154Q / 191I / 297L / 298F / 301R / 352T / 355K, 154Q / 191I / 297N / 298F, 154Q / 191I / 297N / 298F / 352G, 154Q / 191I / 298F / 301R / 348E / 352T / 391R, 154Q / 191I / 298F / 348E / 352T and 154Q / 297L / 298F, wherein the amino acid positions of the polypeptide sequences are numbered with reference to SEQ ID NO:12. In some additional embodiments, the engineered acetate kinase comprises at least one substitution or set of substitutions selected from the group consisting of: 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 / A154Q / T191I / I297N / L298F / A301R / I348E / D352T / V391R. 01R / I348E / D352T / S355K / V391R, V15N / T191I / L298F / D352T / S355K, V15N / I297L / L298F / S355K, I23H / V101I / V102A / I122G / I140P / A14 3C / 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、A154Q / T191I / L298F / A301R / I348E / D352T / V391R、A154Q / T191I / L298F / I348E / D352T 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 listed in Table 3-1. In some additional 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.

[0014] In some embodiments, the present invention provides an engineered acetate kinase, comprising a polypeptide sequence or a 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 a 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, and wherein the engineered acetate kinase comprises at least one substitution or set of substitutions at one or more positions selected from the group consisting of: 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. 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.

[0015] 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 listed in the even-numbered sequences in SEQ ID NO: 2-696. In some further embodiments, the engineered acetate kinase comprises a polypeptide sequence listed in the even-numbered sequences in SEQ ID NO: 2-696. In some further embodiments, the engineered acetate kinase comprises at least one improved property compared to the wild-type Thermotoga maritima acetate kinase. In yet other embodiments, the improved properties include improved activity on substrates compared to the wild-type acetate kinase. In some embodiments, the substrates include nucleoside triphosphates and acetate and / or acetyl phosphate and nucleoside diphosphates. In some other embodiments, substrates include adenosine diphosphate (ADP) and acetyl phosphate and / or adenosine triphosphate (ATP) and acetic acid. In some other embodiments, the improved properties include improved adenosine triphosphate (ATP) production compared to wild-type acetate kinase. In some other embodiments, 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 a kind of engineered acetate kinase provided herein.

[0016] 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 with 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 additional embodiments, the polynucleotide sequence encoding at least one engineered acetate kinase or its functional fragment comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 1. In some other embodiments, the polynucleotide sequence encoding at least one engineered acetate kinase or its functional fragment comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 11. In some other embodiments, the polynucleotide sequence encoding at least one engineered acetate kinase or its functional fragment comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 599. In some other embodiments, the polynucleotide sequence is operably linked to a control sequence. In some embodiments, the polynucleotide sequence is codon optimized. In some other embodiments, the polynucleotide comprises an odd-numbered sequence in SEQ ID NO: 1-695. The present invention also provides an expression vector comprising at least one polynucleotide sequence encoding acetate kinase provided herein. In some embodiments, the expression vector comprises a polynucleotide sequence encoding 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.

[0017] The present invention also provides a method for producing engineered acetate kinase in a host cell, the method comprising culturing a host cell provided herein under suitable conditions, thereby producing at least one engineered acetate kinase. In some embodiments, the method further comprises recovering at least one engineered acetate kinase from the culture and / or host cell. In some other embodiments, the method further comprises the step of purifying at least one engineered acetate kinase.

[0018] Description of the invention

[0019] The present invention provides engineered acetate kinase (AcK), polypeptides with AcK activity and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. Also provided are methods for producing AcK enzymes. The present invention also provides compositions comprising AcK enzymes, and methods for using engineered AcK enzymes. The present invention is particularly useful for the production of pharmaceutical compounds.

[0020] Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as those of ordinary skill in the art to which the present invention belongs. Generally, the nomenclature used herein and the experimental procedures in cell culture, molecular genetics, microbiology, organic chemistry, analytical chemistry and nucleic acid chemistry described below are those well known in the art and generally adopted. Such technology is well known and described in many textbooks and reference works well known to those skilled in the art. Standard techniques or their modified forms 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 herein by reference.

[0021] Although any suitable methods and materials similar or equivalent to those 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 methods, protocols and reagents described, as these can be changed according to the circumstances in which they are used by those skilled in the art. Therefore, the terms that will be defined below are more fully described by reference to the present invention as a whole.

[0022] It should be understood that the general description above and the detailed description below are only exemplary and illustrative, rather than limiting the present invention. The section titles used herein are only for organizational purposes and are not to be construed as limiting the subject matter described. Numerical ranges include numbers that limit the range. Therefore, each numerical range disclosed herein is intended to include each narrower numerical range that falls within such a wider numerical range, as such a narrower numerical range is clearly written out in full herein. It is also intended that each maximum (or minimum) numerical limit disclosed herein includes each lower (or higher) numerical limit, as such lower (or higher) numerical limits are clearly written out herein.

[0023] abbreviation

[0024] Abbreviations for the genetically encoded amino acids are conventional and are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamic acid (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).

[0025] When a three-letter abbreviation is used, unless specifically preceded by "L" or "D" or clear from the context in which the abbreviation is used, the amino acid may be about the α-carbon (C α ) is an amino acid in the L-configuration or D-configuration about the α-carbon. For example, "Ala" represents alanine without specifying the configuration about the α-carbon, while "D-Ala" and "L-Ala" represent D-alanine and L-alanine, respectively. When single-letter abbreviations are used, capital letters represent amino acids in the L-configuration about the α-carbon, and lowercase letters represent amino acids in the D-configuration about the α-carbon. For example, "A" represents L-alanine and "a" represents D-alanine. When a polypeptide sequence is presented as a string of single-letter or three-letter abbreviations (or a mixture thereof), the sequence is presented as amino (N) to carboxyl (C) direction according to common conventions.

[0026] Abbreviations for genetically encoded nucleosides are conventional and are as follows: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). Unless specifically described, abbreviated nucleosides can be ribonucleosides or 2'-deoxyribonucleosides. Nucleosides can be designated as ribonucleosides or 2'-deoxyribonucleosides on an individual or overall basis. When a nucleic acid sequence is presented as a single-letter abbreviation string, the sequence is presented in a 5' to 3' direction according to conventional practice, and the phosphate is not shown.

[0027] definition

[0028] With reference to the present invention, technical and scientific terms used in the description herein shall have the meanings commonly understood by one of ordinary skill in the art, unless specifically defined otherwise. Accordingly, the following terms are intended to have the following meanings.

[0029] As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polypeptide" includes more than one polypeptide.

[0030] Similarly, "comprise, comprises, comprising," "include, includes, and including" are interchangeable and are not intended to be 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).

[0031] It should also be understood that where the term "comprising" is used in the description of various embodiments, those skilled in the art will understand that in some specific instances, the embodiments may alternatively be described using the language "consisting essentially of" or "consisting of."

[0032] As used herein, the term "about" means an acceptable error for a particular value. In some instances, "about" means within 0.05%, 0.5%, 1.0%, or 2.0% of a given value. In some instances, "about" means within 1, 2, 3, or 4 standard deviations of a given value.

[0033] As used herein, "EC" numbers refer 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 that they catalyze.

[0034] As used herein, "ATCC" refers to the American Type Culture Collection, whose biological deposit collections include genes and strains.

[0035] As used herein, "NCBI" refers to the National Center for Biological Information and the sequence databases provided therein.

[0036] As used herein, "acetate kinase" and "AcK" refer to an enzyme that mediates the reversible interconversion of nucleoside triphosphates (e.g., ATP) and acetate to acetyl phosphate and nucleoside diphosphates (e.g., ADP) (EC 2.7.2.1), or a variant enzyme derived from such an AcK enzyme, regardless of whether such variant enzyme retains the same function as the source (i.e., "parent") enzyme.

[0037] "Protein," "polypeptide," and "peptide" are used interchangeably herein to refer to a polymer of at least two amino acids covalently linked by amide bonds, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). Included within this definition are D- and L-amino acids, as well as mixtures of D- and L-amino acids, and polymers comprising mixtures of D- and L-amino acids and D- and L-amino acids.

[0038] "Amino acids" are referred to herein by either their commonly known three letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Likewise, nucleotides may be referred to by their commonly accepted single-letter codes.

[0039] As used herein, "hydrophilic amino acids or residues" refer to amino acids or residues having side chains that exhibit a hydrophobicity 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).

[0040] As used herein, "acidic amino acids or residues" refer to hydrophilic amino acids or residues having side chains that exhibit a pKa value of less than about 6 when the amino acid is contained in a peptide or polypeptide. Acidic amino acids typically have negatively charged side chains at physiological pH due to the loss of hydrogen ions. Genetically encoded acidic amino acids include L-Glu (E) and L-Asp (D).

[0041] 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 contained in a peptide or polypeptide. Basic amino acids generally have positively charged side chains at physiological pH due to association with hydronium ions. Genetically encoded basic amino acids include L-Arg (R) and L-Lys (K).

[0042] 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 a pair of electrons shared by two atoms is held more closely by one of the atoms. Genetically encoded polar amino acids include L-Asn (N), L-Gln (Q), L-Ser (S), and L-Thr (T).

[0043] As used herein, "hydrophobic amino acids or residues" refer to amino acids or residues having side chains that exhibit a hydrophobicity greater than zero according to the standardized 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).

[0044] As used herein, "aromatic amino acid or residue" refers to a hydrophilic or hydrophobic amino acid or residue having a side chain that includes at least one aromatic or heteroaromatic ring. Genetically encoded aromatic amino acids include L-Phe (F), L-Tyr (Y), and L-Trp (W). Although L-His (H) is sometimes classified as a basic residue due to the pKa of its heteroaromatic nitrogen atom, or as an aromatic residue because its side chain includes a heteroaromatic ring, histidine is classified as a hydrophilic residue or as a "constrained residue" (see below) herein.

[0045] As used herein, "constrained amino acid or residue" refers to an amino acid or residue with constrained geometry. 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 has a constrained geometry because it also has a five-membered ring.

[0046] As used herein, "non-polar 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 an electron pair shared by two atoms is usually equally held by each of the two atoms (i.e., the side chain is not polar). Genetically encoded non-polar amino acids include L-Gly (G), L-Leu (L), L-Val (V), L-Ile (I), L-Met (M), and L-Ala (A).

[0047] As used herein, "aliphatic amino acid or residue" refers to a hydrophobic amino acid or residue with an aliphatic hydrocarbon side chain. Genetically encoded aliphatic amino acids include L-Ala (A), L-Val (V), L-Leu (L) and L-Ile (I). It is worth noting that cysteine ​​(or "L-Cys" or "[C]") is unusual because it can form disulfide bridges with other L-Cys (C) amino acids or other sulfonyl or sulfhydryl-containing amino acids. "Cysteine-like residues" include cysteine ​​and other amino acids containing sulfhydryl moieties that can be used to form disulfide bridges. The ability of L-Cys (C) (and other amino acids with -SH side chains) to be present in a peptide in a reduced free-SH or oxidized disulfide-bridged form affects whether L-Cys (C) contributes a net hydrophobic or hydrophilic character to the peptide. Although L-Cys(C) exhibits a hydrophobicity of 0.29 according to Eisenberg's normalized consensus scale (Eisenberg et al., 1984, supra), it should be understood that for the purposes of the present disclosure, L-Cys(C) is classified into its own unique group.

[0048] As used herein, "small amino acid or residue" refers to an amino acid or residue having a side chain comprising a total of three or fewer carbons and / or heteroatoms (excluding α-carbon and hydrogen). According to the above definition, small amino acids or residues can be further classified as aliphatic, non-polar, polar or acidic small amino acids or residues. 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).

[0049] As used herein, "hydroxyl-containing amino acid or residue" refers to an amino acid containing a hydroxyl (-OH) moiety. Genetically encoded hydroxyl-containing amino acids include L-Ser (S), L-Thr (T), and L-Tyr (Y).

[0050] As used herein, "polynucleotide" and "nucleic acid" refer to two or more nucleotides covalently linked together. A polynucleotide may be completely comprised of ribonucleotides (i.e., RNA), completely comprised of 2'deoxyribonucleotides (i.e., DNA), or a mixture of ribonucleotides and 2'deoxyribonucleotides. Although nucleosides are typically linked together via standard phosphodiester linkages, a polynucleotide may include one or more non-standard linkages. A polynucleotide may be single-stranded or double-stranded, or may include both single-stranded and double-stranded regions. In addition, although a polynucleotide typically comprises naturally occurring coding nucleobases (i.e., adenine, guanine, uracil, thymine, and cytosine), it may comprise one or more modified and / or synthesized nucleobases, such as, for example, inosine, xanthine, hypoxanthine, etc. In some embodiments, such modified or synthesized nucleobases are nucleobases encoding amino acid sequences.

[0051] As used herein, "nucleoside" refers to a glycosylamine comprising a nucleoside base (i.e., a nitrogenous base) and a 5-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 nucleoside base, a 5-carbon sugar, and one or more phosphate groups. In some embodiments, nucleosides can be phosphorylated by kinases to produce nucleotides.

[0052] As used herein, "nucleoside diphosphate" refers to a glycosylamine comprising a nucleoside base (i.e., a nitrogenous base), a 5-carbon sugar (e.g., ribose or deoxyribose), and a diphosphate (i.e., pyrophosphate) portion. 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). In some cases, the terms "nucleoside" and "nucleotide" are used interchangeably.

[0053] As used herein, "coding sequence" refers to the portion of a nucleic acid (eg, a gene) that encodes the amino acid sequence of a protein.

[0054] As used herein, the terms "biocatalysis," "biocatalytic," "bioconversion," and "biosynthesis" refer to the use of enzymes to perform chemical reactions on organic compounds.

[0055] As used herein, "wild-type" and "naturally occurring" refer to the form found in nature. For example, a wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a natural source and has not been intentionally modified by human manipulation.

[0056] As used herein, "recombinant," "engineered," "variant," and "non-naturally occurring" when used with respect to cells, nucleic acids, or polypeptides refer to materials that have been modified in a manner that does not otherwise exist in nature or to materials that correspond to the native 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 from synthetic materials and / or through manipulation using recombinant techniques. Non-limiting examples include, among others, recombinant cells that express genes not found in the natural (non-recombinant) form of the cell or that express natural genes that are otherwise expressed at different levels.

[0057] The term "percentage (%) of sequence identity" is used herein to refer to the comparison between polynucleotides or polypeptides, and is determined by comparing two optimally aligned sequences in a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may include additions or deletions (i.e., gaps) compared to the reference sequence for optimal alignment of the two sequences. The percentage can be calculated as follows: determine the number of positions where the same nucleic acid base or amino acid residue appears in the two sequences to produce the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the percentage of sequence identity. Alternatively, the percentage can be calculated as follows: determine the number of positions where the same nucleic acid base or amino acid residue appears in the two sequences or where the nucleic acid base or amino acid residue is aligned with a gap to produce the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the percentage of sequence identity. Those skilled in the art will appreciate that there are many established algorithms that can be used to align 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] ), by 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 is known in the art. Examples of algorithms suitable for determining percent sequence identity and sequence similarity include, but are not limited to, BLAST and BLAST 2.0 algorithms, described by Altschul et al. (see, respectively, Altschul et al., J. Mol. Biol., 215:403-410

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

[1977] ). Software for performing BLAST analysis is available to the public through the website of the National Center for Biotechnology Information. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or satisfy a positive threshold score T when aligned with a word of the same length in the database sequence. 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 to the point where the cumulative alignment score cannot be increased. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for matching residue pairs; always >0) and N (penalty score for mismatched 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 stopped when: the cumulative alignment score drops by an amount X from its maximum achieved value; the cumulative score reaches 0 or less than 0 due to the accumulation of one or more negative scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses the following as defaults: word length (W) of 11, expectation (E) of 10, M=5, N=-4, and comparison of both chains. 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 determinations of sequence alignments and % sequence identity can be made using the BESTFIT or GAP programs in the GCG Wisconsin software package (Accelrys, Madison WI) using the default parameters provided.

[0058] As used herein, "reference sequence" refers to a determined sequence used as a basis for sequence and / or activity comparison. A reference sequence can be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. Typically, a reference sequence is a length of at least 20 nucleotides or amino acid residues, a length of at least 25 residues, a length of at least 50 residues, a length of at least 100 residues, or the total length of a nucleic acid or polypeptide. Because two polynucleotides or polypeptides can each (1) include a similar sequence (i.e., a part of a complete sequence) between the two sequences, and (2) can also include a different (divergent) sequence between the two sequences, a sequence comparison between two (or more) polynucleotides or polypeptides is typically performed by comparing the sequences of the two polynucleotides or polypeptides in a "comparison window" to identify and compare the sequence similarity of a local region. In some embodiments, a "reference sequence" can be based on a primary amino acid sequence, wherein a reference sequence is a sequence that can have one or more variations in a primary sequence.

[0059] As used herein, "comparison window" refers to a conceptual segment of at least about 20 consecutive nucleotide positions or amino acid residues, wherein a sequence can be compared with a reference sequence of at least 20 consecutive nucleotides or amino acids, and wherein the portion of the sequence in the comparison window can include 20% or less additions or deletions (i.e., gaps) compared to the reference sequence (which does not include additions or deletions) for optimal alignment of the two sequences. The comparison window can be longer than 20 consecutive residues, and optionally includes a window of 30, 40, 50, 100 or more.

[0060] As used herein, "corresponding to," "referenced to," or "relative to," when used in the context of numbering a given amino acid or polynucleotide sequence, means that the residues of a given reference sequence are numbered when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue numbers or residue positions of a given polymer are specified with respect to a reference sequence, rather than being specified by the actual numerical positions of the residues within the given amino acid or polynucleotide sequence. For example, given an amino acid sequence, such as an engineered acetate kinase, residue matching between the two sequences can be optimized by introducing spaces to align with the reference sequence. In these cases, the residues in a given amino acid or polynucleotide sequence are numbered with respect to the reference sequence to which it is aligned, despite the presence of spaces.

[0061] As used herein, "substantial identity" refers to a polynucleotide or polypeptide sequence having at least 80% sequence identity, at least 85% identity, at least 89% to 95% sequence identity, or more generally at least 99% sequence identity in a comparison window of at least 20 residue positions, usually in a window of at least 30-50 residues, compared to a reference sequence, wherein the percentage of sequence identity is calculated by comparing the reference sequence in the comparison window with a sequence comprising a deletion or addition totaling 20% ​​or less of the reference sequence. In some specific embodiments applied to polypeptides, the term "substantial identity" means that when optimally aligned, such as by the program GAP or BESTFIT using default gap weights, two polypeptide sequences have at least 80% sequence identity, preferably at least 89% sequence identity, at least 95% sequence identity or more (e.g., 99% sequence identity). In some embodiments, residue positions that are not identical in the compared sequences differ due to conservative amino acid substitutions.

[0062] As used herein, "amino acid difference" and "residue difference" refer to the difference of the amino acid residue at one position of the polypeptide sequence relative to the amino acid residue at the corresponding position in the reference sequence. In some cases, the reference sequence has a histidine tag, but the numbering remains unchanged relative to the equivalent reference sequence without a histidine tag. The position of the amino acid difference is generally referred to as "Xn" herein, where n refers to the corresponding position in the reference sequence on which the residue difference is based. For example, "the residue difference at position X93 compared to SEQ ID NO:4" refers to the difference of the amino acid residue at the polypeptide position corresponding to position 93 of SEQ ID NO:4. Therefore, if the reference polypeptide of SEQ ID NO:4 has serine at position 93, "the residue difference at position X93 compared to SEQ ID NO:4" refers to the amino acid substitution of any residue except serine at the polypeptide position corresponding to position 93 of SEQ ID NO:4. In most examples herein, the specific amino acid residue difference at one position is indicated as "XnY", where "Xn" specifies the corresponding position as described above, and "Y" is the single letter identifier of the amino acid found in the engineered polypeptide (i.e., the residue different from the reference polypeptide). In some examples (e.g., in the tables presented in the Examples), the present invention also provides specific amino acid differences represented by the conventional symbol "AnB", where A is a single-letter identifier for a residue in a reference sequence, "n" is the number of a residue position in a reference sequence, and B is a single-letter identifier for a residue substitution in the sequence of an engineered polypeptide. In some examples, a polypeptide of the present invention may comprise one or more amino acid residue differences relative to a reference sequence, indicated by a column of designated positions where residue differences exist relative to a reference sequence. In some embodiments, when more than one amino acid can be used in a specific residue position of a polypeptide, the various amino acid residues that can be used are separated by " / " (e.g., X307H / X307P or X307H / P). Slashes can also be used to indicate more than one substitution within a given variant (i.e., there is more than one substitution in a given sequence such as in a combinatorial variant). In some embodiments, the present invention includes engineered polypeptide sequences containing one or more amino acid differences, the amino acid differences comprising conservative amino acid substitutions or non-conservative amino acid substitutions. In some additional embodiments, the present invention provides engineered polypeptide sequences comprising both conservative amino acid substitutions and non-conservative amino acid substitutions.

[0063] As used herein, "conservative amino acid substitutions" refer to the replacement of a residue with a different residue having a similar side chain, and thus generally include the replacement of an amino acid in a polypeptide with an amino acid in the same or similar amino acid defined class. For example, but not limited to, in some embodiments, an amino acid with an aliphatic side chain is replaced by another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid with a hydroxyl side chain is replaced by another amino acid with a hydroxyl side chain (e.g., serine and threonine); an amino acid with an aromatic side chain is replaced by another amino acid with an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain is replaced by another amino acid with a basic side chain (e.g., lysine and arginine); an amino acid with an acidic side chain is replaced by another amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid); and / or a hydrophobic amino acid or a hydrophilic amino acid is replaced by another hydrophobic amino acid or a hydrophilic amino acid, respectively.

[0064] As used herein, "non-conservative substitution" refers to the substitution 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, rather than within, the defined groups and affect: (a) the structure of the peptide backbone in the area of ​​the substitution (e.g., proline for glycine), (b) charge or hydrophobicity, or (c) side chain bulk. For example, but not limited to, exemplary non-conservative substitutions may be substitutions of acidic amino acids with basic or aliphatic amino acids; substitutions of aromatic amino acids with small amino acids; and substitutions of hydrophilic amino acids with hydrophobic amino acids.

[0065] As used herein, "deletion" refers to the modification of a polypeptide by removing one or more amino acids from a reference polypeptide. Deletion can include removing 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total amino acids that make up the reference enzyme, or up to 20% of the total amino acids, while retaining enzymatic activity and / or retaining the improved properties of engineered acetate kinase. Deletion can involve the internal portion and / or terminal portion of a polypeptide. In various embodiments, deletion can include continuous segments or can be discontinuous. Deletion in an amino acid sequence is usually represented by "-".

[0066] As used herein, "insertion" refers to the modification of a polypeptide by adding one or more amino acids to a reference polypeptide. The insertion may be in an internal portion of a polypeptide or may be an insertion into the carboxyl or amino terminus. Insertions as used herein include fusion proteins as known in the art. Insertions may be continuous stretches of amino acids or separated by one or more amino acids in a naturally occurring polypeptide.

[0067] The term "amino acid substitution set" or "substitution set" refers to a set of amino acid substitutions in a polypeptide sequence compared 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 listed in the tables provided in the Examples.

[0068] "Functional fragment" and "biologically active fragment" are used interchangeably herein and refer to polypeptides having an amino-terminal deletion and / or a carboxyl-terminal deletion and / or an internal deletion, but wherein the remaining amino acid sequence is identical to the corresponding positions in the sequence to which it is compared (e.g., the full-length engineered acetate kinase of the present invention) and retains substantially all of the activity of the full-length polypeptide.

[0069] As used herein, "isolated polypeptide" refers to a polypeptide that is substantially separated from other contaminants (e.g., proteins, lipids, and polynucleotides) that are naturally associated with it. The term includes 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). The recombinant acetate kinase polypeptide can be present in a cell, in a cell culture medium, or prepared in various forms (such as a lysate or an isolated preparation). Therefore, in some embodiments, the recombinant acetate kinase polypeptide can be an isolated polypeptide.

[0070] 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., on a molar or weight basis, it is more abundant than any other individual macromolecular species in the composition), and when the target species constitutes at least about 50% of the macromolecular species present by mole or % weight, it is generally a substantially purified composition. However, in some embodiments, the composition comprising acetate kinase comprises less than 50% pure (e.g., about 10%, about 20%, about 30%, about 40%, or about 50%) acetate kinase. Typically, a substantially pure acetate kinase composition constitutes 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 present in the composition by mole or % weight. In some embodiments, the target species is purified to substantial homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods), wherein the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ionic species are not considered macromolecular species.In some embodiments, the isolated recombinant acetate kinase polypeptide is a substantially pure polypeptide composition.

[0071] As used herein, "improved enzyme properties" refers to at least one improved property of an enzyme. In some embodiments, the invention provides engineered acetate kinase polypeptides that show improvements in any enzyme property compared to a reference acetate kinase polypeptide and / or a wild-type acetate kinase polypeptide and / or another engineered acetate kinase polypeptide. Therefore, the level of "improvement" can be determined and compared between 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 enzyme 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 tolerance to proteolytic activity (i.e., reduced sensitivity to proteolysis), reduced aggregation, increased solubility, and a changed temperature profile. In other embodiments, the term is used to refer to at least one improved property of acetate kinase. In some embodiments, the invention provides engineered acetate kinase polypeptides that exhibit improvements in any enzymatic property compared to a reference acetate kinase polypeptide and / or a wild-type acetate kinase polypeptide and / or another engineered acetate kinase polypeptide. Thus, the level of "improvement" can be determined and compared between various acetate kinase polypeptides, including wild-type and engineered acetate kinases.

[0072] As used herein, "increased enzyme activity" and "enhanced catalytic activity" refer to the improved properties of engineered polypeptides, which can be expressed as an increase in specific activity (e.g., product produced / time / weight protein) or an increase in the conversion percentage of substrate to product (e.g., using a specified amount of enzyme, the conversion percentage of the starting amount of substrate to product within a specified time period) compared to a reference enzyme. In some embodiments, these terms refer to the improved properties of the engineered acetate kinase polypeptides provided herein, which can be expressed by an increase in specific activity (e.g., product produced / time / weight protein) or an increase in the conversion percentage of substrate to product (e.g., using a specified amount of acetate kinase, the conversion percentage of the starting amount of substrate to product within a specified time period) compared to a reference acetate kinase. In some embodiments, these terms are used to refer to the improved acetate kinases provided herein. Exemplary methods for determining the enzymatic activity of the engineered acetate kinases of the present invention are provided in the Examples. Any property associated with enzyme activity can be affected, including typical enzyme properties K m 、V max or k cat, their alterations can result in increased enzymatic activity. For example, the improvement in enzymatic activity can be from about 1.1 times the enzymatic activity of the corresponding wild-type enzyme to up to 2 times, 5 times, 10 times, 20 times, 25 times, 50 times, 75 times, 100 times, 150 times, 200 times or more enzymatic activity compared to another engineered acetate kinase from which the naturally occurring acetate kinase or acetate kinase polypeptide is derived.

[0073] As used herein, "conversion" refers to the enzymatic conversion (or bioconversion) of one or more substrates into one or more corresponding products. "Percent conversion" refers to the percentage of substrates that are converted into products under specified conditions within a certain period of time. Therefore, the "enzyme activity" or "activity" of an acetate kinase polypeptide can be expressed as the "percent conversion" of substrates into products within a specific period of time.

[0074] An enzyme with "generalist properties" (or "generalist enzymes") refers to an enzyme that exhibits improved activity on a wide range of substrates compared to the parent sequence. A generalist enzyme need not exhibit improved activity for every possible substrate. In some embodiments, the invention provides acetate kinase variants with generalist properties because they exhibit similar or improved activity on a wide range of spatially and electronically different substrates relative to the parent gene. In addition, the generalist enzymes provided herein are engineered to be improved across a wide range of differentiated molecules to increase the production of metabolites / products.

[0075] The term "stringent hybridization conditions" is used herein to refer to conditions under which nucleic acid hybrids are stable. As known to those skilled in the art, the stability of the hybrid is reflected in the melting temperature (T m ). In general, the stability of the hybrid is a function of ionic strength, temperature, G / C content, and the presence of a chaotropic agent. mValues ​​can be calculated using known methods for predicting melting temperatures (see, 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] (erratum, Nucl. Acids Res., 26:6413 ... 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, such as moderately stringent or highly stringent conditions, to the complement of a sequence encoding an engineered acetate kinase of the invention.

[0076] As used herein, "hybridization stringency" refers to hybridization conditions, such as washing conditions, in nucleic acid hybridization. Typically, hybridization reactions are performed under conditions of lower stringency, followed by washings of different but higher stringency. The term "moderate stringency hybridization" refers to conditions that allow target DNA to bind to complementary nucleic acids that have about 60% identity, preferably about 75% identity, about 85% identity, and greater than about 90% identity with the target polynucleotide. Exemplary moderate stringency conditions are conditions equivalent to hybridization at 42°C in 50% formamide, 5×Denhart solution, 5×SSPE, 0.2% SDS, followed by washing at 42°C in 0.2×SSPE, 0.2% SDS. "High stringency hybridization" generally refers to conditions with a thermal melting temperature T as determined for a defined polynucleotide sequence under solution conditions. mConditions that differ by about 10°C or less. In some embodiments, high stringency conditions refer to conditions that allow only hybridization of those nucleic acid sequences that form stable hybrids in 0.018M NaCl at 65°C (i.e., if the hybrid is unstable in 0.018M NaCl at 65°C, it is unstable under high stringency conditions as contemplated herein). High stringency conditions can be provided, for example, by hybridizing under conditions equivalent to 50% formamide, 5×Denhart solution, 5×SSPE, 0.2%SDS at 42°C, followed by washing in 0.1×SSPE and 0.1%SDS at 65°C. Another high stringency condition is hybridization under conditions equivalent to hybridization at 65°C in 5X SSC containing 0.1% (w / v) SDS and washing at 65°C in 0.1×SSC containing 0.1% SDS. Other high stringency hybridization conditions as well as medium stringency conditions are described in the references cited above.

[0077] As used herein, "codon optimized" refers to that the codons of the polynucleotides encoding proteins are changed to those codons preferentially used in a particular organism so that the encoded protein is effectively expressed in the organism of interest. Although the genetic code is degenerate, i.e., most amino acids are represented by several codons referred to as "synonyms" or "synonymous" codons, it is well known that the codon usage of a particular organism is non-random and biased for specific codon triplets. With respect to the aggregated protein coding regions of a given gene, a gene with a common function or ancestral origin, a highly expressed protein versus a low copy number protein and an organism's genome, this codon usage bias may be higher. In some embodiments, the polynucleotides encoding acetate kinase may be codon optimized for optimized production in a host organism selected for expression.

[0078] As used herein, "preferred," "optimal," and "high codon usage bias" codons, when used alone or in combination, refer interchangeably to codons in a protein coding region that are used at a higher frequency than other codons encoding the same amino acid. Preferred codons can be determined based on codon usage in a single gene, a group of genes of a common function or origin, highly expressed genes, codon frequency in aggregating protein coding regions of an entire organism, codon frequency in aggregating protein coding regions of related organisms, or a combination thereof. Codons whose frequency increases with the level of gene expression are generally the optimal codons for expression. Various methods are known for determining codon frequency (e.g., codon usage, relative synonymous codon usage) and codon bias in a particular organism, as well as the effective number of codons used in a gene, including multivariate analysis, such as using cluster analysis or correlation analysis (see, e.g., GCG Codon Preference, Genetics Computer Group Wisconsin Package; Codon W, 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 for many different organisms are available (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 Escherichiacoli and Salmonella, Neidhardt et al. (eds.), ASM Press, Washington DC, p. 2047-2066

[1996] ). The data source for obtaining codon usage can rely on any available nucleotide sequence capable of encoding a protein.These data sets 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] ).

[0079] As used herein, "control sequences" include all components that are necessary or advantageous for the expression of the 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, leader sequences, polyadenylation sequences, propeptide sequences, promoter sequences, signal peptide sequences, start sequences, and transcription terminators. At a minimum, control sequences include promoters and transcription and translation termination signals. Control sequences may be provided with joints for the purpose of introducing specific restriction sites that facilitate connection of the control sequences to the coding region of the nucleic acid sequence encoding the polypeptide.

[0080] "Operably linked" is defined herein as a configuration in which a control sequence is appropriately placed (i.e., in a functional relationship) at a position relative to a polynucleotide of interest such that the control sequence directs or regulates expression of the polynucleotide and / or polypeptide of interest.

[0081] "Promoter sequence" refers to a nucleic acid sequence that is recognized by a host cell for expression of a polynucleotide of interest, such as a coding sequence. The promoter sequence comprises a transcriptional control sequence that mediates the expression of the polynucleotide of interest. The promoter can be any nucleic acid sequence that exhibits transcriptional activity in a selected host cell, including mutants, truncated and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell.

[0082] The phrase "suitable reaction conditions" refers to those conditions in the enzymatic conversion reaction solution (e.g., ranges of enzyme loading, substrate loading, temperature, pH, buffer, cosolvents, etc.) under which the acetate kinase polypeptide of the invention is able to convert a substrate into a desired product compound. Some exemplary "suitable reaction conditions" are provided herein.

[0083] As used herein, "loading," such as in "compound loading" or "enzyme loading," refers to the concentration or amount of a component in the reaction mixture at the start of the reaction.

[0084] As used herein, in the context of an enzymatic conversion reaction process, "substrate" refers to a compound or molecule that is acted upon by an engineered enzyme (eg, an engineered acetate kinase polypeptide) provided herein.

[0085] As used herein, an "increased" yield of a product (e.g., a deoxyribose phosphate analog) produced by a reaction occurs when the presence of a particular component (e.g., acetate kinase) during the reaction results in the production of more product compared to a reaction conducted under the same conditions with the same substrate and other substituents, but in the absence of the component of interest.

[0086] A reaction is said to be "substantially free" of a particular enzyme if the amount of that enzyme is less than about 2%, about 1%, or about 0.1% (wt / wt) compared to other enzymes involved in catalyzing the reaction.

[0087] As used herein, "fractionating" a liquid (e.g., a culture broth) means applying a separation process (e.g., salt precipitation, column chromatography, size exclusion, and filtration), or a combination of such processes, to provide a solution in which the desired protein is present as a percentage of the total protein in the solution that is greater than the percentage in the initial liquid product.

[0088] As used herein, "starting composition" refers to any composition comprising at least one substrate. In some embodiments, the starting composition comprises any suitable substrate.

[0089] As used herein, in the context of an enzymatic conversion process, "product" refers to the compound or molecule resulting from the action performed by the enzyme polypeptide on the substrate.

[0090] As used herein, "equilibrium" as used herein refers to the process of producing a steady-state concentration of a 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 rate constant and reverse rate constant of the chemical or enzymatic reaction.

[0091] As used herein, "alkyl" refers to a saturated hydrocarbon group having 1 to 18 carbon atoms (inclusive), linear or branched, more preferably 1 to 8 carbon atoms (inclusive), and most preferably 1 to 6 carbon atoms (inclusive). Alkyl groups having a specified number of carbon atoms are indicated in brackets (e.g., (C1-C4) alkyl refers to an alkyl group of 1 to 4 carbon atoms).

[0092] As used herein, "alkenyl" refers to a group having 2 to 12 carbon atoms (inclusive), linear or branched, containing at least one double bond but optionally containing more than one double bond.

[0093] As used herein, "alkynyl" refers to a group having 2 to 12 carbon atoms (inclusive), straight or branched, containing at least one triple bond but optionally containing more than one triple bond, and further optionally containing one or more double bonded moieties.

[0094] 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 by the same or different heteroatoms or heteroatom groups. The heteroatoms and / or heteroatom groups that can replace carbon atoms include, but are not limited to -O-, -S-, -SO-, -NRα-, -PH-, -S(O)-, -S(O)2, -S(O)NRα-, -S(O)2NRα-, etc., including combinations thereof, wherein each Rα is independently selected from hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl.

[0095] As used herein, "alkoxy" refers to a group -OR[beta], wherein R[beta] is an alkyl group as defined above, including optionally substituted alkyl groups also as defined herein.

[0096] As used herein, "aryl" refers to an unsaturated aromatic carbocyclic group having 6 to 12 carbon atoms (including endpoints) with a single ring (e.g., phenyl) or more than one condensed ring (e.g., naphthyl or anthracenyl). Exemplary aryl groups include phenyl, pyridyl, naphthyl, etc.

[0097] As used herein, "amino" refers to the group -NH2. Substituted amino refers to the group -NHRδ, NRδRδ and NRδRδRδ, where each Rδ is independently selected from substituted or unsubstituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy, aryl, heteroaryl, heteroarylalkyl, acyl, alkoxycarbonyl, sulfanyl, sulfinyl, sulfonyl, etc. Typical amino groups include, but are not limited to, dimethylamino, diethylamino, trimethylammonium, triethylammonium, methylsulfonylamino, furanyl-oxy-sulfonylamino, etc.

[0098] As used herein, "oxo" refers to =0.

[0099] As used herein, "oxy" refers to a divalent group -O-, which may have various substituents to form different oxy groups, including ethers and esters.

[0100] As used herein, "carboxyl" refers to -COOH.

[0101] As used herein, "carbonyl" refers to -C(O)-, which may have various substituents to form different carbonyl groups including acids, acyl halides, aldehydes, amides, esters, and ketones.

[0102] As used herein, "alkoxycarbonyl" refers to -C(O)ORε, wherein Rε is alkyl as defined herein, which may be optionally substituted.

[0103] As used herein, "aminocarbonyl" refers to -C(O)NH2. Substituted aminocarbonyl refers to -C(O)NRδRδ, wherein the amino group NRδRδ is as defined herein.

[0104] As used herein, "halogen" and "halo" refer to fluorine, chlorine, bromine and iodine.

[0105] As used herein, "hydroxy" refers to -OH.

[0106] As used herein, "cyano" refers to -CN.

[0107] As used herein, "heteroaryl" refers to an aromatic heterocyclic group having 1 to 10 carbon atoms (inclusive) and 1 to 4 heteroatoms (inclusive) selected from oxygen, nitrogen and sulfur in the ring. Such heteroaryl groups may have a single ring (e.g., pyridyl or furyl) or more than one condensed ring (e.g., indolizinyl or benzothienyl).

[0108] As used herein, "heteroarylalkyl" refers to an alkyl group substituted with a heteroaryl group (i.e., a "heteroaryl-alkyl-" group), preferably having 1 to 6 carbon atoms (inclusive) in the alkyl portion and 5 to 12 ring atoms (inclusive) in the heteroaryl portion. Such heteroarylalkyl groups are exemplified by pyridylmethyl and the like.

[0109] As used herein, "heteroarylalkenyl" refers to an alkenyl group substituted with a heteroaryl group (i.e., a "heteroaryl-alkenyl-" group), preferably having from 2 to 6 carbon atoms (inclusive) in the alkenyl portion and from 5 to 12 ring atoms (inclusive) in the heteroaryl portion.

[0110] As used herein, "heteroarylalkynyl" refers to an alkynyl group substituted with a heteroaryl group (i.e., a "heteroaryl-alkynyl-" group), preferably having from 2 to 6 carbon atoms (inclusive) in the alkynyl portion and from 5 to 12 ring atoms (inclusive) in the heteroaryl portion.

[0111] As used herein, "heterocycle", "heterocyclic" and interchangeably "heterocycloalkyl" refer to saturated or unsaturated groups having a single ring or more than one fused ring, having 2 to 10 carbon ring atoms (inclusive) and 1 to 4 heterocyclic atoms (inclusive) selected from nitrogen, sulfur or oxygen within the ring. Such heterocyclic groups can have a single ring (e.g., piperidinyl or tetrahydrofuranyl) or more than one fused ring (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.

[0112] As used herein, "membered ring" is intended to include any cyclic structure. The number before the term "membered" represents the number of backbone atoms that make up the ring. Thus, for example, cyclohexyl, pyridine, pyran, and thiopyran are 6-membered rings, and cyclopentyl, pyrrole, furan, and thiophene are 5-membered rings.

[0113] Unless otherwise indicated, the positions occupied by hydrogen in the foregoing radicals may be further substituted by substituents such as, but not limited to, hydroxy, oxo, nitro, methoxy, ethoxy, alkoxy, substituted alkoxy, trifluoromethoxy, haloalkoxy, fluorine, chlorine, bromine, iodine, halogen, methyl, ethyl, propyl, butyl, alkyl, alkenyl, alkynyl, substituted alkyl, trifluoromethyl, haloalkyl, hydroxyalkyl, alkoxyalkyl, thio, alkylthio, acyl, carboxyl, alkoxycarbonyl, formamido, substituted formamido, alkylsulfonyl, alkylsulfinyl, alkylsulfonylamino, sulfonamido, substituted sulfonamido, cyano, amino , substituted amino, alkylamino, dialkylamino, aminoalkyl, acylamino, amidino, amidoximo, hydroxamoyl, phenyl, aryl, substituted aryl, aryloxy, arylalkyl, arylalkenyl, arylalkynyl, pyridyl, imidazolyl, heteroaryl, substituted heteroaryl, heteroaryloxy, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, substituted cycloalkyl, cycloalkyloxy, pyrrolidinyl, piperidinyl, morpholino, heterocycle, (heterocycle)oxy and (heterocycle)alkyl; and preferred heteroatoms are oxygen, nitrogen and sulfur. It is understood that where open valences exist on these substituents, they may be further substituted with alkyl, cycloalkyl, aryl, heteroaryl and / or heterocyclic groups, where such open valences exist on carbon, they may be further substituted with halogen and oxygen-, nitrogen- or sulfur-bonded substituents, and where more than one such open valence exists, these groups may be joined to form a ring by direct bond formation or by bond formation with new heteroatoms (preferably oxygen, nitrogen or sulfur). It is also understood that the above substitutions may be made provided that replacement of hydrogen with a substituent does not introduce unacceptable instability into the molecules of the invention and is otherwise chemically reasonable.

[0114] As used herein, the term "culturing" refers to the growth of a population of microbial cells under any suitable conditions (eg, using liquid, gel, or solid culture media).

[0115] Recombinant polypeptides can be produced using any suitable method known in the art. The gene encoding the wild-type polypeptide of interest can be cloned into a vector such as a plasmid and expressed in a desired host such as Escherichia coli. Variants of recombinant polypeptides can be produced by various methods known in the art. In fact, there are various different mutagenesis techniques well known to those skilled in the art. In addition, mutagenesis kits can also be obtained from many commercial molecular biology suppliers. The method can be used to make specific substitutions at a determined amino acid (site-directed), specificity (region-specificity) or random mutations in a local region of a gene, or random mutagenesis (e.g., saturation mutagenesis) in the entire gene. Many suitable methods for producing enzyme variants are known to those skilled in the art, including but not limited to, using PCR to single-stranded DNA or double-stranded DNA site-directed mutagenesis, cassette mutagenesis, gene synthesis, error-prone PCR, reorganization, and chemical saturation mutagenesis, or any other suitable method known in the art. Mutagenesis and directed evolution methods can be easily applied to polynucleotides encoding enzymes to produce variant libraries that can be expressed, screened and measured. Any suitable mutagenesis and directed evolution methods can be used in the present invention and are well known in the art (see, e.g., U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, 5,837,458, 5,928,905, 6,096,548, 6,117,679, 6,132,970, 6,165,793, 6,180,406, 6,251,674, 6,265,201、6,277,638、6,287,861、6,287,862、6,291,242、6,297,053、6,303,344、6,309,883、6,319,713、6,319,714、6,323,030、6,326,204、6,335,160、6,335,198、6,344,356、6,352,859 , 6,355,484, 6,358,740, 6,358,742, 6,365,377, 6,365,408, 6,368,861, 6,372,497, 6,337,186, 6,376,246, 6,379,964, 6,387,702, 6,391,552, 6,391,640, 6,395,547, 6,406,855, 6,406,9 10. 6,413,745, 6,413,774, 6,420,175, 6,423,542, 6,426,224, 6,436,675, 6,444,468, 6,455,253, 6,479,652, 6,482,647, 6,483,011, 6,484,105, 6,489,146, 6,500,617, 6,500,639, 6,506,602、6,506,603、6,518,065、6,519,065、6,521,453、6,528,311、6,537,746、6,573,098、6,576,467、6,579,678、6,586,182、6,602,986、6,605,430、 6,613,514, 6,653,072, 6,686,515, 6,703,240, 6,716,631, 6,825,001, 6,902,922, 6,917,882, 6,946,296, 6,961,664, 6,995,017, 7,024,312, 7,05 8,515、7,105,297、7,148,054、7,220,566、7,288,375、7,384,387、7,421,347、7,430,477、7,462,469、7,534,564、7,620,500、7,620,502、7,629,17 0, 7,702,464, 7,747,391, 7,747,393, 7,751,986, 7,776,598, 7,783,428, 7,795,030, 7,853,410, 7,868,138, 7,783,428, 7,873,477, 7,873,499, 7, 904,249, 7,957,912, 7,981,614, 8,014,961, 8,029,988, 8,048,674, 8,058,001, 8,076,138, 8,108,150, 8,170,806, 8,224,580, 8,377,681, 8,383,346, 8,457,903, 8,504,498, 8,589,085, 8,762,066, 8,768,871, 9,593,326, and all related U.S. and PCT and non-U.S. counterpart applications; 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] ; WO 95 / 22625; WO 97 / 0078; WO 97 / 35966; WO 98 / 27230; WO 00 / 42651; WO 01 / 75767; and WO 2009 / 152336, all of which are incorporated herein by reference).

[0116] In some embodiments, the enzyme clones obtained after mutagenesis are screened by subjecting the enzyme preparation to a determined temperature (or other assay conditions) and measuring the amount of enzyme activity remaining after heat treatment or other suitable assay conditions. The clones containing the polynucleotide encoding the polypeptide are then isolated from the gene, sequenced to identify changes in the nucleotide sequence (if any), and used to express the enzyme in a host cell. Measuring the enzyme activity from the expression library can be performed using any suitable method known in the art (e.g., standard biochemical techniques, such as HPLC analysis).

[0117] After the variants are generated, they can be screened for any desired property (e.g., high or increased activity, or low or decreased activity, increased thermal activity, increased thermal stability, and / or acidic pH stability, etc.). In some embodiments, "recombinant acetate kinase polypeptides" (also referred to herein as "engineered acetate kinase polypeptides," "variant acetate kinases," "acetate kinase variants," and "acetate kinase combination variants") can be used. In some embodiments, "recombinant acetate kinase polypeptides" (also referred to herein as "engineered acetate kinase polypeptides," "variant acetate kinases," "acetate kinase variants," and "acetate kinase combination variants") can be used.

[0118] As used herein, "vector" is a DNA construct for introducing a DNA sequence into a cell. In some embodiments, a vector is an expression vector operably linked to a suitable control sequence capable of achieving expression of a polypeptide encoded in a 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 transgenic) to drive expression in a host cell, and in some embodiments, also comprises a transcription terminator sequence.

[0119] As used herein, the term "expression" includes any step involved in the production of the 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.

[0120] As used herein, the term "production" refers to the production of proteins and / or other compounds from a cell. It is intended that the term encompass any step involved in the production of 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 a polypeptide from a cell.

[0121] As used herein, an amino acid or nucleotide sequence (e.g., a promoter sequence, a signal peptide, a terminator sequence, etc.) is "heterologous" if the two sequences are not associated in nature with another sequence to which it is operably linked. For example, a "heterologous polynucleotide" is any polynucleotide that is introduced into a host cell by laboratory techniques, and includes polynucleotides that are removed from a host cell, subjected to laboratory manipulation, and then reintroduced into a host cell.

[0122] As used herein, the terms "host cell" and "host strain" refer to a suitable host for an expression vector comprising a DNA as 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 as known in the art.

[0123] The term "analog" means a polypeptide having more than 70% sequence identity, but less than 100% sequence identity (e.g., more than 75%, 78%, 80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) with a reference polypeptide. In some embodiments, an analog means a polypeptide comprising one or more non-naturally occurring amino acid residues (including but not limited to homoarginine, ornithine and norvaline) and naturally occurring amino acids. In some embodiments, an analog also includes one or more D-amino acid residues and non-peptide connections between two or more amino acid residues.

[0124] The term "effective amount" means an amount sufficient to produce the desired result. One of ordinary skill in the art can determine what the effective amount is by using routine experimentation.

[0125] The terms "isolated" and "purified" are used to refer to a molecule (e.g., an isolated nucleic acid, polypeptide, etc.) or other component that is removed from at least one other component with which it is naturally associated. The term "purified" does not require absolute purity, but is intended as a relative definition.

[0126] As used herein, "stereoselectivity" refers to the preferential formation of one stereoisomer relative to another stereoisomer in a chemical or enzymatic reaction. Stereoselectivity can be partial, in which case the formation of one stereoisomer is superior to that of another stereoisomer, or stereoselectivity can be complete, in which case only one stereoisomer is formed. When the stereoisomers are enantiomers, stereoselectivity is referred to as enantioselectivity, i.e., the fraction of one enantiomer in the sum of the two (usually reported as a percentage). Alternatively, the art generally reports it as enantiomeric excess ("ee") (usually a percentage) calculated therefrom according to the following formula: [major enantiomer-minor enantiomer] / [major enantiomer+minor enantiomer]. In the case where the stereoisomers are diastereomers, stereoselectivity is referred to as diastereoselectivity, i.e., the fraction of one diastereomer in a mixture of two diastereomers (usually reported as a percentage), which is generally optionally reported as diastereomeric excess ("de"). Enantiomeric excess and diastereomeric excess are types of stereoisomer excess.

[0127] As used herein, the terms "regioselectivity" and "regioselective reaction" refer to reactions in which one direction of bond formation or breaking occurs in preference to all other possible directions. A reaction can be completely (100%) regioselective if the discrimination is complete, substantially regioselective (at least 75%) if the reaction product at one site is preferred over the reaction products at other sites, or partially regioselective (x%, where the percentage is set depending on the reaction of interest).

[0128] As used herein, "chemoselectivity" refers to the preferential formation of one product over another in a chemical or enzymatic reaction.

[0129] As used herein, "pH stable" refers to an acetate kinase polypeptide that maintains similar activity (e.g., more than 60% to 80%) after exposure to high or low pH (e.g., 4.5-6 or 8 to 12) for a period of time (e.g., 0.5-24 hours) compared to the untreated enzyme.

[0130] As used herein, "thermostable" refers to an acetate kinase polypeptide that maintains similar activity (e.g., more than 60% to 80%) after exposure to the same elevated temperature for a period of time (e.g., 0.5h-24h) compared to a wild-type enzyme exposed to the same elevated temperature (e.g., 40°C to 80°C).

[0131] As used herein, "solvent stable" refers to an acetate kinase polypeptide that maintains similar activity (e.g., more than 60% to 80%) after exposure to the same solvent at the same concentration for a period of time (e.g., 0.5h-24h) compared to the wild-type enzyme exposed to different concentrations (e.g., 5%-99%) of the solvent (ethanol, isopropanol, dimethyl sulfoxide [DMSO], tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene, butyl acetate, methyl tert-butyl ether, etc.).

[0132] As used herein, "thermostable and solvent stable" refers to an acetate kinase polypeptide that is both thermostable and solvent stable.

[0133] As used herein, "optional" and "optionally" mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances when the event or circumstance occurs and instances in which the event or circumstance does not occur. One of ordinary skill in the art will understand that for any molecule described as containing one or more optional substituents, only sterically feasible and / or synthetically feasible compounds are intended to be included.

[0134] As used herein, "optionally substituted" refers to all subsequent modifiers in one or a series of chemical groups. For example, in the term "optionally substituted arylalkyl", the "alkyl" part and the "aryl" part of the molecule may be substituted or may not be substituted, and for a series of "optionally substituted alkyl, cycloalkyl, aryl and heteroaryl", the alkyl group, cycloalkyl group, aryl group and heteroaryl group may be substituted or may not be substituted independently of each other. DETAILED DESCRIPTION OF THE INVENTION

[0136] The present invention provides engineered acetate kinase (AcK), polypeptides with AcK activity and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. Also provided are methods for producing AcK enzymes. The present invention also provides compositions comprising AcK enzymes, and methods for using engineered AcK enzymes. The present invention is particularly useful for the production of pharmaceutical compounds.

[0137] In some embodiments, the present invention provides enzymes suitable for recycling ATP from ADP to produce phosphorylated glycerol derivatives and glyceraldehyde derivatives having bulky substituents on the C2 carbon of glycerol, particularly phosphorylated ethynyl-glycerol and ethynyl-glyceraldehyde, which are intermediates in the in vitro enzymatic synthesis of non-natural nucleoside analogs represented by compound (1).

[0138]

[0139] Compound (1)

[0140] The production of phosphorylated glyceraldehyde derivatives such as compound (5) can be difficult. However, the corresponding non-phosphorylated glyceraldehyde derivative (6) can be prepared by oxidation of a glycerol derivative (7) with alcohol oxidase. After the formation of glyceraldehyde, glyceraldehyde can be phosphorylated to the desired intermediate (5) by pantothenate kinase (PanK), as shown in Scheme 1.

[0141]

[0142] Scheme I

[0143] The generation of phosphorylated intermediate (5) utilizes ATP as a phosphate donor. As depicted in Scheme II, the resulting ADP is then recycled to ATP by AcK using acetyl phosphate. The improved AcK enzyme of the present invention has improved activity for recycling ATP from ADP using acetyl phosphate.

[0144]

[0145] Solution II

[0146] In some embodiments, the engineered AcK polypeptide of the present disclosure is part of a multi-enzyme system to produce 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, pentose phosphomutase, purine nucleoside phosphorylase, alcohol oxidase, aldolase, and / or sucrose phosphorylase.

[0147] Engineered AcK peptide

[0148] The invention provides engineered AcK polypeptides, polynucleotides encoding the polypeptides, methods for preparing the polypeptides, and methods for using the polypeptides. When describing a polypeptide, it should be understood that it also describes a polynucleotide encoding the polypeptide. In some embodiments, the invention provides an engineered, non-naturally occurring AcK enzyme with improved properties compared to the wild-type AcK enzyme. Any suitable reaction conditions can be used for the present invention. In some embodiments, methods are used to analyze the improved properties of the engineered polypeptide for phosphorylation reactions. In some embodiments, as further described below and in the examples, reaction conditions are changed according to the concentration or amount, pH, including temperature and reaction time, of engineered AcK, one or more substrates, one or more buffers, cofactors, one or more solvents and / or the conditions of the engineered AcK polypeptide being fixed on a solid support.

[0149] In some embodiments, additional reaction components or additional techniques are utilized to supplement reaction conditions. In some embodiments, these include taking steps to stabilize the enzyme or prevent enzyme inactivation, reduce product inhibition, shift the reaction equilibrium to desired product formation.

[0150] In some other embodiments, any of the above-described methods for converting a substrate compound into a product compound may also include one or more steps selected from the following: extraction, separation, purification, crystallization, filtration and / or lyophilization of one or more product compounds. Methods, techniques and protocols for extracting, separating, purifying and / or crystallizing one or more products from a biocatalytic reaction mixture produced by the methods provided herein are known to those of ordinary skill and / or are obtained by routine experimentation. In addition, illustrative methods are provided in the examples hereinafter.

[0151] Engineered AcK polynucleotides encoding engineered polypeptides, expression vectors and host cells

[0152] The present invention provides polynucleotides encoding 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 produce a recombinant polynucleotide capable of expressing a polypeptide. In some embodiments, an expression construct comprising at least one heterologous polynucleotide encoding one or more engineered enzyme polypeptides is introduced into an appropriate host cell to express one or more corresponding enzyme polypeptides.

[0153] As will be apparent to the technician, the availability of protein sequences and the knowledge of the codons corresponding to various amino acids provide descriptions of all polynucleotides that can encode the subject polypeptide. The degeneracy of the genetic code (wherein the same amino acid is encoded by optional or synonymous codons) allows the preparation of a very large number of nucleic acids, all of which encode engineered enzymes (e.g., AcK) polypeptides. Therefore, the present invention provides methods and compositions for selecting and combining each and every possible variation of the preparable enzyme polynucleotides for producing the enzyme polypeptides described herein based on possible codon options, and all such variations are considered to be specifically disclosed for any polypeptide described herein, including the amino acid sequences presented in the embodiments (e.g., in each table).

[0154] In some embodiments, codons are preferably optimized to be utilized by the selected host cell for protein production. For example, preferred codons used in bacteria are typically used for expression in bacteria. Therefore, the codon-optimized polynucleotides encoding engineered enzyme polypeptides comprise preferred codons at about 40%, 50%, 60%, 70%, 80% or greater than 90% of the codon positions in the full-length coding region.

[0155] In some embodiments, the enzyme polynucleotide encodes an engineered polypeptide having enzymatic activity and 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 with a reference sequence selected from SEQ ID NO provided herein, or an amino acid sequence of any variant (e.g., those 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 one or more reference polynucleotides 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 other embodiments, the reference polypeptide sequence comprises SEQ ID NO: 600.

[0156] In some embodiments, polynucleotide can be selected from any polynucleotide sequence provided herein or its complementary sequence or the polynucleotide sequence of any variant enzyme polypeptide provided herein and hybridize under high stringency conditions.In some embodiments, the polynucleotide encoding that can hybridize under high stringency conditions comprises the enzyme polypeptide that has the amino acid sequence of one or more residue differences compared with the reference sequence.

[0157] In some embodiments, the polynucleotide of any separation in the engineered enzyme polypeptide of encoding this paper is manipulated in various ways to promote the expression of enzyme polypeptide.In some embodiments, the polynucleotide of encoding enzyme polypeptide constitutes expression vector, wherein there is one or more control sequence to regulate the expression of enzyme polynucleotide and / or polypeptide.According to the expression vector used, the manipulation of the polynucleotide of separation before the polynucleotide of separation is inserted into the vector can be desired or necessary.The technology of utilizing recombinant DNA method to modify polynucleotide and nucleotide sequence is well known in the art.In some embodiments, control sequence comprises, among others, promoter, leader sequence, polyadenylation sequence, propeptide sequence, signal peptide sequence and transcription terminator.In some embodiments, suitable promoter is selected based on the selection of host cell. For bacterial host cells, suitable promoters for directing transcription of the nucleic acid constructs of the present disclosure include, but are not limited to, promoters obtained from the Escherichia coli lac operon, the Streptomyces coelicolor agarase gene (dagA), the Bacillus subtilis levansucrase gene (sacB), the Bacillus licheniformis alpha-amylase gene (amyL), the Bacillus stearothermophilus maltogenic amylase gene (amyM), the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), the Bacillus licheniformis penicillinase gene (penP), the Bacillus subtilis xylA and xylB genes, and prokaryotic β-lactamase genes (see, e.g., Villa-Kamaroff et al., Proc. Natl Acad. Sci. USA 75:3727-3731

[1978] ), and 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, but are not limited to, promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, niger acid-stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline proteinase, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum. oxysporum) trypsin-like protease (see, e.g., WO96 / 00787), and the NA2-tpi promoter (a hybrid of the promoters from the Aspergillus niger neutral α-amylase gene and the Aspergillus oryzae triosephosphate isomerase gene), and mutants, truncated, and hybrid promoters thereof. Exemplary yeast cell promoters can be derived from the genes for 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. Other useful promoters for yeast host cells are known in the art (see, e.g., Romanos et al., Yeast 8:423-488

[1992] ).

[0158] In some embodiments, the control sequence is also a suitable transcription terminator sequence (i.e., a sequence recognized by the host cell to terminate transcription). In some embodiments, the terminator sequence is operably connected to the 3' end of the nucleic acid sequence encoding the enzyme polypeptide. Any suitable terminator that is functional in the selected host cell can be used in the present invention. Exemplary transcription terminators for filamentous fungal host cells can be obtained from the following genes: Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger α-glucosidase, and Fusarium oxysporum trypsin-like protease. Exemplary terminators for yeast host cells can be obtained from the following genes: 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, for example, Romanos et al., above).

[0159] In some embodiments, the control sequence is also a suitable leader sequence (i.e., the non-translated region of the 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 selected host cell can be used in the present invention. Exemplary leaders for filamentous fungal host cells are obtained from the following genes: Aspergillus oryzae TAKA amylase and Aspergillus nidulans triosephosphate isomerase. Suitable leaders for yeast host cells are obtained from the following genes: Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae α-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0160] In some embodiments, the control sequence is also a polyadenylation sequence (i.e., a sequence operably linked to the 3' end of the nucleic acid sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to the transcribed mRNA). Any suitable polyadenylation sequence that is functional in the host cell of choice may be used in the present invention. Exemplary polyadenylation sequences for filamentous fungal host cells include, but are not limited to, the following genes: 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] ).

[0161] In some embodiments, the control sequence is also a signal peptide (i.e., a coding region encoding an amino acid sequence that is linked to the amino terminus of a polypeptide and directs the encoded polypeptide to the secretory pathway of the cell). In some embodiments, the 5' end of the coding sequence of the nucleic acid sequence inherently contains a signal peptide coding region that is naturally linked in translation reading frame to a segment of the coding region encoding 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 that directs the expressed polypeptide to the secretory pathway of the selected host cell can be used for expression of one or more engineered polypeptides. Effective signal peptide coding regions for bacterial host cells include, but are not limited to, those obtained from the following genes: Bacillus NClB 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, those obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase. Useful signal peptides for yeast host cells include, but are not limited to, those from the genes for Saccharomyces cerevisiae alpha factor and Saccharomyces cerevisiae invertase.

[0162] In some embodiments, the control sequence is also a propeptide coding region encoding an amino acid sequence positioned at the amino terminus of a polypeptide. The polypeptide produced is referred to as a "proenzyme", "propolypeptide" or "zymogen". The propolypeptide can be converted into a mature active polypeptide by catalyzing or autocatalyzing the cleavage of the propeptide from the propolypeptide. The propeptide coding region can be obtained from any suitable source including but not limited to the following genes: Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae α-factor, Rhizomucor miehei aspartic protease and Myceliophthora thermophila lactase (see, e.g., WO 95 / 33836). When both the signal peptide and propeptide regions are present at the amino terminus of a polypeptide, the propeptide region is positioned adjacent to the amino terminus of the polypeptide and the signal peptide region is positioned adjacent to the amino terminus of the propeptide region.

[0163] In some embodiments, regulatory sequences are also utilized. These sequences promote the regulation of polypeptide expression relative to host cell growth. Examples of regulatory systems are those that cause the expression of genes to be turned 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 lac, tac and trp operator systems. In yeast host cells, suitable regulatory systems include but are not limited to ADH2 systems or GAL1 systems. In filamentous fungi, suitable regulatory sequences include but are not limited to TAKA alpha-amylase promoters, Aspergillus niger glucoamylase promoters and Aspergillus oryzae glucoamylase promoters.

[0164] On the other hand, the present invention relates to a recombinant expression vector comprising a polynucleotide encoding an engineered enzyme polypeptide and one or more expression regulatory regions such as a promoter and terminator, a replication origin, etc., according to the type of host to be introduced. In some embodiments, various nucleic acids and control sequences described herein are linked together to produce a recombinant expression vector comprising one or more convenient restriction sites to allow insertion or replacement of the nucleic acid sequence encoding the enzyme polypeptide at such a site. Alternatively, in some embodiments, the nucleic acid sequence of the present invention is expressed by inserting the nucleic acid sequence or a nucleic acid construct comprising the sequence into a suitable vector for expression. In some embodiments relating to the generation of an expression vector, the coding sequence is located in a vector such that the coding sequence is operably linked to a suitable control sequence for expression.

[0165] The recombinant expression vector can be any suitable vector (e.g., plasmid or virus) that can be easily subjected to recombinant DNA procedures and cause expression of the enzyme polynucleotide sequence. The choice of vector generally depends on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can be a linear plasmid or a closed circular plasmid.

[0166] 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, an extrachromosomal element, a minichromosome, or an artificial chromosome). The vector may include any means for ensuring self-replication. In some selectable embodiments, the vector is a vector that, when introduced into a host cell, is integrated into the genome and replicated with one or more chromosomes into which it is integrated. In addition, in some embodiments, a single vector or plasmid is utilized, or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, and / or a transposon.

[0167] In some embodiments, the expression vector comprises one or more selectable markers that allow easy selection of transformed cells. A "selectable marker" is a gene whose product provides antimicrobial or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, etc. Examples of bacterial selectable markers include, but are not limited to, dal genes from 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. oryzae), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), and equivalents thereof.

[0168] On the other hand, the present invention provides a host cell, the host cell comprising at least one polynucleotide encoding at least one engineered enzyme polypeptide of the present invention, the polynucleotide being operably connected to one or more control sequences for expressing one or more engineered enzymes in the host cell. The host cell suitable for expressing the polypeptide encoded by the expression vector of the present invention is well known in the art, and includes but is not limited to bacterial cells, such as Escherichia coli, Vibrio fluvialis, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (Pichia pastoris) (ATCC accession number 201178)); insect cells, such as Drosophila (Drosophila) S2 and Spodoptera (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 selection markers include, but are not limited to, the dal gene from Bacillus subtilis or Bacillus licheniformis, or markers that confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol and / or tetracycline resistance.

[0169] In some embodiments, the expression vectors of the invention contain elements that allow the vector to be integrated into the genome of the host cell or that allow the vector to replicate autonomously 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 for integration of the vector into the genome by homologous or nonhomologous recombination.

[0170] In some alternative embodiments, the expression vector comprises other nucleotide sequences for guiding integration into the genome of the host cell by homologous recombination. Other nucleotide sequences enable the carrier to be integrated into the host cell genome at one or more precise positions in one or more chromosomes. In order to increase the possibility of integration at the precise position, the integration element preferably comprises a sufficient number of nucleotides, such as 100 to 10,000 base pairs, preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000 base pairs, which are highly homologous to the corresponding target sequence, to improve the possibility of homologous recombination. The integration element can be any sequence homologous to the target sequence in the genome of the host cell. In addition, the integration element can be a non-coding or encoding nucleic acid sequence. On the other hand, the carrier can be integrated into the genome of the host cell by non-homologous recombination.

[0171] For autonomous replication, the vector may also include an origin of replication so that the vector can replicate autonomously in the host cell in question. Examples of bacterial origins of replication are the P15A ori that allows replication in Escherichia coli, or the origin of replication of plasmids pBR322, pUC19, pACYCl77 (the plasmid has a P15A ori) or pACYC184, and the origin of replication of pUB110, pE194 or pTA1060 that allows replication in Bacillus (Bacillus). Examples of origins of replication for use in yeast host cells are the 2 μm origin of replication, ARS1, ARS4, a combination of ARS1 and CEN3, and a combination of ARS4 and CEN6. The origin of replication may be an origin of replication with a mutation that allows it to function temperature-sensitively in a host cell (see, e.g., Ehrlich, Proc. Natl. Acad. Sci. USA 75: 1433

[1978] ).

[0172] In some embodiments, more than one copy of the nucleic acid sequence of the present invention is inserted into the host cell to increase the production of the gene product. The increase in the number of nucleic acid sequence copies can be obtained by integrating at least one additional copy of the sequence into the host cell genome, or by including an amplifiable selection marker gene in the nucleic acid sequence, wherein cells containing an amplified copy of the selection marker gene and thereby containing an additional copy of the nucleic acid sequence can be selected by culturing cells in the presence of a suitable selection agent.

[0173] Many expression vectors useful in the present invention are commercially available. Suitable commercial expression vectors include, but are not limited to, p3xFLAG™ TM Expression vector (Sigma-Aldrich Chemicals), which includes 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), and 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] ).

[0174] Therefore, in some embodiments, a vector comprising a sequence encoding at least one variant acetate kinase is transformed into a host cell to allow the propagation of the vector and the expression of one or more variant acetate kinases. In some embodiments, the variant acetate kinase is post-translationally modified to remove the signal peptide, and can be cracked after secretion in some cases. In some embodiments, the host cell of the conversion described above is cultivated under conditions that allow expression of one or more variant acetate kinases in a suitable nutrient medium. Any suitable culture medium that can be used to cultivate host cells can be used for the present invention, including but not limited to minimal culture medium or composite culture medium containing suitable supplements. In some embodiments, host cells grow in HTP culture medium. Suitable culture medium can be obtained from multiple commercial suppliers, or can be prepared according to disclosed formulas (e.g., in the catalog of the American Type Culture Collection).

[0175] On the other hand, the present invention provides host cells comprising polynucleotides encoding improved acetate kinase polypeptides provided herein, the polynucleotides being operably connected to one or more control sequences for expressing acetate kinase in host cells. Host cells for expressing acetate kinase polypeptides encoded by expression vectors of the present invention are well known in the art, and include but are not limited to bacterial cells such as Escherichia 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 number 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. Suitable culture medium and growth conditions for host cells described above are well known in the art.

[0176] Polynucleotides for expressing acetate kinase can be introduced into cells by various methods known in the art. Techniques include, among others, electroporation, biolistic particle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion. Various methods for introducing polynucleotides into cells are known to those skilled in the art.

[0177] 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 imperfecti. In some embodiments, the fungal host cell is a yeast cell and a filamentous fungal cell. The filamentous fungal host cell of the present invention includes all filamentous forms of Eumycotina and Oomycota. The feature of filamentous fungi is a vegetative mycelium, in which the cell wall is composed of chitin, cellulose, and other complex polysaccharides. The filamentous fungal host cell of the present invention is morphologically different from yeast.

[0178] In some embodiments of the invention, the filamentous fungal host cell is of any suitable genus and species, including, but not limited to, Achlya, Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Cephalosporium, Chrysosporium, Cochliobolus, Corynascus, Cryphonectria, Cryptococcus, Coprinus, Coriolus, Diplodia, Endothis, Fusarium, Gibberella, Gliocladium, Humicola, Hypocrea. , Myceliophthora, Mucor, Neurospora, Penicillium, Podospora, Phlebia, Piromyces, Pyricularia, Rhizomucor, Rhizopus, Schizophyllum, Scytalid ium, Sporotrichum, Talaromyces, Thermoascus, Thielavia, Trametes, Tolypocladium, Trichoderma, Verticillium, and / or Volvariella, and / or sexual or asexual forms, and synonyms, basal synonyms, or taxonomic equivalents thereof.

[0179] 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 cell is 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, Pichia spp. lactis, Candida albicans or Yarrowia lipolytica.

[0180] In some embodiments of the invention, the host cell is an algal cell, such as Chlamydomonas (eg, C. reinhardtii) and Phormidium (Phormidium sp. ATCC 29409).

[0181] 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-variable bacterial cells. Any suitable bacterial organism can be used in the present invention, including but not limited to Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Acinetobacter, Acidothermus, Arthrobacter, Azobacter, Bacillus, Bifidobacterium, Brevibacterium ), Butyrivibrio, Buchnera, Campestris, Campylobacter, Clostridium, Corynebacterium, Chromatium, Coprococcus, Escherichia, Enterococcus, Enterobacter, Erwinia, Fuso bacterium, Faecalibacterium, Francisella, Flavobacterium, Geobacillus, Haemophilus, Helicobacter, Klebsiella, Lactobacillus, Lactococcus, Ilyobacter, Micrococcus, Microbar 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, Azotobacter, 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 strains of bacteria are known and suitable for the present invention. In some embodiments of the present invention, the bacterial host cell 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. roseoparqffinus, A. sulfurus, and A. ureafaciens). In some embodiments of the invention, the bacterial host cell is a Bacillus species (e.g., B. thuringensis, B. anthracis,In some embodiments, the host cell is an industrial Bacillus strain, including but not limited to Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis, Bacillus megaterium, Bacillus brevis, Bacillus 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 Bacillus subtilis, Bacillus pumilus, B. licheniformis, B. megaterium, 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 bacillus host cell is bacillus subtilis, bacillus licheniformis, bacillus megaterium, bacillus stearothermophilus and / or bacillus amyloliquefaciens. In some embodiments, the bacterial host cell is a clostridium species (for example, acetobutylicum clostridium (C.acetobutylicum), tetanus clostridium E88 (C.tetani E88), ivory coast clostridium (C.lituseburense), C.saccharobutylicum, perfringens clostridium (C.perfringens) and Bai Shi clostridium (C.beijerinckii)). In some embodiments, the bacterial host cell is a coryneform bacteria species (for example, glutamicum coryneform bacteria (C.glutamicum) and acetoacetic acid coryneform bacteria (C.acetoacidophilum)). In some embodiments, the bacterial host cell is an Escherichia species (for example, Escherichia coli). In some embodiments, the host cell is Escherichia coli W3110. In some embodiments, the bacterial host cell is an Erwinia species (e.g., Erwinia uredovora, Erwinia carotovora, Erwinia ananas, Erwinia herbicola, E. punctata, and E. terreus). In some embodiments, the bacterial host cell is a Pantoea species (e.g., Pantoea citrea and Pantoea agglomerans). In some embodiments, the bacterial host cell is a Pseudomonas species (e.g., Pseudomonas putida, Pseudomonas aeruginosa,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. fungicidicus, S. griseus, and S. lividans). In some embodiments, the bacterial host cell is a Zymomonas species (eg, Z. mobilis and Z. lipolytica).

[0182] Many prokaryotic and eukaryotic strains that can be used in the present invention are readily available to the public from a number of culture collections, such as the American Type Culture Collection (ATCC), the German Collection of Microorganisms and Fungi (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DSM), the Netherlands Central Agricultural Research Center (Centraalbureau Voor Schimmelcultures, CBS), and the U.S. Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

[0183] In some embodiments, host cells are genetically modified to have the characteristics of improved protein secretion, protein stability and / or protein expression and / or other properties desired for secretion.Genetic modification can be achieved by genetic engineering techniques and / or typical microbiological techniques (for example, chemical or UV mutagenesis and subsequent selection).In fact, in some embodiments, the combination of recombinant modification and typical selection techniques is used to produce host cells.Using recombinant technology, nucleic acid molecules can introduce, lack, suppress or modify in a manner that causes the increase in the output of one or more acetate kinase variants in the host cell and / or in the culture medium.For example, knocking out Alp1 function produces cells with protease defects, and knocking out pyr5 function produces cells with pyrimidine defect phenotype.In a genetic engineering method, homologous recombination is used to induce the genetic modification of targeting by specific targeting genes in vivo, to suppress the expression of coded proteins.In alternative methods, siRNA, antisense and / or ribozyme technology can be used to suppress gene expression.The method of protein expression in various reduction cells known in the art includes but is not limited to the disappearance of all or part of the gene of the coded protein, and site-specific mutagenesis to destroy the expression or activity of gene product. (See, e.g., Chaveroche et al., Nucl. Acids Res., 28:22e97

[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] , each of which is incorporated herein by reference). Random mutagenesis followed by screening for desired mutations may also be used (See, e.g., Combier et al., FEMS Microbiol. Lett., 220:141-8

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

[2003] , both incorporated by reference).

[0184] The introduction of a vector or DNA construct into a 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 commonly used techniques known in the art. In some embodiments, the E. coli expression vector pCK100900i (see, U.S. Pat. No. 9,714,437, hereby incorporated by reference) can be used.

[0185] In some embodiments, the engineered host cells of the invention (i.e., "recombinant host cells") are cultured in conventional nutrient media modified appropriately to activate promoters, select transformants, or amplify acetate kinase polynucleotides. Culture conditions, such as temperature, pH, etc., are those previously used with the host cells selected for expression and are well known to those skilled in the art. As described, many standard references and textbooks are available for the culture and production of many cells, including those of bacterial, plant, animal (especially mammalian) and archaeal origin.

[0186] In some embodiments, the cells expressing the variant acetate kinase polypeptide of the present invention are grown under batch or continuous fermentation conditions. Typical "batch fermentation" is a closed system, wherein the composition of the culture medium is set at the beginning of the fermentation, and is not affected by artificial changes during the fermentation. A variation of the batch system is "fed-batch fermentation", which can also be used in the present invention. In this variation, as the fermentation proceeds, the substrate is added in increments. When catabolite repression may inhibit the metabolism of the cell, and when there is a limited amount of substrate in the desired culture medium, the fed-batch system is useful. Batch fermentation and fed-batch fermentation are common and well known in the art. "Continuous fermentation" is an open system, wherein a determined fermentation medium is continuously added to a bioreactor, and an equal amount of conditioned medium is taken out simultaneously for processing. Continuous fermentation usually maintains the culture at a constant high density, wherein the cell is mainly in logarithmic phase growth. Continuous fermentation systems attempt to maintain steady-state growth conditions. The method for regulating the nutrients and growth factors used for continuous fermentation processes and the technology for maximizing the product formation rate are well known in the field of industrial microbiology.

[0187] In some embodiments of the invention, a cell-free transcription / translation system can be used to produce one or more variant acetate kinases. Several systems are commercially available and methods are well known to those skilled in the art.

[0188] The present invention provides methods for preparing variant acetate kinase polypeptides or biologically active fragments thereof. In some embodiments, the method comprises: 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 also 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 also provides a method for preparing a variant acetate kinase polypeptide, the method comprising culturing a host cell transformed with a variant acetate kinase polynucleotide under conditions suitable for producing the variant acetate kinase polypeptide, and recovering the variant acetate kinase polypeptide. Typically, the acetate kinase polypeptide is recovered or separated from the host cell culture medium, 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, destroyed by physical or chemical means, and the resulting crude extract is retained for further purification. Microbial cells for protein expression can be destroyed by any convenient method, including but not limited to freeze-thaw cycles, sonication, mechanical destruction, and / or the use of cell lysing agents, as well as many other suitable methods well known to those skilled in the art.

[0189] The engineered acetate kinase 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, among others, lysozyme treatment, sonication, filtration, salting out, ultracentrifugation, and chromatography. A suitable solution for lysis and efficient protein extraction from bacteria such as E. coli is available under the trade name CelLytic B TM(Sigma-Aldrich) is commercially available. Therefore, in some embodiments, the obtained polypeptide is recovered / separated and optionally purified by any of various methods known in the art. For example, in some embodiments, the polypeptide is separated 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, as required, a protein refolding step is used to complete the construction of the mature protein. In addition, in some embodiments, high performance liquid chromatography (HPLC) is used in the final purification step. For example, in some embodiments, methods known in the art can be used in the present invention (see, for example, 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). In fact, any suitable purification method known in the art can be used in the present invention.

[0190] Chromatographic techniques used to separate acetate kinase polypeptides include, but are not limited to, reverse phase chromatography, high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, and affinity chromatography. The conditions used to purify 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.

[0191] In some embodiments, affinity techniques can be used to isolate improved acetate kinase. For affinity chromatography purification, any antibody that specifically binds to the acetate kinase polypeptide can be used. In order to produce antibodies, various host animals can be immunized by injection of acetate kinase, including but not limited to rabbits, mice, rats, etc. The acetate kinase polypeptide can be attached to a suitable carrier such as BSA by means of side chain functional groups or linkers attached to side chain functional groups. Depending on the host species, various adjuvants can be used to enhance the immune response, 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 limpets. Keyholelimpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacillus Calmette-Guérin) and Corynebacterium parvum.

[0192] In some embodiments, the acetate kinase variant is prepared and used in the form of cells expressing the enzyme, as a crude extract, or as an isolated or purified preparation. In some embodiments, the acetate kinase variant is prepared as a lyophilized agent, a powder form (e.g., acetone powder), or as an enzyme solution. In some embodiments, the acetate kinase variant is in the form of a substantially pure preparation.

[0193] In some embodiments, the acetate kinase polypeptide is connected 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), reversed silica, or metals such as gold or platinum. The configuration of the substrate can be in the form of beads, balls, particles, granules, gels, membranes, or surfaces. The surface can be flat, substantially flat, or non-flat. The solid support can be porous or non-porous, and can have swelling or non-swelling characteristics. The solid support can be configured in the form of a hole, depression, or other container, vessel, feature, or position. More than one support can be configured on the array at multiple locations, and the positions can be addressed by the automatic delivery of reagents or by detection methods and / or instruments.

[0194] In some embodiments, immunological methods are used to purify acetate kinase variants. In one method, antibodies 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) generated using conventional methods are immobilized on beads, mixed with cell culture medium under conditions where the variant acetate kinase is bound, and precipitated. In a related method, immunochromatography can be used.

[0195] In some embodiments, variant acetate kinase is expressed as a fusion protein comprising a non-enzyme portion. In some embodiments, the variant acetate kinase sequence is fused to a purification-promoting domain. As used herein, the term "purification-promoting domain" refers to a domain that mediates the purification of a polypeptide fused thereto. Suitable purification domains include, but are not limited to, metal chelating peptides, histidine-tryptophan modules that allow purification on immobilized metals, sequences that bind glutathione (e.g., GST), hemagglutinin (HA) tags (corresponding to epitopes derived from influenza hemagglutinin proteins; see, e.g., Wilson et al., Cell 37:767

[1984] ), maltose binding protein sequences, FLAG epitopes used in FLAGS extension / affinity purification systems (e.g., systems available from Immunex Corp), etc. It is contemplated that an expression vector for the compositions and methods described herein provides expression of a fusion protein comprising a polypeptide of the present invention fused to a polyhistidine region 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. The pGEX vector (Promega) can also be used to express exogenous polypeptides as fusion proteins with glutathione S-transferase (GST). In general, 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-fusion proteins) followed by elution in the presence of free ligand.

[0196] Therefore, in another aspect, the invention provides a method for producing an engineered enzyme polypeptide, wherein the method comprises culturing a host cell capable of expressing a polynucleotide encoding an engineered enzyme polypeptide under conditions suitable for expression of the polypeptide. In some embodiments, the method further comprises the step of separating and / or purifying the enzyme polypeptide as described herein.

[0197] Suitable culture media and growth conditions for host cells are well known in the art. It is contemplated that any suitable method for introducing a polynucleotide for expressing an enzyme polypeptide into a cell can be used in the present invention. Suitable techniques include, but are not limited to, electroporation, biolistic particle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion.

[0198] Various features and embodiments of the invention are illustrated in the following representative examples, which are intended to be illustrative rather than limiting.

[0199] experiment

[0200] The following examples are provided, including the results of experiments and acquisitions, for illustrative purposes only and should not be construed as limiting the present invention. In fact, many reagents and equipment described below have various suitable sources. It is not intended that the present invention be limited to any particular source of any reagent or equipment item.

[0201] In the experimental disclosure hereinafter, the following abbreviations are used: M (mole / liter); mM (millimol / liter), uM and μM (micromol / liter); nM (nanomoles / liter); mol (mole); gm and g (gram); mg (milligram); ug and μg (microgram); L and l (liter); ml and mL (milliliter); cm (centimeter); mm (millimeter); um and μm (micrometer); sec. (second); min(s) (minute); h(s) and hr(s) (hour); U (unit); MW (molecular weight); rpm (revolutions per minute); psi 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, broth); SFP (shake flask powder); CDS (coding sequence); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); nt (nucleotide; polynucleotide); aa (amino acid; polypeptide); Escherichia coli W3110 (a commonly used laboratory strain of E. coli, available from Coli Genetic Stock Center [CGSC], New Haven, CT); HTP (high throughput); HPLC (high pressure liquid chromatography); HPLC-UV (HPLC-ultraviolet visible detector); 1H NMR (proton nuclear magnetic resonance spectroscopy); FIOPC (fold increase relative to positive control); Sigma and Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO); Difco (Difco Laboratories, BDDiagnostic Systems, Detroit, MI); Microfluidics (Microfluidics, Westwood, MA); Life Technologies (Life Technologies, Fisher Scientific, part of 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). .

[0202] Example 1

[0203] Production of engineered polypeptides in pCK110900

[0204] A polynucleotide (SEQ ID NO: 1) encoding a naturally occurring wild-type polypeptide having acetate kinase activity from Thermotoga maritima (SEQ ID NO: 2) was cloned into the pCK110900 vector system (see, e.g., U.S. Pat. No. 9,714,437, which is hereby incorporated by reference in its entirety), and then expressed in E. coli W3110fhuA under the control of the lac promoter.

[0205] In a 96-well format, a single colony was picked and grown in 180 μL of LB containing 1% glucose and 30 μg / mL CAM at 30°C, 200 rpm, 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, 85% humidity. When the optical density (OD 600 ) reached 0.6-0.8, by adding IPTG to a final concentration of 1mM to induce the expression of the acetate kinase gene. After induction, continue to grow for 18-20 hours. The cells are harvested by centrifugation at 4000rpm for 10 minutes at 4°C, and the culture medium is discarded. The cell pellet is stored at -80°C until ready for use. Before the assay, the cell pellet is resuspended in 400μL of lysis buffer containing 50mM potassium phosphate, pH 7.5 with 1g / L lysozyme and 0.5g / L PMBS. At room temperature, the plate is shaken for 2 hours at medium speed on a microtiter plate shaker. The plate is then centrifuged at 4°C for 20 minutes at 4000rpm, and the clarified supernatant is used in the HTP assay reaction described below.

[0206] The shake flask procedure can be used to produce engineered acetate kinase polypeptide shake flask powder (SFP) that can be used for secondary screening assays and / or can be used in the biocatalytic methods described herein. Compared with the cell lysate used in the HTP assay, the shake flask powder (SFP) preparation of the enzyme provides a more purified engineered enzyme preparation (e.g., up to 30% of the total protein), and also allows the use of a more concentrated enzyme solution. In order to start culturing, a single E. coli colony containing a plasmid encoding an engineered polypeptide of interest is inoculated into 5 mL of LB containing 30 μg / ml CAM and 1% glucose. The culture is grown overnight (at least 16 hours) at 30°C with 250 rpm shaking in an incubator. The grown culture is diluted into 250 mL of TB containing 30 μg / mL CAM in a 1 L shake flask to a final OD of 0.05. 600 Grow 250 mL of culture at 30 °C and 250 rpm until OD 600 Reach 0.6-0.8. Expression of the acetate kinase gene is induced by adding IPTG to a final concentration of 1 mM, and growth is continued for another 18-20 hours. The cells are harvested by transferring the culture to a pre-weighed centrifuge bottle and then centrifuged at 7000 rpm for 10 minutes at 4°C. The supernatant is discarded and the remaining cell pellet is weighed. In some embodiments, the cells are stored at -80°C until ready for use. For lysis, the cell pellet is resuspended in 6 mL of cold 50 mM potassium phosphate buffer, pH 7.5 per 1 g of cell pellet. Use 110L The resuspended cells were lysed by a processor system (Microfluidics). Cell debris was removed by centrifugation at 10,000 rpm for 60 minutes at 4°C. 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 provided a dry shake flask powder containing the crude engineered polypeptide.

[0207] Example 2

[0208] Evolution and screening of engineered polypeptides derived from SEQ ID NO: 2 for improved acetate kinase (AcK) activity

[0209] The polynucleotides (SEQ ID NO: 1) encoding the polypeptides with acetate kinase activity of SEQ ID NO: 2 are used to generate the engineered polypeptides of Table 2-1. These polypeptides show improved acetate kinase activity (e.g., the ability to promote ATP recycling via ADP and acetyl phosphate, as measured by the % conversion of acetylene glyceraldehyde to acetylene glyceraldehyde phosphate product under ATP limiting conditions) under the desired conditions compared to the starting polypeptides. Engineered polypeptides with amino acid sequences of even-numbered sequence identifiers are generated from the "skeleton" amino acid sequence of SEQ ID NO: 2 as described below together with the HTP assay and analysis methods described in Table 2-2.

[0210] 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 and analytical methods that measure the ability of the polypeptides to promote ATP recycling via ADP and acetyl phosphate (as measured via conversion of acetylglyceraldehyde substrate to acetylglyceraldehyde phosphate product under ATP limiting conditions).

[0211] Enzyme assay was performed in 96-well format with 50 μL total volume / well, including HTP enzyme lysate, 20 g / L acetyl glyceraldehyde, 2 equivalents of acetyl phosphate, 0.3 g / L ATP, 0.5 g / L engineered pantothenate kinase (SEQ ID NO: 698), 10 mM MgCl2, 100 mM potassium phosphate, pH 7.5, final concentration. The reaction was performed by adding the following to each well: (i) 25 μL of 40 g / L acetyl glyceraldehyde, 300 mM acetyl phosphate, 0.6 g / L ATP, 1 g / L engineered pantothenate kinase (SEQ ID NO: 698), 20 mM MgCl2, 200 mM potassium phosphate solution (adjusting the pH of the mixture to 7.8), (ii) 25 μL of 0.3% (v / v) diluted AcK HTP lysate. The reaction plate was heat sealed and shaken overnight at 30 ° C with 600 rpm.

[0212] After overnight incubation (~16 hours), 100 μL of 50mM 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 400 rpm for 20-30 minutes at 25 ° C. Before UPLC analysis using the method described in Table 2-2 below, the sample was further diluted 4x in methanol. The data shown in Table 2-1 reflect the activity relative to SEQ ID NO: 2, which is calculated as the peak area of ​​compound 5 formed by the variant enzyme under specified reaction conditions compared to the peak area of ​​compound 5 formed by SEQ ID NO: 2.

[0213] Selected hit variants were grown in 250 mL shake flasks and enzyme powders were produced. The activity of the enzyme powders was evaluated using an assay similar to that described above, with 0.0002 g / L-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 MgCl2, 100 mM potassium phosphate, pH 6.8, 600 rpm, 30°C, 3 hours.

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223] Example 3

[0224] Evolution and screening of engineered polypeptides derived from SEQ ID NO: 12 for improved acetate kinase (AcK) activity

[0225] The engineered polynucleotides (SEQ ID NO: 11) encoding the polypeptides with acetate kinase activity of SEQ ID NO: 12 were used to generate the engineered polypeptides of Table 3-1. These polypeptides exhibit improved acetate kinase activity (e.g., the ability to promote ATP recycling via ADP and acetyl phosphate, as measured by the % conversion of acetylene glyceraldehyde to acetylene glyceraldehyde phosphate product under ATP limiting conditions) under the desired conditions compared to the starting polypeptides. Engineered polypeptides with amino acid sequences with even-numbered sequence identifiers were generated from the "skeleton" amino acid sequence of SEQ ID NO: 12 as described below together with the HTP assay and analysis methods described in Table 2-2.

[0226] 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 and analytical methods that measure the ability of the polypeptides to promote ATP recycling via ADP and acetyl phosphate (as measured via conversion of acetylglyceraldehyde substrate to acetylglyceraldehyde phosphate product under ATP limiting conditions).

[0227] Enzyme assay was performed in 96-well format with 50 μL total volume / well, including HTP enzyme lysate, 20 g / L acetyl glyceraldehyde, 2 equivalents of acetyl phosphate, 0.3 g / L ATP, 0.5 g / L engineered pantothenate kinase (SEQ ID NO: 700), 10 mM MgCl2, 100 mM potassium phosphate, pH 7.5, final concentration. The reaction was performed by adding the following to each well: (i) 25 μL of 40 g / L acetyl glyceraldehyde, 300 mM acetyl phosphate, 0.6 g / L ATP, 1 g / L engineered pantothenate kinase (SEQ ID NO: 700), 20 mM MgCl2, 200 mM potassium phosphate solution (adjusting the pH of the mixture to 7.8), (ii) 25 μL of 0.3% (v / v) diluted AcK HTP lysate. The reaction plate was heat sealed and shaken overnight at 30 ° C with 600 rpm.

[0228] After incubation overnight (~16 hours), 100 μL of 50mM 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 400 rpm for 20-30 minutes at 25 ° C. Before UPLC analysis using the method described in Table 2-2, the sample was further diluted 4x in methanol. The data shown in Table 3-1 reflect the activity relative to SEQ ID NO: 12, which is calculated as the peak area of ​​compound 5 formed by the variant enzyme under specified reaction conditions compared to the peak area of ​​compound 5 formed by SEQ ID NO: 12.

[0229] The hit variants were grown in 250 mL shake flasks and enzyme powders were produced. The activity of the enzyme powders was evaluated using an assay similar to that described above, with 0.0002 g / L-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 MgCl2, 100 mM potassium phosphate, pH 6.8, 600 rpm, 30°C, 3 hours.

[0230]

[0231]

[0232] Example 4

[0233] Evolution and screening of engineered polypeptides derived from SEQ ID NO: 600 for improved acetate kinase (AcK) activity

[0234] The polynucleotide (SEQ ID NO: 599) encoding the polypeptide with acetate kinase activity of SEQ ID NO: 600 is used to generate the engineered polypeptides of Table 4-1. These polypeptides show improved acetate kinase activity under desired conditions (e.g., the ability to promote ATP recycling via ADP and acetyl phosphate, as measured by the % conversion of acetylene glycerol to acetylene glycerol phosphate product under ATP limiting conditions) compared to the starting polypeptide. Engineered polypeptides with amino acid sequences with even-numbered sequence identifiers were generated from the "skeleton" amino acid sequence of SEQ ID NO: 600 described below together with the HTP assay and analysis methods described in Table 4-2.

[0235] 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 and analytical methods that measure the ability of the polypeptides to promote ATP recycling via ADP and acetyl phosphate (as measured via conversion of ethynylglycerol substrate to ethynylglycerol phosphate product under ATP limiting conditions).

[0236] Enzyme assay was performed in a 96-well format with a total volume of 50 μL / well, including HTPase lysate, 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 MgCl2, 50 mM Bis Tris buffer pH 7.0, final concentration. The reaction was performed by adding the following to each well: (i) 40 μL of 62.5 g / L ethynylglycerol, 1.5625 equivalents of mM acetyl phosphate, 0.125 mol% L ATP, 0.625 g / L engineered pantothenate kinase (SEQ ID NO: 702), 12.5 mM MgCl2, 50 mM Bis Tris buffer pH 7.0. The pH of the final master mix was pH = 6.8 (ii) 10 μL of 50x diluted AcK HTP lysate. The reaction plates were heat sealed and shaken at 600 rpm at 30°C overnight.

[0237] After incubation overnight (~18 hours), in order to generate chromophore-containing species and enable simple reaction monitoring, the reaction samples were derivatized using tripotassium 5,5',5"-[2,2',2"-nitrile tris(methylene-tris(1H-benzimidazole-2,1-diyl)]tripalanic acid hydrate ((BimC4A)3) under the following conditions: 5 equivalents of benzyl azide, 5 mol% copper sulfate, 7.5 mol% (BimC4A)3, 20 mol% sodium ascorbate, 9:1 water:DMSO to achieve click chemistry. After the reaction, 5uL of the reaction was combined with 220uL of (BimC4A)3 derivatization solution in a new 96-well plate. The click chemistry reaction was incubated at 45°C for 1 hour. The 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).

[0238] Hit variants were grown in 250 mL shake flasks and enzyme powders were produced. The activity of enzyme powders was evaluated using a similar assay to that described above with 0.0125 g / L-0.05 g / L SF powder, 50 g / L ethynylglycerol, 1.25 equivalents of acetyl phosphate, 0.1 mol% ATP, 0.25 g / L-1.5 g / L engineered pantothenate kinase (SEQ ID NO: 702), 10 mM MgCl2, 100 mM Bis Tris buffer pH 7.0 (final master mix pH = 6.8), 600 rpm, 30 ° C, 18 hours. Shake flask powder test data are shown in Table 4-1.

[0239]

[0240]

[0241]

[0242] 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 for all purposes.

[0243] While various specific embodiments have been illustrated and described, it will be appreciated that various changes can be made without departing from the spirit and scope of the invention.

Claims

1. An engineered acetate kinase, wherein the polypeptide sequence of the engineered acetate kinase is the same as SEQ ID NO: 2 is E298L / T / V / F / Q / K / W; or is a combination of E298L / T / V / F / Q / K / W and 1-9 amino acid residue differences selected from K405E, A135L, N136A / V, I140P, A142V, A143C, L242T, D316R, T372F, R374L / E, V101I, V102A, I122G, I23H, N395K, V15N, A154Q, T191I, I297N / L, S355K, I164H, E70K, L298F, D352T / G, V391R, L51F, A301R, I348E, E29D and Q162H.

2. The engineered acetate kinase according to claim 1, wherein the difference in amino acid residues between the polypeptide sequence of the engineered acetate kinase and SEQ ID NO: 2 is E298L / T / V / F / Q / K / W; or a combination of E298L / T / V / F / Q / K / W and K405E, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:

2.

3. The engineered acetate kinase of claim 1, wherein the amino acid residue differences between the polypeptide sequence of the engineered acetate kinase and SEQ ID NO: 12 are 1-9 amino acid residue differences selected from the group consisting of A135L, N136A / V, I140P, A142V, A143C, L242T, D316R, T372F, R374L / E, V101I, V102A, I122G, I23H, N395K, V15N, A154Q, T191I, I297N / L, S355K, I164H, E70K, L298F, D352T / G, V391R, L51F, A301R, I348E, E29D, and Q162H, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:

12.

4. The engineered acetate kinase of claim 1, wherein the amino acid residue difference between the polypeptide sequence of the engineered acetate kinase and SEQ ID NO: 600 is 1-3 amino acid residue differences selected from L242T, R374L, L298F, I23H and E70K, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:

600.

5. The engineered acetate kinase of claim 1, wherein the engineered acetate kinase is an engineered acetate kinase variant listed in the even-numbered sequences of SEQ ID NOs: 4-14, 22, 64, or 592-696.

6. The engineered acetate kinase of any one of claims 1-5, wherein the engineered acetate kinase comprises at least one improved property compared to wild-type Thermotoga maritima acetate kinase.

7. The engineered acetate kinase of claim 6, wherein the improved property comprises improved activity towards a substrate compared to wild-type acetate kinase.

8. The engineered acetate kinase of claim 7, wherein the substrate comprises adenosine diphosphate and acetyl phosphate.

9. The engineered acetate kinase of claim 6, wherein the improved property comprises improved adenosine triphosphate production compared to wild-type acetate kinase.

10. The engineered acetate kinase of any one of claims 1-5, wherein the engineered acetate kinase is purified.

11. The engineered acetate kinase of any one of claims 1-5, wherein the engineered acetate kinase is part of a multi-enzyme system for producing nucleoside analogs.

12. A composition comprising at least one engineered acetate kinase according to any one of claims 1-11.

13. A polynucleotide sequence encoding at least one engineered acetate kinase according to any one of claims 1-11.

14. The polynucleotide sequence of claim 13, wherein the polynucleotide sequence is operably linked to a control sequence.

15. The polynucleotide sequence according to any one of claims 13-14, wherein the polynucleotide sequence is codon optimized.

16. The polynucleotide sequence of claim 13, wherein the polynucleotide sequence is an odd-numbered sequence in SEQ ID NO: 3-13, 21, 63 or 591-695.

17. An expression vector comprising at least one polynucleotide sequence according to any one of claims 13 to 16.

18. A host cell comprising at least one expression vector according to claim 17.

19. A host cell comprising at least one polynucleotide sequence according to any one of claims 13-16.

20. A method for producing an engineered acetate kinase in a host cell, the method comprising culturing the host cell according to claim 18 or 19 under suitable conditions, thereby producing at least one engineered acetate kinase.

21. The method of claim 20, further comprising recovering at least one engineered acetate kinase from the culture and / or host cell.

22. The method of claim 20 or 21, further comprising the step of purifying the at least one engineered acetate kinase.

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