Engineered pantothenate kinase variant enzymes

Abstract

The present invention provides engineered pantothenate kinase (PanK), polypeptides having PanK activity and polynucleotides encoding these enzymes, and vectors and host cells comprising these polynucleotides and polypeptides. Also provided are methods for producing PanK enzymes. The present invention also provides compositions comprising PanK enzymes, and methods for using engineered PanK enzymes. 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 / 695,587, filed July 9, 2018, and U.S. Provisional Patent Application Serial No. 62 / 822,301, filed March 22, 2019, both of which are incorporated herein by reference in their entirety for all purposes. Field of the Invention

[0002] This invention provides engineered pantothenic acid kinase (PanK), polypeptides having PanK activity, and polynucleotides encoding these enzymes, as well as vectors and host cells containing these polynucleotides and polypeptides. A method for producing PanK enzymes is also provided. This invention further provides compositions containing PanK enzymes and methods for using engineered PanK enzymes. This 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 list was submitted along with the specification via EFS-Web as an ASCII-formatted text file, named "CX2-177WO2_ST25.txt", created on June 26, 2019, and measuring 553 kilobytes in size. The sequence list submitted via EFS-Web is part of the specification and is incorporated herein by reference in its entirety. Background of the Invention

[0006] Retroviruses, known as human immunodeficiency virus (HIV), are the pathogens of acquired immunodeficiency syndrome (AIDS), a complex disease involving the progressive destruction of the immune system and the degeneration of the central and peripheral nervous systems in affected individuals. A common feature of retroviral replication is the reverse transcription of the viral RNA genome by a virus-encoded reverse transcriptase to produce DNA copies of the HIV sequence required for viral replication. Some compounds, such as MK-8591, are known reverse transcriptase inhibitors and are used to treat AIDS and similar diseases. While some compounds are known to inhibit HIV reverse transcriptase, there remains a need in the art for additional compounds that can inhibit this enzyme more effectively and thus improve efficacy against AIDS.

[0007] Due to their similarity to natural nucleosides used in DNA synthesis, nucleoside analogs such as MK-8591 (Merck) are effective inhibitors of HIV reverse transcriptase. The binding of reverse transcriptase to these analogs halts DNA synthesis by inhibiting the progressive nature of reverse transcriptase. This enzyme stagnation leads to premature termination of the DNA molecule, rendering it ineffective. However, producing nucleoside analogs using standard chemical synthesis techniques can be challenging due to their chemical complexity. SUMMARY OF THE INVENTION

[0009] This invention provides engineered pantothenic acid kinase (PanK), polypeptides having PanK activity, and polynucleotides encoding these enzymes, as well as vectors and host cells containing these polynucleotides and polypeptides. A method for producing PanK enzymes is also provided. This invention further provides compositions containing PanK enzymes and methods for using engineered PanK enzymes. This invention is particularly useful for the production of pharmaceutical compounds.

[0010] In some embodiments, the present invention provides an engineered pantothenic acid 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 with SEQ ID NO:2, SEQ ID NO:30, SEQ ID NO:60, SEQ ID NO:132, SEQ ID NO:222, SEQ ID NO:230, SEQ ID NO:240 and / or SEQ ID NO:276, wherein the engineered pantothenic acid 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 referenced to SEQ ID NO:2, SEQ ID NO:30, SEQ ID NO:60, SEQ ID NO:132, SEQ ID NO:222, SEQ ID NO:230, SEQ ID NO:240 and / or SEQ ID NO:276.

[0011] In some embodiments, the present invention provides an engineered pantothenic 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 with SEQ ID NO:2, wherein the engineered pantothenic kinase comprises at least one substitution or set of substitutions in the polypeptide sequence, and wherein the amino acid positions of the polypeptide sequence refer to the number SEQ ID NO:2. In some embodiments, the engineered pantothenic acid 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 with SEQ ID NO:2, and wherein the engineered pantothenic acid kinase comprises at least one substitution or set of substitutions at one or more positions selected from the following: 54 / 240 / 277 / 281, 240, 240 / 277, 240 / 277 / 281, 240 / 277 / 281 / 282, 240 / 281, 240 / 281 / 282, 277 and 277 / 281, and wherein the amino acid positions of the polypeptide sequence refer to the numbering of SEQ ID NO:2. In some other embodiments, the engineered pantothenic acid kinase comprises at least one substitution or set of substitutions selected from the following: 54S / 240W / 277M / 281M, 240F, 240F / 277I, 240F / 277I / 281L, 240F / 277M, 240F / 277M / 281M, 240F / 277M / 281M / 282M, 240F / 281M, 240F / 281M / 282M, 240W / 277I / 281M, 240W / 281M, 277I / 281L, 277I / 281M, 277M, and 277M / 281M, wherein the amino acid positions of the polypeptide sequence are referenced to SEQ ID NO:2. In some additional embodiments, the engineered pantothenic acid kinase comprises at least one substitution or set of substitutions selected from the following: I54S / Y240W / L277M / I281M, Y240F, Y240F / L277I, Y240F / L277I / I281L, Y240F / L277M, Y240F / L277M / I281M, Y240F / L277M / I281M / N282M, Y240F / I281M, Y240F / I281M / N282M, Y240W / L277I / I281M, Y240W / I281M, L277I / I281L, L277I / I281M, L277M, and L277M / I281M, wherein the amino acid positions of the polypeptide sequence are as per SEQ ID. NO:2.In some embodiments, the engineered pantothenic acid kinase comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the same polypeptide sequence as at least one engineered pantothenic acid kinase variant listed in Table 2-1.

[0012] In some other embodiments, the engineered pantothenic acid kinase comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the same polypeptide sequence as SEQ ID NO:2. In some other embodiments, the engineered pantothenic acid kinase is a variant engineered polypeptide listed in SEQ ID NO:2.

[0013] In some embodiments, the present invention provides an engineered pantothenic acid 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 with SEQ ID NO:30, wherein the engineered pantothenic acid kinase comprises at least one substitution or set of substitutions in the polypeptide sequence, and wherein the amino acid positions of the polypeptide sequence refer to the number in SEQ ID NO:30. In some embodiments, the engineered pantothenic acid kinase comprises a sequence identical to SEQ ID NO:30. NO:30 has a polypeptide sequence or functional fragment thereof with 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 pantothenic acid kinase contains at least one substitution or set of substitutions at one or more positions selected from the following: 13, 13 / 14 / 19 / 22, 13 / 14 / 22, 13 / 14 / 22 / 37, 13 / 14 / 22 / 106 / 247, 13 / 14 / 218 / 247 / 305, 13 / 19, 13 / 22, 15 / 26 / 283, 15 / 27, 15 / 27 / 283, 15 / 27 / 283 / 305, 15 / 27 / 305, 15 / 283, 19 / 22 / 26 / 69, 19 / 29, 20, 20 / 24, 20 / 24 / 25 / 69 / 75, 20 / 24 / 30 / 75, 20 / 70 / 75, 22 / 218, 22 / 218 / 271, 24, 24 / 30, 24 / 30 / 75, 24 / 75 / 86 / 134, 26, 26 / 29, 27, 27 / 78, 27 / 78 / 123 / 283, 27 / 283, 29 and 75, wherein the amino acid positions of the polypeptide sequences are referenced to SEQ ID NO:30.In some other embodiments, the engineered pantothenic acid kinase comprises at least one substitution or set of substitutions selected from the following: 13D, 13D / 14E / 19T / 22G, 13D / 14E / 22G, 13D / 14E / 22G / 37L, 13D / 14E / 22T / 106A / 247M, 13D / 14E / 218E / 247M / 305T, 13D / 19T, 13D / 22T, 15L / 26L / 283H, 15L / 27N, 15L / 27N / 283H, 15L / 27N / 283L, 15L / 27N / 283L / 305I, 15L / 27N / 305I, 15L / 283L, 19 R / 22D / 26P / 69Y, 19R / 29S, 20M, 20M / 24A, 20M / 24A / 30R / 75E, 20M / 24S / 25T / 69T / 75E, 20M / 70D / 75E, 22G / 218E, 22G / 218E / 271S, 24A, 24A / 30R, 24S / 30R / 75E, 24S / 75E / 86H / 134L, 26L, 26P, 26P / 29S, 27N, 27N / 78A, 27N / 78A / 123W / 283H, 27N / 283H, 27N / 283L, 29S and 75E, wherein the amino acid positions of the polypeptide sequences are referenced to SEQ ID NO:30.In some other embodiments, the engineered pantothenic acid kinase comprises at least one substitution or set of substitutions selected from the following: L13D, L13D / Q14E / Q19T / A22G, L13D / Q14E / A22G, L13D / Q14E / A22G / A37L, L13D / Q14E / A22T / R106A / F247M, L13D / Q14E / F218E / F2 47M / A305T, L13D / Q19T, L13D / A22T, F15L / S26L / W283H, F15L / V27N, F15L / V27N / W283H , F15L / V27N / W283L, F15L / V27N / W283L / A305I, F15L / V27N / A305I, F15L / W283L, Q19R / A22D / S26P / N69Y, Q19R / M29S, W20M, W20M / R24A, W20M / R24A / T30R / V75E, W20M / R24S / D 25T / N69T / V75E, W20M / L70D / V75E, A22G / F218E, A22G / F218E / I271S, R24A, R24A / T30R The peptide sequences are R24S / T30R / V75E, R24S / V75E / R86H / Q134L, S26L, S26P, S26P / M29S, V27N, V27N / Q78A, V27N / Q78A / L123W / W283H, V27N / W283H, V27N / W283L, M29S, and V75E, wherein the amino acid positions of the peptide sequences are as specified in SEQ ID NO:30. In some embodiments, the engineered pantothenic kinase comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the same peptide sequence as at least one engineered pantothenic kinase variant listed in Table 4-1. In some other embodiments, the engineered pantothenic acid kinase comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the same polypeptide sequence as SEQ ID NO:30. In some other embodiments, the engineered pantothenic acid kinase is a variant engineered polypeptide listed in SEQ ID NO:30.

[0014] In some embodiments, the present invention provides an engineered pantothenic acid 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 with SEQ ID NO:60, wherein the engineered pantothenic acid kinase comprises at least one substitution or set of substitutions in the polypeptide sequence, and wherein the amino acid positions of the polypeptide sequence refer to the number in SEQ ID NO:60. In some embodiments, the engineered pantothenic acid kinase comprises a sequence identical to SEQ ID NO:60. NO:60 has a polypeptide sequence or functional fragment thereof with 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 pantothenic acid kinase contains at least one substitution or set of substitutions at one or more positions selected from the following: 14 / 19 / 41 / 157 / 161 / 261, 19 / 22 / 41 / 44 / 54 / 119 / 157 / 261 / 298 / 308, 19 / 22 / 54 / 15 7 / 169, 22 / 106 / 218, 41, 41 / 44 / 54 / 119 / 120 / 157 / 169 / 261, 41 / 44 / 54 / 119 / 120 / 157 / 261 / 298 / 308, 41 / 44 / 54 / 119 / 120 / 161 / 169 / 261 / 298, 41 / 44 / 54 / 119 / 298 / 305, 41 / 44 / 161 / 169 / 261, 41 / 44 / 169 / 261 / 298 / 308, 41 / 44 / 169 / 261 / 308, 4 1 / 54 / 119 / 157 / 169 / 261, 41 / 119 / 161 / 169 / 261 / 308, 41 / 119 / 161 / 308, 44 / 54 / 119 / 120 / 157 / 161 / 169, 44 / 54 / 119 / 120 / 157 / 161 / 261, 44 / 54 / 119 / 120 / 169 / 261, 44 / 54 / 119 / 157 / 161 / 261 / 298, 44 / 54 / 119 / 169, 44 / 76 / 119 / 157 / 161, 44 / 1 19 / 120 / 261, 44 / 119 / 157 / 161, 44 / 119 / 161 / 261 / 298, 44 / 157 / 161 / 169, 44 / 157 / 298, 44 / 261 / 298 / 308, 44 / 261 / 308, 54 / 119 / 157 / 161 / 169, 54 / 157 / 161 / 261 / 308, 119 / 157 / 161, 119 / 157 / 161 / 169 / 261, 119 / 169 / 261, 119 / 261 / 298 / 308,120 / 157 / 261, 157, 157 / 161 / 169 / 261, 157 / 161 / 308, 157 / 169 / 261 / 298 / 308, 157 / 308, 250, 302 and 310, and the amino acid positions of the polypeptide sequences therein refer to SEQ ID NO:60. In some other embodiments, the engineered pantothenic acid kinase comprises at least one substitution or set of substitutions selected from the following: 14E / 19T / 41C / 157M / 161K / 261P, 19A / 22G / 54V / 157M / 169Q, 19T / 22G / 41C / 44P / 54V / 119A / 157M / 261G / 298R / 308I, 22G / 106A / 218E, 41C / 44P / 54W / 119P / 298R / 305T, 41C / 44P / 161R / 169Q / 261G, 41C / 44Q / 54V / 119A / 120K / 157M / 169Q / 261P、41C / 44Q / 54V / 119P / 120K / 157M / 261G / 298R / 308V、41C / 44Q / 54V / 119P / 120K / 161K / 169Q / 261G / 298R、41C / 44Q / 169Q / 261 P / 298R / 308V, 41C / 44Q / 169Q / 261P / 308V, 41C / 54W / 119P / 157M / 169Q / 261G, 41C / 119P / 161R / 169Q / 261G / 308V, 41C / 119P / 161R / 308V, 41R、44P / 54V / 119P / 157M / 161R / 261P / 298R、44P / 54W / 119A / 120K / 169Q / 261G、44P / 76Q / 119P / 157M / 161K、44P / 119K / 157M / 161K、44P / 119P / 120K / 261P、44P / 157M / 298R、44Q / 54V / 119A / 120K / 157M / 161R / 169Q、44Q / 54V / 119K / 120K / 157M / 161R / 261G、44Q / 54W / 119P / 16 9Q、44Q / 119P / 161R / 261P / 298R、44Q / 157M / 161K / 169Q、44Q / 261G / 298R / 308V、44Q / 261P / 308V、54V / 157M / 161K / 261G / 308I、54W / 119A / 157M / 161K / 169Q, 119K / 157M / 161R / 169Q / 261P, 119P / 157M / 161K, 119P / 261G / 298R / 308I, 119Q / 169Q / 261G, 120K / 157M / 261P, 157M,157M / 161R / 169Q / 261G, 157M / 161R / 308V, 157M / 169Q / 261P / 298R / 308I, 157M / 308V, 250R, 302A and 310S, wherein the amino acid positions of the polypeptide sequences are as specified in SEQ ID NO:60. In some other embodiments, the engineered pantothenic acid kinase comprises at least one substitution or set of substitutions selected from the following: Q14E / Q19T / G41C / V157M / S161K / N261P, Q19A / A22G / I54V / V157M / N169Q, Q19T / A22G / G41C / E44P / I54V / R119A / V157M / N261G / S298R / A308I, A22G / R106A / F218E, G41C / E44P / I54W / R119P / S298R / A305T, G41C / E44P / S161R / N 169Q / N261G, G41C / E44Q / I54V / R119A / R120K / V157M / N169Q / N261P, G41C / E44Q / I54V / R119P / R120K / V157M / N261G / S298R / A308V, G41 C / E44Q / I54V / R119P / R120K / S161K / N169Q / N261G / S298R, G41C / E44Q / N169Q / N261P / S298R / A308V, G41C / E44Q / N169Q / N261P / A308V, G41C / I54W / R119P / V157M / N169Q / N261G, G41C / R119P / S161R / N169Q / N261G / A308V, G41C / R119P / S161R / A308V, G41R, E44P / I54V / R1 19P / V157M / S161R / N261P / S298R, E44P / I54W / R119A / R120K / N169Q / N261G, E44P / L76Q / R119P / V157M / S161K, E44P / R119K / V157M / S16 1K, E44P / R119P / R120K / N261P, E44P / V157M / S298R, E44Q / I54V / R119A / R120K / V157M / S161R / N169Q, E44Q / I54V / R119K / R120K / V157M / S161R / N261G、E44Q / I54W / R119P / N169Q、E44Q / R119P / S161R / N261P / S298R、E44Q / V157M / S161K / N169Q、E44Q / N261G / S298R / A308V、E44Q / N261P / A308V, I54V / V157M / S161K / N261G / A308I, I54W / R119A / V157M / S161K / N169Q, R1 19K / V157M / S161R / N169Q / N261P, R119P / V157M / S161K, R119P / N261G / S298R / A308I, R119Q / N The peptide sequences are 169Q / N261G, R120K / V157M / N261P, V157M, V157M / S161R / N169Q / N261G, V157M / S161R / A308V, V157M / N169Q / N261P / S298R / A308I, V157M / A308V, G250R, T302A, and E310S, wherein the amino acid positions of the peptide sequences are as per SEQ ID NO:60. In some embodiments, the engineered pantothenic kinase comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical peptide sequences to at least one engineered pantothenic kinase variant listed in Table 5-1. In some other embodiments, the engineered pantothenic acid kinase comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the same polypeptide sequence as SEQ ID NO:60. In some other embodiments, the engineered pantothenic acid kinase is a variant engineered polypeptide listed in SEQ ID NO:60.

[0015] In some embodiments, the present invention provides an engineered pantothenic 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 with SEQ ID NO:132, wherein the engineered pantothenic kinase contains at least one substitution or set of substitutions in the polypeptide sequence, and wherein the amino acid positions of the polypeptide sequence refer to the number SEQ ID NO:132. In some embodiments, the engineered pantothenic acid 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 with SEQ ID NO:132, and wherein the engineered pantothenic acid kinase comprises at least one substitution or set of substitutions at one or more positions selected from: 24 / 48, 64, 71, 123, 125, 134 and 180, wherein the amino acid positions of the polypeptide sequence refer to SEQ ID NO:132. In some further embodiments, the engineered pantothenic acid kinase comprises at least one substitution or set of substitutions selected from 24A / 48M, 64L, 71Q, 123H, 123W, 125G, 125S, 134R and 180E, wherein the amino acid positions of the polypeptide sequence refer to SEQ ID NO:132. In some additional embodiments, the engineered pantothenic acid kinase comprises at least one substitution or set of substitutions selected from R24A / L48M, F64L, R71Q, L123H, L123W, T125G, T125S, Q134R, and Y180E, wherein the amino acid positions of the polypeptide sequence are as per SEQ ID NO:132. In some embodiments, the engineered pantothenic acid 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 pantothenic acid kinase variant listed in Table 6-1. In some other embodiments, the engineered pantothenic acid kinase comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the same polypeptide sequence as SEQ ID NO:132. In some other embodiments, the engineered pantothenic acid kinase is a variant engineered polypeptide listed in SEQ ID NO:132.

[0016] In some other embodiments, the present invention provides an engineered pantothenic acid 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 with SEQ ID NO:132, wherein the engineered pantothenic acid kinase contains at least one substitution or set of substitutions in the polypeptide sequence, and wherein the amino acid positions of the polypeptide sequence refer to SEQ ID NO:132. In some embodiments, the engineered pantothenic acid 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 with SEQ ID NO:132, and wherein the engineered pantothenic acid kinase comprises at least one substitution or set of substitutions at one or more positions selected from 92 / 301, 125, and 180, wherein the amino acid positions of the polypeptide sequence refer to SEQ ID NO:132. In some further embodiments, the engineered pantothenic acid kinase comprises at least one substitution or set of substitutions selected from 92G / 301M, 125G, 125S, and 180E, wherein the amino acid positions of the polypeptide sequence refer to SEQ ID NO:132. In some additional embodiments, the engineered pantothenic acid kinase comprises at least one substitution or set of substitutions selected from S92G / L301M, T125G, T125S, and Y180E, wherein the amino acid positions of the polypeptide sequence refer to SEQ ID NO:132. In some embodiments, the engineered pantothenic acid kinase comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the sequence of at least one engineered pantothenic acid kinase variant listed in Table 6-2. In some additional embodiments, the engineered pantothenic acid kinase comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the polypeptide sequence of SEQ ID NO:132. In some other embodiments, the engineered pantothenic acid kinase is a variant engineered peptide listed in SEQ ID NO:132.

[0017] In some embodiments, the present invention provides an engineered pantothenic acid 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 with SEQ ID NO:222, wherein the engineered pantothenic acid kinase contains at least one substitution or set of substitutions in the polypeptide sequence, and wherein the amino acid positions of the polypeptide sequence refer to the number SEQ ID NO:222. In some embodiments, the engineered pantothenic acid 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 with SEQ ID NO:222, and wherein the engineered pantothenic acid kinase comprises at least one substitution or set of substitutions at one or more positions selected from 83 / 84 / 305, 143 and 154, wherein the amino acid positions of the polypeptide sequence refer to SEQ ID NO:222. In some further embodiments, the engineered pantothenic acid kinase comprises at least one substitution or set of substitutions selected from 83P / 84H / 305T, 143G and 154P, wherein the amino acid positions of the polypeptide sequence refer to SEQ ID NO:222. In some additional embodiments, the engineered pantothenic acid kinase comprises at least one substitution or set of substitutions selected from N83P / G84H / A305T, K143G, and H154P, wherein the amino acid positions of the polypeptide sequence refer to SEQ ID NO:222. In some embodiments, the engineered pantothenic acid kinase comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the sequence of at least one engineered pantothenic acid kinase variant listed in Table 7-1. In some additional embodiments, the engineered pantothenic acid kinase comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the polypeptide sequence of SEQ ID NO:222. In some other embodiments, the engineered pantothenic acid kinase is a variant engineered peptide listed in SEQ ID NO:222.

[0018] In some embodiments, the present invention provides an engineered pantothenic acid 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 with SEQ ID NO:230, wherein the engineered pantothenic acid kinase contains at least one substitution or set of substitutions in the polypeptide sequence, and wherein the amino acid positions of the polypeptide sequence refer to the number SEQ ID NO:230. In some embodiments, the engineered pantothenic acid 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 with SEQ ID NO:230, and wherein the engineered pantothenic acid kinase comprises at least one substitution or set of substitutions at one or more positions selected from the following: 13 / 169 / 213 / 247 / 283 / 288, 16 / 247, 49 / 247, 64 / 104 / 154 / 284, 75 / 104 / 284, 169 / 247 and 247, wherein the amino acid positions of the polypeptide sequence refer to the numbering of SEQ ID NO:230. In some other embodiments, the engineered pantothenic acid kinase comprises at least one substitution or set of substitutions selected from the following: 13H / 169S / 213E / 247L / 283S / 288T, 16E / 247L, 49D / 247L, 64I / 104F / 154P / 284V, 75Q / 104F / 284V, 169S / 247L, and 247L, wherein the amino acid positions of the polypeptide sequence are as specified in SEQ ID NO:230. In some additional embodiments, the engineered pantothenic acid kinase comprises at least one substitution or set of substitutions selected from the following: L13H / N169S / D213E / F247L / H283S / Q288T, D16E / F247L, E49D / F247L, F64I / T104F / H154P / L284V, V75Q / T104F / L284V, N169S / F247L, and F247L, wherein the amino acid positions of the polypeptide sequence are as per SEQ ID NO:230. In some embodiments, the engineered pantothenic acid 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 pantothenic acid kinase variant listed in Table 8-1.In some other embodiments, the engineered pantothenic acid kinase comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the same polypeptide sequence as SEQ ID NO:230. In some other embodiments, the engineered pantothenic acid kinase is a variant engineered polypeptide listed in SEQ ID NO:230.

[0019] In some other embodiments, the present invention provides an engineered pantothenic 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 with SEQ ID NO:240, wherein the engineered pantothenic kinase comprises at least one substitution or set of substitutions in the polypeptide sequence, and wherein the amino acid positions of the polypeptide sequence refer to the number SEQ ID NO:240. In some embodiments, the engineered pantothenic 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 with SEQ ID NO:240, and wherein the engineered pantothenic kinase contains at least one substitution or set of substitutions at one or more positions selected from the following: 8, 8 / 13 / 14 / 83 / 247, 8 / 64 / 213 / 247, 8 / 64 / 247, 8 / 247 / 283, 10 and 264 / 276, wherein the amino acid positions of the polypeptide sequence refer to the numbering of SEQ ID NO:240. In some other embodiments, the engineered pantothenic acid kinase comprises at least one substitution or set of substitutions selected from the following: 8E / 13H / 14A / 83P / 247L, 8E / 64I / 213E / 247L, 8E / 64I / 247L, 8E / 247L / 283S, 8W, 10S, and 264R / 276S, wherein the amino acid positions of the polypeptide sequence are as specified in SEQ ID NO:240. In some additional embodiments, the engineered pantothenic acid kinase comprises at least one substitution or set of substitutions selected from the following: L8E / L13H / Q14A / N83P / F247L, L8E / F64I / D213E / F247L, L8E / F64I / F247L, L8E / F247L / H283S, L8W, T10S, and K264R / T276S, wherein the amino acid positions of the polypeptide sequence are as per SEQ ID NO:240. In some embodiments, the engineered pantothenic acid 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 pantothenic acid kinase variant listed in Table 9-1. In some other embodiments, the engineered pantothenic acid kinase comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the same polypeptide sequence as SEQ ID NO:240.In some other embodiments, the engineered pantothenic acid kinase is a variant engineered peptide listed in SEQ ID NO:240.

[0020] In some embodiments, the present invention provides an engineered pantothenic acid 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 with SEQ ID NO:276, wherein the engineered pantothenic acid kinase contains at least one substitution or set of substitutions in the polypeptide sequence, and wherein the amino acid positions of the polypeptide sequence refer to the number SEQ ID NO:276. In some embodiments, the engineered pantothenic 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 with SEQ ID NO:276, and wherein the engineered pantothenic kinase comprises at least one substitution or set of substitutions at one or more positions selected from the following: 11 / 13 / 23 / 61 / 85 / 304, 13 / 19 / 61, 23 / 61 and 23 / 61 / 304, wherein the amino acid positions of the polypeptide sequence refer to the number SEQ ID NO:276. In some other embodiments, the engineered pantothenic acid kinase comprises at least one substitution or set of substitutions selected from 1Q / 13R / 23V / 61V / 85V / 304G, 13R / 19T / 61I, 23V / 61I / 304G, and 23V / 61V, wherein the amino acid positions of the polypeptide sequence are referenced to SEQ ID NO:276. In some other embodiments, the engineered pantothenic acid kinase comprises at least one substitution or set of substitutions selected from P11Q / L13R / L23V / L61V / Q85V / S304G, L13R / Q19T / L61I, L23V / L61I / S304G, and L23V / L61V, wherein the amino acid positions of the polypeptide sequence are referenced to SEQ ID NO:276. In some embodiments, the engineered pantothenic acid 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 pantothenic acid kinase variant listed in Table 10-1. In some further embodiments, the engineered pantothenic acid 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 SEQ ID NO:276. In some further embodiments, the engineered pantothenic acid kinase is a variant engineered polypeptide listed in SEQ ID NO:276.

[0021] In some further embodiments, the engineered pantothenic acid kinase comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the same polypeptide sequence as at least one engineered pantothenic acid kinase variant listed in the even-numbered sequences of SEQ ID NO:2-270. In some further embodiments, the engineered pantothenic acid kinase comprises the polypeptide sequence listed in the even-numbered sequences of SEQ ID NO:2-270. In some further embodiments, the engineered pantothenic acid kinase comprises at least one improved property compared to wild-type E. coli pantothenic acid kinase. In still other embodiments, the improved property includes improved activity against a substrate compared to wild-type pantothenic acid kinase. In some further embodiments, the substrate comprises 2,4-dinitrophenylhydrazine and at least one aldehyde. In some further embodiments, the improved property includes improved production of phospho-ethynylglyceraldehyde compared to wild-type pantothenic acid kinase. In some other embodiments, the engineered pantothenic acid kinase is purified. The present invention also provides compositions comprising at least one engineered pantothenic acid kinase provided herein. In some embodiments, the present invention provides compositions comprising one engineered pantothenic acid kinase provided herein.

[0022] The present invention also provides a polynucleotide sequence encoding at least one engineered pantothenic acid kinase provided herein. In some embodiments, the polynucleotide sequence encoding at least one engineered pantothenic acid kinase comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more of the sequence identity with SEQ ID NO:1, SEQ ID NO:29, SEQ ID NO:59, SEQ ID NO:131, SEQ ID NO:221, SEQ ID NO:229, SEQ ID NO:239 and / or SEQ ID NO:275. In some embodiments, the polynucleotide sequence of the engineered pantothenic acid kinase contains at least one substitution at one or more positions. In some further embodiments, the polynucleotide sequence encoding at least one engineered pantothenic acid kinase or a functional fragment thereof comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the sequence identity with SEQ ID NO:1. In some further embodiments, the polynucleotide sequence encoding at least one engineered pantothenic acid kinase or a functional fragment thereof comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the sequence identity with SEQ ID NO:29. In some further embodiments, the polynucleotide sequence encoding at least one engineered pantothenic acid kinase or a functional fragment thereof comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the sequence identity with SEQ ID NO:59. In some further embodiments, the polynucleotide sequence encoding at least one engineered pantothenic acid kinase or a functional fragment thereof comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the sequence identity with SEQ ID NO:131. In some other embodiments, the polynucleotide sequence encoding at least one engineered pantothenic acid kinase or a functional fragment thereof includes 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:221.In some other embodiments, the polynucleotide sequence encoding at least one engineered pantothenic acid kinase or a functional fragment thereof comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the sequence identity with SEQ ID NO:229. In some other embodiments, the polynucleotide sequence encoding at least one engineered pantothenic acid kinase or a functional fragment thereof comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the sequence identity with SEQ ID NO:239. In some further embodiments, the polynucleotide sequence encoding at least one engineered pantothenic acid kinase or a functional fragment thereof comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the sequence identity with SEQ ID NO:275. In some further embodiments, the polynucleotide sequence is operatively linked to a control sequence. In some embodiments, the polynucleotide sequence is codon-optimized. In some further embodiments, the polynucleotide comprises the odd-numbered sequences in SEQ ID NO:1-269. The present invention also provides an expression vector comprising at least one polynucleotide sequence encoding the pantothenic acid kinase provided herein. In some embodiments, the expression vector comprises a polynucleotide sequence encoding the pantothenic acid 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 the pantothenic acid kinase provided herein.

[0023] The present invention also provides a method for producing engineered pantothenic acid kinase in host cells, the method comprising culturing the host cells provided herein under suitable conditions to produce at least one engineered pantothenic acid kinase. In some embodiments, the method further includes recovering at least one engineered pantothenic acid kinase from the culture and / or host cells. In some other embodiments, the method further includes the step of purifying at least one engineered pantothenic acid kinase.

[0024] Invention Description

[0025] This invention provides engineered pantothenic acid kinase (PanK), polypeptides having PanK activity, and polynucleotides encoding these enzymes, as well as vectors and host cells containing these polynucleotides and polypeptides. A method for producing PanK enzymes is also provided. This invention further provides compositions containing PanK enzymes and methods for using engineered PanK enzymes. This invention is particularly useful for the production of pharmaceutical compounds.

[0026] Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein and the experimental procedures described below in cell culture, molecular genetics, microbiology, organic chemistry, analytical chemistry, and nucleic acid chemistry are those well known and commonly employed in the art. Such techniques are well known and have been described in numerous textbooks and references familiar to those skilled in the art. Standard techniques or modifications thereof are used for chemical synthesis and chemical analysis. All patents, patent applications, articles, and publications mentioned herein (both above and below) are hereby expressly incorporated by reference.

[0027] While any suitable methods and materials similar to or equivalent to those described herein may be used in the practice of this invention, some methods and materials are described herein. It should be understood that the invention is not limited to the specific methods, schemes, and reagents described, as these can be varied depending on how they are used by those skilled in the art. Therefore, the terminology defined below is described more fully by reference to the invention as a whole.

[0028] It should be understood that the general description above and the detailed description below are exemplary and illustrative only, and not limiting of the invention. Section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter. Numerical ranges include numbers that define the range. Therefore, each numerical range disclosed herein is intended to include every narrower numerical range falling within such a wider numerical range, as such narrower numerical ranges are clearly stated in full herein. It is also intended that each largest (or smallest) numerical limit disclosed herein includes every lower (or higher) numerical limit, as such lower (or higher) numerical limits are clearly stated herein.

[0029] abbreviation

[0030] The abbreviations for amino acids used in genetic coding 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).

[0031] 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 The amino acid sequence is represented by either the L- or D-configuration of the α-carbon. For example, "Ala" indicates alanine without specifying the configuration with respect to the α-carbon, while "D-Ala" and "L-Ala" indicate D-alanine and L-alanine, respectively. When using single-letter abbreviations, uppercase letters indicate amino acids with the L-configuration with respect to the α-carbon, and lowercase letters indicate amino acids with the D-configuration with respect to the α-carbon. For example, "A" indicates L-alanine and "a" indicates D-alanine. When a polypeptide sequence is presented as a string of single-letter or three-letter abbreviations (or a combination thereof), the sequence is presented in the amino (N) to carboxyl (C) direction according to conventional practice.

[0032] The abbreviations used for genetically encoded nucleosides are conventional and follow these rules: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). Unless specifically described, the abbreviation for nucleosides can be either ribonucleoside or 2'-deoxyribonucleoside. Nucleosides can be designated as ribonucleoside or 2'-deoxyribonucleoside based on their individual or collective designation. When nucleic acid sequences are presented as single-letter abbreviation strings, the sequences are presented in the 5' to 3' orientation according to conventional practice, and phosphate groups are not shown.

[0033] definition

[0034] With reference to this invention, the technical and scientific terms used in the description herein will have the meanings commonly understood by one of ordinary skill in the art, unless otherwise specifically defined. Therefore, the following terms are intended to have the following meanings.

[0035] Unless the context clearly indicates otherwise, the singular forms "a," "an," and "the" as used herein include plural referents. Thus, a reference to "a polypeptide," for example, includes more than one polypeptide.

[0036] Similarly, “comprise, comprise, comprising” and “include, include, and including” are interchangeable and not intended to be restrictive. Therefore, 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).

[0037] It should also be understood that, where the term “comprising” is used in the description of various implementation schemes, those skilled in the art will understand that, in some specific instances, the language of “substantially consisting of” or “consisting of” may optionally be used to describe the implementation scheme.

[0038] As used herein, the term “about” refers to the acceptable error for a particular value. In some instances, “about” means within 0.05%, 0.5%, 1.0%, or 2.0% of the given value range. In some instances, “about” means within 1, 2, 3, or 4 standard deviations of the given value.

[0039] As used in this article, "EC" designation refers to the enzyme nomenclature system of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). This IUBMB biochemical classification is a numerical classification system for enzymes based on enzyme-catalyzed chemical reactions.

[0040] As used in this article, "ATCC" refers to the American Type Culture Collection, whose biological collections include genes and strains.

[0041] As used herein, "NCBI" refers to the National Center for Biological Information and the sequence databases provided therein.

[0042] As used herein, “pantothenic acid kinase” or “PanK” refers to an enzyme that phosphorylates pantothenic acid to form 4'-phosphopantothenic acid (EC2.7.1.33) or a variant of such PanK enzyme, regardless of whether such variant enzyme retains the same function as the source (i.e., “parent”) enzyme.

[0043] The terms “protein,” “polypeptide,” and “peptide” are used interchangeably herein to refer to a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modifications (e.g., glycosylation or phosphorylation). This definition includes D-amino acids and L-amino acids, mixtures of D-amino acids and L-amino acids, and polymers comprising D-amino acids and L-amino acids, as well as mixtures of D-amino acids and L-amino acids.

[0044] Amino acids are referred to in this article either by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Biochemistry Nomenclature Committee. Similarly, nucleotides are referred to by their generally accepted single-letter codes.

[0045] As used herein, “hydrophilic amino acid or residue” refers to an amino acid or residue with a side chain exhibiting less than zero hydrophobicity according to the normalized common 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).

[0046] As used herein, “acidic amino acid or residue” refers to a hydrophilic amino acid or residue having a side chain exhibiting 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).

[0047] As used herein, "basic amino acid or residue" refers to a hydrophilic amino acid or residue having a side chain exhibiting a pKa value greater than about 6 when the amino acid is contained in a peptide or polypeptide. Basic amino acids typically have positively charged side chains at physiological pH due to association with hydrated hydrogen ions. Genetically encoded basic amino acids include L-Arg(R) and L-Lys(K).

[0048] 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 common electron pair of 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).

[0049] As used herein, “hydrophobic amino acid or residue” refers to an amino acid or residue with a side chain exhibiting hydrophobicity greater than zero according to the normalized common 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).

[0050] As used herein, “aromatic amino acid or residue” refers to a hydrophilic or hydrophobic amino acid or residue having a side chain comprising 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 its 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 a “constrained residue” (see below) herein.

[0051] As used herein, “restricted amino acid or residue” refers to an amino acid or residue having a restricted geometry. In this context, restricted residues include L-Pro(P) and L-His(H). Histidine has a restricted geometry because it has a relatively small imidazole ring. Proline has a restricted geometry because it also has a five-membered ring.

[0052] As used herein, "nonpolar amino acid or residue" refers to a hydrophobic amino acid or residue having a side chain that is uncharged at physiological pH and has a bond in which the electron pair shared by the two atoms is usually held equally by the two atoms (i.e., the side chain is not polar). Genetically encoded nonpolar amino acids include L-Gly (G), L-Leu (L), L-Val (V), L-Ile (I), L-Met (M), and L-Ala (A).

[0053] As used herein, “aliphatic amino acid or residue” refers to a hydrophobic amino acid or residue having an aliphatic hydrocarbon side chain. Genetically encoded aliphatic amino acids include L-Ala (A), L-Val (V), L-Leu (L), and L-Ile (I). Notably, cysteine ​​(or “L-Cys” or “[C]”) is uncommon because it can form disulfide bridges with other L-Cys (C) amino acids or other amino acids containing sulfonyl or thiol groups. “Cysine-like residues” include cysteine ​​and other amino acids containing a thiol moiety 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 the peptide in a reduced free -SH or oxidized disulfide bridging form affects whether L-Cys (C) contributes net hydrophobic or hydrophilic characteristics to the peptide. Although L-Cys(C) exhibits a hydrophobicity of 0.29 according to Eisenberg’s normalized common scale (Eisenberg et al., 1984, above), it should be understood that for the purposes of this disclosure, L-Cys(C) is classified as its own distinct group.

[0054] 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). Based on the above definition, small amino acids or residues can be further classified as aliphatic, nonpolar, 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).

[0055] As used in this article, "hydroxyl-containing amino acids or residues" refers to amino acids containing a hydroxyl (-OH) moiety. Genetically encoded hydroxyl-containing amino acids include L-Ser (S), L-Thr (T), and L-Tyr (Y).

[0056] As used herein, "polynucleotide" and "nucleic acid" refer to two or more nucleotides covalently linked together. A polynucleotide can be completely comprised of ribonucleotides (i.e., RNA), completely comprised of 2' deoxyribonucleotides (i.e., DNA), or a mixture comprising ribonucleotides and 2' deoxyribonucleotides. Although nucleosides are typically linked together via standard phosphodiester linkages, a polynucleotide can include one or more non-standard linkages. A polynucleotide can be single-stranded or double-stranded, or can 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 can comprise one or more modified and / or synthetic nucleobases, such as, for example, inosine, xanthine, hypoxanthine, etc. In some embodiments, such modified or synthetic nucleobases are nucleobases encoding amino acid sequences.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] As used herein, the terms "biocatalysis," "biocatalytic," "bioconversion," and "biosynthesis" refer to the use of enzymes to perform chemical reactions on organic compounds.

[0061] As used herein, "wild-type" and "naturally occurring" refer to forms found in nature. For example, a wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a natural source and has not been intentionally modified by human manipulation.

[0062] As used herein, when referring to cells, nucleic acids, or peptides, "recombinant," "engineered," "variant," and "non-natural" mean materials that have been modified in a manner not originally found in nature, or materials in their natural or organic form. In some embodiments, the cells, nucleic acids, or peptides are identical to naturally occurring cells, nucleic acids, or peptides, but are produced or derived from synthetic materials and / or manipulated using recombinant techniques. Non-limiting examples include, among others, recombinant cells expressing genes not found in naturally (non-recombinant) cells or expressing natural genes originally expressed at different levels.

[0063] The term "sequence identity percentage (%)" is used herein to refer to comparisons between polynucleotides or peptides and is determined by comparing two best-aligned sequences in a comparison window. The portion of the polynucleotide or peptide sequence in the comparison window compared to a reference sequence may include additions or deletions (i.e., vacancies) for optimal alignment of the two sequences. The percentage can be calculated by determining the number of positions in the two sequences where the same nucleic acid base or amino acid residue appears to generate the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the sequence identity percentage. Alternatively, the percentage can be calculated by determining the number of positions in the two sequences where the same nucleic acid base or amino acid residue appears or where the nucleic acid base or amino acid residue is aligned with a vacancy to generate the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the sequence identity percentage. Those skilled in the art will understand that many established algorithms exist for aligning two sequences. The best alignment of sequences for comparison can be performed by any suitable method, including but not limited to Smith and Waterman’s local homology algorithm (Smith and Waterman, Adv. Appl. Math., 2:482

[1981] ), by Needleman and Wunsch’s homology alignment algorithm (Needleman and Wunsch, J. Mol. Biol., 48:443

[1970] ), by Pearson and Lipman’s similarity search method (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 sequence identity percentages and sequence similarity include, but are not limited to, the BLAST and BLAST 2.0 algorithms, described by Altschul et al. (see 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 publicly available from the website of the National Center for Biotechnology Information (NCBI). 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 compared to words of the same length in a database sequence. T is referred to as the neighbor word score threshold (see Altschul et al., above).These initial neighboring word hits act as seeds to initiate the search for longer HSPs containing them. Word hits then extend in both directions along each sequence until the cumulative alignment score can no longer increase. For nucleotide sequences, the cumulative score is calculated using parameters M (reward score for matching residue pairs; always > 0) and N (penalty score for mismatched residues; always < 0). For amino acid sequences, a score matrix is ​​used to calculate the cumulative score. Word hit extension in each direction stops when: the cumulative alignment score decreases by an amount X from its maximum value; the cumulative score reaches 0 or less due to the accumulation of one or more negatively scored residues; or either sequence end 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) 11, expected value (E) 10, M = 5, N = -4, and comparison of two strands. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (See, Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915

[1989] ). Exemplary 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.

[0064] As used herein, a “reference sequence” refers to a defined sequence used as the basis for sequence and / or activity comparisons. A reference sequence can be a subset of a larger sequence, such as a segment of a full-length gene or polypeptide sequence. Typically, a reference sequence is at least 20 nucleotides or amino acid residues long, at least 25 residues long, at least 50 residues long, at least 100 residues long, or the full length of a nucleic acid or polypeptide. Because two polynucleotides or polypeptides can each (1) include sequences similar to each other (i.e., a portion of the complete sequence), and (2) can also include sequences different from each other, sequence comparisons between two (or more) polynucleotides or polypeptides are typically performed by comparing the sequences of the two polynucleotides or polypeptides in a “comparison window” to identify and compare sequence similarity in local regions. In some embodiments, a “reference sequence” can be based on a primary amino acid sequence, where the reference sequence is a sequence that may have one or more variations in the primary sequence.

[0065] As used herein, a “comparison window” refers to a conceptual segment of at least about 20 consecutive nucleotide positions or amino acid residues, wherein the 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, compared with the reference sequence (which does not contain additions or deletions), may include 20% or less of additions or deletions (i.e., vacancies) for optimal alignment of the two sequences. The comparison window may be longer than 20 consecutive residues and optionally includes windows of 30, 40, 50, 100, or longer.

[0066] As used herein, when referring to the numbering of a given amino acid or polynucleotide sequence, "corresponding to," "reference," or "relative to" means that residues in a specified reference sequence are numbered when the given amino acid or polynucleotide sequence is compared to a reference sequence. In other words, the residue numbers or positions of a given polymer are assigned with respect to a reference sequence, rather than by the actual numerical positions of residues within the given amino acid or polynucleotide sequence. For example, given an amino acid sequence, such as an engineered pantothenic acid kinase, the amino acid sequence can be optimized for residue matching between two sequences by introducing vacancies to align with a reference sequence. In these cases, residues in a given amino acid or polynucleotide sequence are numbered with respect to the reference sequence with which they are aligned, despite the presence of vacancies.

[0067] As used herein, “substantial identity” refers to a polynucleotide or polypeptide sequence having at least 80%, at least 85%, at least 89% to 95%, or more typically at least 99% sequence identity compared to a reference sequence within a comparison window of at least 20 residue positions, typically between 30 and 50 residue positions. The percentage of sequence identity is calculated by comparing the reference sequence with sequences containing 20% ​​or less of deletions or additions totaling the reference sequence within the comparison window. In some specific embodiments applied to polypeptides, the term “substantial identity” means that two polypeptide sequences share at least 80% sequence identity, preferably at least 89%, at least 95%, or more (e.g., 99%) when optimally aligned using default vacancy weights, such as via procedures like GAP or BESTFIT. In some embodiments, the different residue positions in the compared sequences differ due to conserved amino acid substitutions.

[0068] As used herein, "amino acid difference" and "residue difference" refer to the difference in the amino acid residue at one position of a 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 relative to an equivalent reference sequence without a histidine tag, the numbering remains unchanged. The position of the amino acid difference herein is generally referred to as "Xn", 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 in the amino acid residue at the polypeptide position corresponding to SEQ ID NO:4, position 93. Therefore, if the reference polypeptide of SEQ ID NO:4 has serine at position 93, then "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 SEQ ID NO:4, position 93. 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 instances (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 the residue position in the reference sequence, and B is a single-letter identifier for the residue substitution in the sequence of the engineered polypeptide. In some instances, 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 at which residue differences exist relative to the 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 further embodiments, the present invention provides engineered polypeptide sequences comprising both conservative amino acid substitutions and non-conservative amino acid substitutions.

[0069] As used herein, “conservative amino acid substitution” means replacing a residue with a different residue having a similar side chain, and therefore generally includes replacing an amino acid in a polypeptide with an amino acid from the same or similar amino acid definition class. For example, but not limited to, in some embodiments, an amino acid having an aliphatic side chain is replaced by another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid having a hydroxyl side chain is replaced by another amino acid having a hydroxyl side chain (e.g., serine and threonine); an amino acid having an aromatic side chain is replaced by another amino acid having an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid having a basic side chain is replaced by another amino acid having a basic side chain (e.g., lysine and arginine); an amino acid having an acidic side chain is replaced by another amino acid having an acidic side chain (e.g., aspartic acid or glutamic acid); and / or a hydrophobic or hydrophilic amino acid is replaced by another hydrophobic or hydrophilic amino acid, respectively.

[0070] 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 can use amino acids between rather than within defined groups and affect (a) the structure of the peptide backbone in the substituted region (e.g., proline replacing glycine), (b) charge or hydrophobicity, or (c) side-chain volume. For example, but not limited to, exemplary non-conservative substitutions could be the substitution of an acidic amino acid with a basic or aliphatic amino acid; the substitution of an aromatic amino acid with a small amino acid; and the substitution of a hydrophilic amino acid with a hydrophobic amino acid.

[0071] As used herein, "deletion" refers to a modification of a peptide by removing one or more amino acids from a reference peptide. Deletions may include the removal of one or more amino acids, two or more amino acids, five or more amino acids, ten or more amino acids, fifteen or more amino acids, or twenty or more amino acids, up to 10% or up to 20% of the total number of amino acids constituting the reference enzyme, while preserving enzyme activity and / or the improved properties of the engineered pantothenic acid kinase. Deletions may involve internal and / or terminal portions of the peptide. In various embodiments, deletions may include continuous segments or may be discontinuous. Deletions in the amino acid sequence are typically indicated by a "-".

[0072] As used herein, “insertion” refers to a modification of a polypeptide by adding one or more amino acids to a reference polypeptide. Insertions can be located within the polypeptide or at the carboxyl or amino terminus. Insertions as used herein include fusion proteins as known in the art. Insertions can be continuous segments of amino acids or separated by one or more amino acids in a naturally occurring polypeptide.

[0073] The term "amino acid substitution set" or "substitution set" refers to a group of amino acid substitutions in a polypeptide sequence compared to a reference sequence. The 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, the substitution set refers to the set of amino acid substitutions present in any variant pantothenic acid kinase listed in the tables provided in the examples.

[0074] "Functional fragment" and "biologically active fragment" are used interchangeably herein to refer to polypeptides having an amino-terminal deletion and / or a carboxy-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 being compared (e.g., a full-length engineered pantothenate kinase of the invention) and which retains substantially all of the activity of the full-length polypeptide.

[0075] As used herein, "isolated polypeptide" refers to a polypeptide that is substantially separate from other contaminants naturally associated with it, such as proteins, lipids, and polynucleotides. This term includes polypeptides that have been removed or purified from their natural environment or expression system (e.g., within host cells or via in vitro synthesis). Recombinant pantothenic acid kinase polypeptides can be present in cells, in cell culture media, or prepared in various forms, such as lysates or isolated products. Therefore, in some embodiments, the recombinant pantothenic acid kinase polypeptide may be an isolated polypeptide.

[0076] As used herein, "substantially pure polypeptide" or "purified protein" refers to a composition in which the polypeptide is the dominant substance (i.e., it is more abundant than any other individual macromolecule in the composition on a molar or weight basis) and is generally substantially purified when the target substance constitutes at least about 50% of the present macromolecules by molar or % weight. However, in some embodiments, compositions containing pantothenic acid kinase contain less than 50% pure (e.g., about 10%, about 20%, about 30%, about 40%, or about 50%) pantothenic acid kinase. Typically, substantially pure pantothenic acid kinase compositions constitute about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more by molar or % weight of all macromolecules present in the composition. In some embodiments, the target substance is purified to substantially homogeneity (i.e., contaminant substances are not detectable in the composition by conventional detection methods), wherein the composition consists substantially of a single macromolecule. Solvent species, small molecules (<500 Daltons), and elemental ionic species are not considered macromolecular species.In some embodiments, an isolated recombinant pantothenate kinase polypeptide is a substantially pure polypeptide composition.

[0077] As used herein, "improved enzyme properties" refers to at least one improved property of an enzyme. In some embodiments, the present invention provides engineered pantothenic kinase peptides exhibiting improved enzymatic properties compared to a reference pantothenic kinase peptide and / or a wild-type pantothenic kinase peptide and / or another engineered pantothenic kinase peptide. Therefore, the level of "improvement" can be determined and compared among various pantothenic kinase peptides, including wild-type and engineered pantothenic kinases. Improved properties include, but are not limited to, properties such as: increased protein expression, increased thermoactivity, increased thermal stability, 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 chemoselectivity, improved solvent stability, increased tolerance to acidic pH, increased tolerance to proteolytic activity (i.e., reduced sensitivity to proteolysis), reduced aggregation, increased solubility, and altered temperature profile. In other embodiments, the term is used to refer to at least one improved property of pantothenic kinase. In some embodiments, the present invention provides engineered pantothenic kinase peptides that exhibit improved enzymatic properties compared to a reference pantothenic kinase peptide and / or a wild-type pantothenic kinase peptide and / or another engineered pantothenic kinase peptide. Therefore, the level of “improvement” among various pantothenic kinase peptides, including wild-type and engineered pantothenic kinases, can be determined and compared.

[0078] As used herein, “increased enzyme activity” and “enhanced catalytic activity” refer to improved properties of the engineered peptide, which can be expressed as an increase in specific activity (e.g., product / time / weight protein produced) or a percentage conversion of substrate to product (e.g., percentage conversion of a given amount of substrate to product using a given amount of enzyme over a specified time period). In some embodiments, the term refers to improved properties of the engineered pantothenic kinase peptide provided herein, which can be expressed as an increase in specific activity (e.g., product / time / weight protein produced) or a percentage conversion of substrate to product (e.g., percentage conversion of a given amount of pantothenic kinase to product over a specified time period using a given amount of pantothenic kinase) compared to a reference pantothenic kinase. In some embodiments, these terms are used to refer to the improved pantothenic kinase provided herein. Exemplary methods for determining the enzyme activity of the engineered pantothenic kinase of the present invention are provided in the examples. Any properties associated with enzyme activity, including typical enzyme properties Km, Vmax, or kcat, can be affected, and changes to these can lead to increased enzyme activity. For example, improvements in enzyme activity can range from approximately 1.1 times the activity of the corresponding wild-type enzyme to up to 2, 5, 10, 20, 25, 50, 75, 100, 150, 200, or greater compared to another engineered pantothenic acid kinase derived from naturally occurring pantothenic acid kinase or pantothenic acid kinase peptide.

[0079] As used herein, “conversion” refers to the enzymatic conversion (or bioconversion) of one or more substrates into one or more corresponding products. “Conversion percentage” refers to the percentage of substrate that is converted into a product under specified conditions within a certain time period. Therefore, the “enzyme activity” or “activity” of a pantothenic acid kinase peptide can be expressed as the “conversion percentage” of the substrate to the product within a specific time period.

[0080] Enzymes with "generalist properties" (or "generalist enzymes") are enzymes that exhibit improved activity for a wide range of substrates compared to their parental sequences. Generalist enzymes do not necessarily exhibit improved activity for every possible substrate. In some embodiments, the present invention provides pantothenic kinase variants with generalist properties because they exhibit similar or improved activity for a wide range of spatially and electronically different substrates relative to their parent genes. Furthermore, the generalist enzymes provided herein are engineered to be modified across a wide range of differentiated molecules to increase the production of metabolites / products.

[0081] The term "strict hybridization conditions" is used herein to refer to conditions under which nucleic acid hybrids are stable. As those skilled in the art know, the stability of a hybrid is reflected in its melting temperature (Tm). Typically, the stability of a hybrid is a function of ionic strength, temperature, G / C content, and the presence of a ionizing agent. The Tm value of a polynucleotide can be calculated using known methods for predicting melting temperature (see, for example, 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] (errata, Nucl. Acids Res.) Res., 19:698

[1991] ; Sambrook et al., above; 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 the polypeptide disclosed herein and hybridizes under defined conditions, such as moderately or highly stringent conditions, with a complementary sequence encoding the engineered pantothenic acid kinase of the present invention.

[0082] As used herein, “hybridization strictness” refers to the hybridization conditions, such as washing conditions, in nucleic acid hybridization. Typically, hybridization reactions are carried out under conditions of lower strictness, followed by different but higher-strictness washing. The term “moderately strict hybridization” refers to conditions that allow target DNA to bind to complementary nucleic acids having approximately 60% identity with the target DNA, preferably approximately 75% or approximately 85% identity, and greater than approximately 90% identity with the target polynucleotide. An exemplary moderately strict condition is equivalent to hybridization at 42°C in 50% formamide, 5× Denhart solution, 5× SSPE, and 0.2% SDS, followed by washing at 42°C in 0.2× SSPE and 0.2% SDS. “Highly strict hybridization” generally refers to conditions that differ from the pyrolysis temperature Tm, as determined for the defined polynucleotide sequence under solution conditions, by approximately 10°C or less. In some implementations, high stringency conditions refer to hybridization conditions that only allow hybridization of nucleic acid sequences that form stable hybrids in 0.018 M NaCl at 65 °C (i.e., if a hybrid is unstable in 0.018 M NaCl at 65 °C, it is unstable under high stringency conditions as considered herein). High stringency conditions can be provided, for example, by hybridization under conditions equivalent to hybridization in 50% formamide, 5× Denhart solution, 5× SSPE, and 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 in 5× SSC containing 0.1% (w / v) SDS at 65 °C and washing in 0.1× SSC containing 0.1% SDS at 65 °C. Other high stringency hybridization conditions, as well as moderate stringency conditions, are described in the references cited above.

[0083] As used herein, "codon-optimized" refers to the codon change of a polynucleotide encoding a protein to those codons preferentially used in a particular organism, thereby enabling efficient expression of the encoded protein in the organism of interest. Although the genetic code is degenerate, meaning that most amino acids are represented by a few codons called "synonyms," it is well known that codon use in a particular organism is non-random and biased towards specific codon triplets. This codon use bias can be even greater for a given gene, genes with common functions or ancestral origins, highly expressed proteins compared to low-copy-number proteins, and protein-coding regions of an organism's genome. In some embodiments, polynucleotides encoding pantothenic acid kinase can be codon-optimized for optimized production in a selected host organism for expression.

[0084] As used herein, the terms "preferred," "optimal," and "high codon usage bias," when used alone or in combination, can interchangeably refer to codons used in protein-coding regions 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 with a common function or origin, codon usage in highly expressed genes, codon frequencies in aggregated protein-coding regions throughout an organism, codon frequencies in aggregated protein-coding regions of a related organism, or combinations 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 frequencies (e.g., codon usage, relative synonymous codon usage) and codon preferences and the effective number of codons used in a gene in a particular organism, including multivariate analysis, such as using cluster analysis or correlation analysis (see, for example, GCG CodonPreference, 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 are available for many different organisms (see, for example, Wada et al., Nucl. Acids Res., 20:2111-2118

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

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

[1996] ). The data source for obtaining codon usage can depend on any available nucleotide sequence capable of encoding a protein.These datasets include nucleic acid sequences of proteins that are actually known to encode expressed proteins (e.g., complete protein-coding sequences - CDS), expressed sequence tags (ESTS), or predicted coding regions of genome sequences (see, for example, 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] ).

[0085] As used herein, "control sequence" includes all components essential 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, polyadenylated sequences, propeptide sequences, promoter sequences, signal peptide sequences, initiation sequences, and transcription terminators. At a minimum, control sequences include promoters as well as transcription and translation termination signals. For the purpose of introducing specific restriction sites, control sequences may be provided with adapters that facilitate the attachment of the control sequence to the coding region of the nucleic acid sequence encoding the polypeptide.

[0086] "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 the polynucleotide of interest, such that the control sequence directs or regulates the expression of the polynucleotide of interest and / or polypeptide.

[0087] A "promoter sequence" is a nucleic acid sequence that is recognized by the host cell for the expression of a polynucleotide of interest, such as a coding sequence. A promoter sequence contains the transcriptional control sequence that mediates the expression of the polynucleotide of interest. A promoter can be any nucleic acid sequence that exhibits transcriptional activity in a selected host cell, including mutant, truncated, and heterozygous promoters, and can be derived from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to those of the host cell.

[0088] The phrase "suitable reaction conditions" refers to those conditions (e.g., ranges of enzyme loading, substrate loading, temperature, pH, buffer solution, solubilizer, etc.) in the enzymatic conversion reaction solution under which the pantothenic acid kinase peptide of the present invention can convert the substrate into the desired product compound. Some exemplary "suitable reaction conditions" are provided herein.

[0089] As used in this article, “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.

[0090] As used herein, in the context of enzymatic conversion reactions, "substrate" refers to a compound or molecule acted upon by an engineered enzyme (e.g., an engineered pantothenic acid kinase polypeptide) provided herein.

[0091] As used herein, the “increased” yield of the product (e.g., deoxyribose phosphate analogue) produced by the reaction occurs when a specific component (e.g., pantothenic acid kinase) present during the reaction results in the production of more product compared to a reaction carried out under the same conditions with the same substrate and other substitutes, but without the component of interest.

[0092] If a particular enzyme is present in less than about 2%, about 1%, or about 0.1% (wt / wt) of other enzymes involved in the catalytic reaction, the reaction is said to be "substantially free" of that enzyme.

[0093] As used herein, “fractionating” liquids (e.g., cultured broths) refer to solutions provided by applying separation processes (e.g., salt precipitation, column chromatography, size exclusion, and filtration) or combinations thereof, in which the desired percentage of protein in the solution is greater than that in the initial liquid product.

[0094] As used herein, "starting composition" means any composition comprising at least one substrate. In some embodiments, the starting composition comprises any suitable substrate.

[0095] As used in this article, in the context of enzymatic conversion processes, "product" refers to a compound or molecule produced from the action of an enzyme polypeptide on a substrate.

[0096] As used herein, “equilibrium” refers to a process that produces a steady-state concentration of a chemical substance in a chemical or enzymatic reaction, as determined by the forward and reverse rate constants of the chemical or enzymatic reaction (e.g., the interconversion of two substances A and B), including the interconversion of stereoisomers.

[0097] As used herein, "alkyl" means a saturated hydrocarbon group having 1 to 18 carbon atoms (including endpoints), whether straight-chain or branched, more preferably 1 to 8 carbon atoms (including endpoints), and most preferably 1 to 6 carbon atoms (including endpoints). Alkyl groups having a specified number of carbon atoms are indicated in parentheses (e.g., (C1-C4) alkyl means alkyl with 1 to 4 carbon atoms).

[0098] As used herein, "alkenyl" means a straight or branched group having 2 to 12 carbon atoms (including the endpoints) and containing at least one double bond but optionally more than one double bond.

[0099] As used herein, “alkynyl” means a straight or branched group having 2 to 12 carbon atoms (including the endpoints), containing at least one triple bond but optionally more than one triple bond, and optionally also containing one or more double bonded portions.

[0100] As used herein, "heteroalkyl", "heteroalkenyl" and "heteroalkynyl" refer to alkyl, alkenyl and alkynyl groups as defined herein in which one or more carbon atoms are each independently replaced by the same or different heteroatoms or heteroatom groups. Heteroatoms and / or heteroatom groups that may 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-, and the like, including combinations thereof, wherein each Rα is independently selected from hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl.

[0101] As used herein, “alkoxy” refers to the group -ORβ, where Rβ is an alkyl group as defined above, including optionally substituted alkyl groups as defined herein.

[0102] As used herein, "aryl" refers to an unsaturated aromatic carbocyclic group having 6 to 12 carbon atoms (including the endpoints) having a monocyclic (e.g., phenyl) or more than one fused ring (e.g., naphthyl or anthracene). Exemplary aryl groups include phenyl, pyridyl, naphthyl, etc.

[0103] As used herein, "amino" refers to the group -NH2. Substituted amino refers to the groups -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, and the like. Typical amino groups include, but are not limited to, dimethylamino, diethylamino, trimethylammonium, triethylammonium, methylsulfonylamino, furanyl-oxy-sulfonylamino, and the like.

[0104] As used herein, "oxo" refers to =0.

[0105] As used in this article, "oxygen group" refers to a divalent group -O-, which can have various substituents to form different oxygen groups, including ethers and esters.

[0106] As used herein, "carboxyl" refers to -COOH.

[0107] As used herein, "carbonyl" refers to -C(O)-, which can have various substituents to form different carbonyl groups, including acids, acyl halides, aldehydes, amides, esters, and ketones.

[0108] As used herein, “alkoxycarbonyl” refers to -C(O)ORε, where Rε is an alkyl group as defined herein, which may optionally be substituted.

[0109] As used herein, "aminocarbonyl" refers to -C(O)NH2. The substituted aminocarbonyl group refers to -C(O)NRδRδ, where the amino group NRδRδ is as defined herein.

[0110] As used in this article, "halogen" and "halo" refer to fluorine, chlorine, bromine, and iodine.

[0111] As used in this article, "hydroxyl group" refers to -OH.

[0112] As used in this article, "cyano" refers to -CN.

[0113] As used herein, “heteroaryl” refers to an aromatic heterocyclic group having one to ten carbon atoms (including the endpoints) and one to four heteroatoms (including the endpoints) selected from oxygen, nitrogen, and sulfur within the ring. Such heteroaryl groups may have a monocyclic ring (e.g., pyridyl or furanyl) or more than one fused ring (e.g., indolizinyl or benzothiophene).

[0114] 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 (including the endpoints) in the alkyl moiety and 5 to 12 ring atoms (including the endpoints) in the heteroaryl moiety. Such heteroarylalkyl groups are exemplified by pyridylmethyl groups, etc.

[0115] As used herein, “heteroaryl alkenyl” refers to an alkenyl group substituted with a heteroaryl group (i.e., a heteroaryl-alkenyl-group), preferably having 2 to 6 carbon atoms (including the endpoints) in the alkenyl moiety and 5 to 12 ring atoms (including the endpoints) in the heteroaryl moiety.

[0116] As used herein, “heteroarylynyl” refers to an ynyl group substituted with a heteroaryl group (i.e., a heteroaryl-ynyl- group), preferably having 2 to 6 carbon atoms (including the endpoints) in the ynyl moiety and 5 to 12 ring atoms (including the endpoints) in the heteroaryl moiety.

[0117] 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 from 2 to 10 carbon ring atoms (inclusive) and from 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.

[0118] As used herein, “membered ring” refers to any ring structure. The number preceding the term “membered” indicates the number of skeleton atoms that make up the ring. Thus, for example, cyclohexyl, pyridine, pyran, and thioran are 6-membered rings, while cyclopentyl, pyrrole, furan, and thiophene are 5-membered rings.

[0119] Unless otherwise indicated, the positions occupied by hydrogen in the foregoing groups may be further substituted with substituents such as, but not limited to, hydroxy, oxo, nitro, methoxy, ethoxy, alkoxy, substituted alkoxy, trifluoromethoxy, haloalkoxy, fluoro, chloro, bromo, iodo, 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 will be understood that where open valences exist on these substituents, they may be further substituted with alkyl, cycloalkyl, aryl, heteroaryl, and / or heterocyclic groups, and where these 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 a new heteroatom (preferably oxygen, nitrogen, or sulfur). It will also be understood that the above substitutions may be made provided that replacement of hydrogen with a substituent does not introduce unacceptable instability into the molecule of the invention and is otherwise chemically reasonable.

[0120] As used herein, the term “culture” refers to the growth of a microbial cell population under any suitable conditions (e.g., using liquid, gel, or solid culture media).

[0121] Recombinant peptides can be generated using any suitable method known in the art. A gene encoding the wild-type peptide of interest can be cloned into a vector such as a plasmid and expressed in a desired host such as *E. coli*. Variants of recombinant peptides can be generated using a variety of methods known in the art. In fact, a wide variety of different mutagenesis techniques are well known to those skilled in the art. Furthermore, mutagenesis kits are available from many commercial molecular biology vendors. Methods can be used to make specific substitutions at defined amino acid sites, specific (region-specific) or random mutations in local regions of a gene, or random mutagenesis throughout the gene (e.g., saturation mutagenesis). Many suitable methods for generating enzyme variants are known to those skilled in the art, including but not limited to site-directed mutagenesis of single-stranded or double-stranded DNA using PCR, cassette mutagenesis, gene synthesis, error-prone PCR, shuffling, and chemical saturation mutagenesis, or any other suitable method known in the art. Mutagenesis and directed evolution methods can be readily applied to polynucleotides encoding enzymes to generate libraries of variants that can be expressed, screened, and assayed. 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, 6,352,85 9、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, 7,0 58,515, 7,105,297, 7,148,054, 7,220,566, 7,288,375, 7,384,387, 7,421,347, 7,430,477, 7,462,469, 7,534,564, 7,620,500, 7,620,502, 7,629 170, 7,702,464, 7,747,391, 7,747,393, 7,751,986, 7,776,598, 7,783,428, 7,795,030, 7,853,410, 7,868,138, 7,783,428, 7,873,477, 7,873,499 7,904,249, 7,957,912, 7,981,614, 8,014,961, 8,029,988, 8,048,674, 8,058,001, 8,076,138, 8,108,150, 8,170,806, 8,224,580, 8,377,681, 8,383,346, 8,457,903, 8,504,498, 8,589,085, 8,762,066, 8,768,871, 9,593,326, and all relevant U.S. and PCT and non-U.S. corresponding 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 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.

[0122] In some embodiments, the enzyme clones obtained after the mutagenesis treatment are screened by subjecting the enzyme preparation to a determined temperature (or other assay conditions) and measuring the amount of enzyme activity remaining after the 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).

[0123] After 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 pantothenate kinase polypeptides" (also referred to herein as "engineered pantothenate kinase polypeptides," "variant pantothenate kinases," "pantothenate kinase variants," and "pantothenate kinase combination variants") can be used. In some embodiments, "recombinant pantothenate kinase polypeptides" (also referred to herein as "engineered pantothenate kinase polypeptides," "variant pantothenate kinases," "pantothenate kinase variants," and "pantothenate kinase combination variants") can be used.

[0124] As used herein, a “vector” is a DNA construct for introducing a DNA sequence into a cell. In some embodiments, a vector is an expression vector operatively linked to a suitable control sequence capable of enabling the expression of a polypeptide encoded in the DNA sequence in a suitable host. In some embodiments, the “expression vector” has a promoter sequence operatively linked to a DNA sequence (e.g., a transgene) to drive expression in a host cell, and in some embodiments, also includes a transcription terminator sequence.

[0125] As used herein, the term "expression" includes any step involved in peptide production, including but not limited to transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also covers the secretion of peptides from cells.

[0126] As used herein, the term "production" refers to the generation of proteins and / or other compounds from cells. It is intended that the term encompass any step involved in peptide production, including but not limited to transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses the secretion of peptides from cells.

[0127] As used herein, two sequences are “heterologous” if an amino acid or nucleotide sequence (e.g., a promoter sequence, signal peptide, terminator sequence, etc.) and another sequence to which it is operatively linked do not associate in nature. For example, “heterologous polynucleotide” is any polynucleotide introduced into a host cell through laboratory techniques, and includes polynucleotides that have been removed from a host cell, subjected to laboratory manipulation, and then reintroduced into a host cell.

[0128] As used herein, the terms “host cell” and “host strain” refer to a suitable host for an expression vector containing DNA (e.g., a polynucleotide encoding a variant of pantothenic acid kinase) provided herein. 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.

[0129] The term "analyte" refers to a polypeptide that shares more than 70% but less than 100% sequence identity with a reference polypeptide (e.g., more than 75%, 78%, 80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity). In some embodiments, an analogue refers to a polypeptide comprising one or more non-naturally occurring amino acid residues (including but not limited to homoarginine, ornithine, and valine) and naturally occurring amino acids. In some embodiments, an analogue also includes one or more D-amino acid residues and non-peptide linkages between two or more amino acid residues.

[0130] 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.

[0131] The terms "isolated" and "purified" are used to refer to molecules (e.g., isolated nucleic acids, polypeptides, etc.) or other components that are removed from at least one other component with which they are naturally associated. The term "purified" does not require absolute purity, but is intended as a relative definition.

[0132] As used herein, “stereoselectivity” refers to the preferential formation of one stereoisomer relative to another in a chemical or enzymatic reaction. Stereoselectivity can be partial, in which case the formation of one stereoisomer is preferred over the other, or it can be complete, in which case only one stereoisomer is formed. When the stereoisomers are enantiomers, stereoselectivity is called enantiomerism, which is the fraction (usually reported as a percentage) of one enantiomer in the sum of the two. Alternatively, it is usually reported in the art as an enantiomer excess (“ee”) (usually a percentage) calculated from the following formula: [major enantiomer - minor enantiomer] / [major enantiomer + minor enantiomer]. When the stereoisomers are diastereomers, stereoselectivity is called diastereoselectivity, which is the fraction (usually reported as a percentage) of one diastereomer in a mixture of two diastereomers, and is usually optionally reported as a diastereomer excess (“de”). Enantiomeric excess and diastereomeric excess are types of stereoisomeric excess.

[0133] As used herein, the terms “regioselectivity” and “regioselective reaction” refer to a reaction in which one direction of bond formation or breaking occurs preferentially over all other possible directions. A reaction may be fully (100%) regioselective if the distinction is absolute, substantially regioselective (at least 75%) if the reaction product at one site is superior to the reaction product at other sites, or partially regioselective (x%), where the percentage depends on the reaction of interest.

[0134] As used herein, "chemoselectivity" refers to the preferential formation of one product over another in a chemical or enzymatic reaction.

[0135] As used herein, “pH stable” refers to a pantothenic acid kinase peptide that maintains similar activity (e.g., more than 60% to 80%) after exposure to high or low pH (e.g., 4.5–6 or 8–12) for a period of time (e.g., 0.5–24 hours) compared to untreated enzymes.

[0136] As used herein, “thermally stable” refers to a pantothenic kinase peptide 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., 0.5 h to 24 h) compared to a wild-type enzyme exposed to elevated temperatures (e.g., 40 °C to 80 °C).

[0137] As used herein, "solvent stable" refers to a pantothenate kinase polypeptide that maintains similar activity (e.g., greater than 60% to 80%) after exposure to the same solvent at the same concentration for a period of time (e.g., 0.5 h to 24 h) 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.).

[0138] As used in this article, "thermally stable and solvent stable" refers to pantothenic acid kinase peptides that are both thermally and solvent stable.

[0139] 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.

[0140] As used herein, "optionally substituted" refers to all subsequent modifiers in a chemical group or a series of chemical groups. For example, in the term "optionally substituted arylalkyl", the "alkyl" portion and the "aryl" portion of the molecule may or may not be substituted, and for a series of "optionally substituted alkyl, cycloalkyl, aryl and heteroaryl", the alkyl group, cycloalkyl group, aryl group and heteroaryl group independently of each other may or may not be substituted. Detailed Description of the Invention

[0142] This invention provides engineered pantothenic acid kinase (PanK), polypeptides having PanK activity, and polynucleotides encoding these enzymes, as well as vectors and host cells containing these polynucleotides and polypeptides. A method for producing PanK enzymes is also provided. This invention further provides compositions containing PanK enzymes and methods for using engineered PanK enzymes. This invention is particularly useful for the production of pharmaceutical compounds.

[0143] In some embodiments, the present invention provides enzymes suitable for producing the following: phosphorylated glycerol derivatives and glyceraldehyde derivatives having bulky substituents on the C2 carbon of glycerol, in particular phosphorylated ethynyl-glycerol and ethynyl-glyceraldehyde, which are intermediates in the in vitro enzymatic synthesis of non-natural nucleoside analogs represented by compound (1).

[0144]

[0145] 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 oxidizing a glycerol derivative (7) with alcohol oxidase. After glyceraldehyde is formed, it can be phosphorylated by PanK to the desired intermediate (5), as shown in Scheme 1.

[0146]

[0147] Engineered PanK peptide

[0148] This invention provides engineered PanK peptides, polynucleotides encoding the peptides, methods for preparing the peptides, and methods for using the peptides. When describing a peptide, it should be understood that it also describes the polynucleotides encoding the peptide. In some embodiments, this invention provides engineered, non-naturally occurring PanK enzymes with improved properties compared to wild-type PanK enzymes. Any suitable reaction conditions can be used in this invention. In some embodiments, methods are used to analyze the improved properties of the engineered peptides undergoing isomerization reactions. In some embodiments, as further described below and in the examples, reaction conditions are varied based on the engineered PanK, one or more substrates, one or more buffers, one or more solvents, the concentration or amount of cofactors, pH, conditions including temperature and reaction time, and / or the conditions under which the engineered PanK peptide is immobilized 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, or shift the reaction equilibrium toward desired product formation.

[0150] In some further embodiments, any of the above-described methods for converting a substrate compound into a product compound may further comprise one or more steps selected from the group consisting of extraction, separation, purification, crystallization, filtration, and / or lyophilization of the 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 can be obtained through routine experimentation. In addition, illustrative methods are provided in the Examples below.

[0151] Engineered PanK polynucleotides encoding engineered peptides, expression vectors, and host cells

[0152] The present invention provides polynucleotides encoding the engineered enzyme polypeptides described herein. In some embodiments, the polynucleotides are operably linked to one or more heterologous regulatory sequences that control gene expression to produce a recombinant polynucleotide capable of expressing the 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 skilled artisan, the availability of protein sequences and knowledge of the codons corresponding to the various amino acids provides a description of all polynucleotides capable of encoding the subject polypeptides. The degeneracy of the genetic code, in which the same amino acid is encoded by alternative or synonymous codons, allows the preparation of a very large number of nucleic acids, all of which encode engineered enzyme (e.g., PanK) polypeptides. Thus, the present invention provides methods and compositions for generating each and every possible variation of a preparable enzyme polynucleotide encoding the enzyme polypeptides described herein by selecting combinations based on the 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 Examples (e.g., in the various Tables).

[0154] In some embodiments, codons are preferably optimized for use in the selected host cell for protein production. For example, preferred codons used in bacteria are typically used for expression in bacteria. Thus, codon-optimized polynucleotides encoding engineered enzyme polypeptides contain preferred codons at approximately 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 the SEQ ID NO provided herein or any variant thereof (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 variants disclosed in the examples. In some embodiments, the reference polypeptide sequence includes SEQ ID NO:2, while in other embodiments, the reference polypeptide sequence includes SEQ ID NO:30, SEQ ID NO:60, SEQ ID NO:132, SEQ ID NO:222, SEQ ID NO:230, SEQ ID NO:240 and / or SEQ ID NO:276.

[0156] In some embodiments, the polynucleotide is capable of hybridizing under highly stringent conditions with a reference polynucleotide sequence selected from any polynucleotide sequence provided herein, or its complementary sequence, or with a polynucleotide sequence encoding any variant enzyme polypeptide provided herein. In some embodiments, the polynucleotide capable of hybridizing under highly stringent conditions encodes an enzyme polypeptide comprising an amino acid sequence that differs from the reference sequence by one or more residues.

[0157] In some embodiments, the isolated polynucleotide encoding any of the engineered enzyme peptides described herein is manipulated in various ways to promote the expression of the enzyme peptide. In some embodiments, the polynucleotide encoding the enzyme peptide constitutes an expression vector in which one or more control sequences are present to regulate the expression of the enzyme polynucleotide and / or peptide. Depending on the expression vector used, manipulation of the isolated polynucleotide prior to insertion into the vector may be desired or necessary. Techniques for modifying polynucleotide and nucleic acid sequences using recombinant DNA methods are well known in the art. In some embodiments, the control sequences include, among others, a promoter, a leader sequence, a polyadenylation sequence, a propeptide sequence, a signal peptide sequence, and a transcription terminator. In some embodiments, a suitable promoter is selected based on the choice of host cell. For bacterial host cells, suitable promoters for guiding the transcription of nucleic acid constructs of this disclosure include, but are not limited to, promoters obtained from: the *Escherichia coli* lac operon, the *Streptomyces coelicolor* agarose glycosylase gene (dagA), the *Bacillus subtilis* fructan sucrase gene (sacB), the *Bacillus licheniformis* α-amylase gene (amyL), the *Bacillus thermophilus* maltose amylase gene (amyM), the *Bacillus amyloliquefaciens* α-amylase gene (amyQ), the *Bacillus licheniformis* penicillinase gene (penP), the *Bacillus subtilis* xylA and xylB genes, and prokaryotic β-lactamase genes (see, for example, 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 α-amylase, niger acid-stable α-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 trypsin-like protease (see, e.g., WO 98 / 05266). 96 / 00787), and the NA2-tpi promoter (a hybrid of the promoters from the Aspergillus niger neutral α-amylase gene and the Aspergillus oryzae triose phosphate isomerase gene), and mutants, truncations, and hybrid promoters thereof. Exemplary yeast cell promoters can be derived from the following genes: 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 linked to the 3' end of the nucleic acid sequence encoding the enzyme polypeptide. Any suitable terminator having a function in the host cell of selection 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 untranslated region of mRNA important for translation by the host cell). In some embodiments, the leader sequence is operatively linked to the 5' end of the nucleic acid sequence encoding the enzyme polypeptide. Any suitable leader sequence that is functional in a selected host cell can be used in this invention. Exemplary leader sequences for filamentous fungal host cells are obtained from the genes of Aspergillus oryzae TAKA amylase and Aspergillus nidulans triphosphate isomerase. Suitable leader sequences for yeast host cells are obtained from the genes of 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 operatively linked to the 3' end of a nucleic acid sequence and recognized by the host cell during transcription as a signal to add polyadenylation residues to the transcribed mRNA). Any suitable polyadenylation sequence that is functional in a selected host cell can be used in this invention. Exemplary polyadenylation sequences for filamentous fungal host cells include, but are not limited to, genes such as Aspergillus oryzae TAKA amylase, Aspergillus niger glucosylase, Aspergillus nidulans o-aminobenzoic acid synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger α-glucosidase. Useful polyadenylation sequences for yeast host cells are known (see, for example, 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 of an amino acid sequence that is linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell's secretory pathway). In some embodiments, the 5' end of the coding sequence of the nucleic acid sequence inherently contains a signal peptide coding region that is naturally linked in a translation reading frame to a segment of the coding region of the secreted polypeptide. Alternatively, in some embodiments, the 5' end of the coding sequence contains a signal peptide coding region foreign to the coding sequence. Any suitable signal peptide coding region in the secretory pathway that directs the expressed polypeptide into the selected host cell can be used for the expression of one or more engineered polypeptides. Valid signal peptide coding regions for bacterial host cells include, but are not limited to, those obtained from the following genes: Bacillus NClB 11837 maltose amylase, Bacillus stearothermophilus α-amylase, Bacillus subtilis protease, Bacillus stearothermophilus β-lactamase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM), and Bacillus subtilis prsA. Other signal peptides are known in the art (see, for example, Simonen and Palva, Microbiol. Rev., 57:109-137

[1993] ). In some embodiments, signal peptide coding regions effective for filamentous fungal host cells include, but are not limited to, signal peptide coding regions derived from the following genes: Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucosylase, Rhizopus oryzae aspartic protease, Humicola insolens cellulase, and Humicola lanuginosa lipase. Useful signal peptides for yeast host cells include, but are not limited to, those derived from the following genes: Saccharomyces cerevisiae α-factor and Saccharomyces cerevisiae invertase.

[0162] In some embodiments, the control sequence is also a propeptide coding region encoding an amino acid sequence located at the N-terminus of the polypeptide. The resulting polypeptide is referred to as a "proenzyme," "propolypeptide," or "zymogen." The propeptide can be converted into a mature, active polypeptide by catalytic or autocatalytic cleavage of the propeptide. The propeptide coding region can be obtained from any suitable source, including but not limited to: Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae α-factor, Rhizopus oryzae aspartic protease, and Myceliophthora thermophila lactase (see, for example, WO 95 / 33836). When both the signal peptide and the propeptide region are present at the N-terminus of the polypeptide, the propeptide region is located immediately adjacent to the N-terminus of the polypeptide, and the signal peptide region is located immediately adjacent to the N-terminus of the propeptide region.

[0163] In some implementations, regulatory sequences are also utilized. These sequences promote the regulation of peptide expression relative to host cell growth. Examples of regulatory systems are those that cause gene expression 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, the lac, tac, and trp operon systems. In yeast host cells, suitable regulatory systems include, but are not limited to, the ADH2 system or the GAL1 system. In filamentous fungi, suitable regulatory sequences include, but are not limited to, the TAKA α-amylase promoter, the Aspergillus niger glucosylase promoter, and the Aspergillus oryzae glucosylase promoter.

[0164] On the other hand, the present invention relates to a polynucleotide comprising an engineered enzyme polypeptide, and a recombinant expression vector comprising one or more expression regulatory regions such as promoters and terminators, origins of replication, etc., depending on the type of host to which it is 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 substitution of a nucleic acid sequence encoding an enzyme polypeptide at such sites. 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 containing the sequence into a suitable vector for expression. In some embodiments involving the production of an expression vector, the coding sequence is located in the vector such that the coding sequence is operatively linked to a suitable control sequence for expression.

[0165] Recombinant expression vectors can be any suitable vector (e.g., plasmids or viruses) that facilitates recombinant DNA procedures and induces the expression of the enzyme's polynucleotide sequence. The choice of vector typically depends on its compatibility with the host cell to which it will be introduced. Vectors can be linear plasmids or closed circular plasmids.

[0166] In some embodiments, the expression vector is an autonomously replicating vector (i.e., a vector that exists as an extrachromosomal entity and whose replication is independent of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome). The vector may comprise any means for ensuring self-replication. In some selectable embodiments, the vector is one that, when introduced into a host cell, is integrated into the genome and replicates together with the 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, and / or transposons, that together comprise the total DNA to be introduced into the genome of the host cell are utilized.

[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, the 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] In another aspect, the present invention provides a host cell comprising at least one polynucleotide encoding at least one engineered enzyme polypeptide of the present invention, the polynucleotide being operatively linked to one or more control sequences for expressing one or more engineered enzymes in the host cell. Suitable host cells for expressing polypeptides encoded by the expression vector of the present invention are well known in the art and include, but are not limited to, bacterial cells such as *Escherichia coli*, *Vibrio fluvialis*, *Streptomyces*, and *Salmonella typhimurium* cells; fungal cells such as yeast cells (e.g., *Saccharomyces cerevisiae* or *Pichiapastoris* (ATCC Registry No. 201178)); insect cells such as *Drosophila* S2 and *Spodoptera* Sf9 cells; animal cells such as CHO, COS, BHK, 293, and Bowes melanoma cells; and plant cells. Exemplary host cells also include various Escherichia coli strains (e.g., W3110 (ΔfhuA) and BL21). Examples of bacterial selection markers include, but are not limited to, the dal genes 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 vector of the present invention includes elements that allow the vector to integrate into the genome of a host cell or allow the vector to replicate autonomously in the cell independently of the genome. In some embodiments involving integration into the host cell genome, the vector depends on a nucleic acid sequence encoding a polypeptide or any other element of the vector used to integrate the vector into the genome via homologous or non-homologous recombination.

[0170] In some alternative embodiments, the expression vector contains additional nucleic acid sequences to guide integration into the host cell's genome via homologous recombination. These additional nucleic acid sequences enable the vector to integrate into the host cell's genome at one or more precise locations on one or more chromosomes. To increase the likelihood of integration at precise locations, the integrative element preferably contains 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, that are highly homologous to the corresponding target sequence to enhance the likelihood of homologous recombination. The integrative element can be any sequence homologous to the target sequence in the host cell's genome. Furthermore, the integrative element can be a non-coding or coding nucleic acid sequence. On the other hand, the vector can integrate into the host cell's genome via non-homologous recombination.

[0171] For autonomous replication, the vector may also contain an origin of replication, enabling the vector to replicate autonomously in the host cell in question. Examples of bacterial origins of replication are P15A ori, which allows replication in *Escherichia coli*, or the origins of replication of plasmids pBR322, pUC19, pACYCl77 (which contains P15A ori), or pACYC184, and the origins of replication of pUB110, pE194, or pTA1060, which allow replication in *Bacillus*. Examples of origins of replication used in yeast host cells are 2 μm origins of replication, ARS1, ARS4, combinations of ARS1 and CEN3, and combinations of ARS4 and CEN6. The origin of replication may be a mutation that enables it to function temperature-sensitively in the host cell (see, for example, 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 a host cell to increase the production of the gene product. The increase in the copy number of the nucleic acid sequence can be achieved 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 thus additional copies of the nucleic acid sequence can be selected by culturing cells in the presence of a suitable selectant.

[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 vectors (Sigma-Aldrich Chemicals) include a CMV promoter and hGH polyadenylation site for expression in mammalian host cells, and a pBR322 origin of replication and 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, for example, Lathe et al., Gene 57:193-201

[1987] ).

[0174] Therefore, in some embodiments, a vector containing a sequence encoding at least one variant of pantothenic acid kinase is transformed into host cells to allow for the proliferation of the vector and the expression of one or more variants of pantothenic acid kinase. In some embodiments, the variant pantothenic acid kinase is post-translational modified to remove the signal peptide and, in some cases, may be cleaved after secretion. In some embodiments, the transformed host cells described above are cultured in a suitable nutrient medium under conditions that allow for the expression of one or more variants of pantothenic acid kinase. Any suitable culture medium that can be used to culture host cells may be used in this invention, including but not limited to basal or combined media containing suitable supplements. In some embodiments, host cells are grown in HTP medium. Suitable media may be available from multiple commercial suppliers or may be prepared according to publicly available formulations (e.g., in the catalogue of the U.S. Center for Type Culture Collection).

[0175] In another aspect, the present invention provides a host cell comprising a polynucleotide encoding an improved pantothenic acid kinase polypeptide provided herein, said polynucleotide being operatively linked to one or more control sequences for expressing pantothenic acid kinase in the host cell. Host cells for expressing the pantothenic acid kinase polypeptide encoded by the expression vector of the present invention are well known in the art and include, but are not limited to, bacterial cells such as *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 *Noctus* Sf9 cells; animal cells such as CHO, COS, BHK, 293, and Bowes melanoma cells; and plant cells. Suitable culture media and growth conditions for the host cells described above are well known in the art.

[0176] The polynucleotide used to express pantothenic acid kinase can be introduced into cells by a variety of 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, algal 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. Filamentous fungi are characterized by vegetative mycelia, wherein 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 present invention, the filamentous fungal host cell can be 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*, *Endothia*, *Fusarium*, *Gibberella*, *Gliocladium*, *Humicola*, and *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 present invention, the host cell is a yeast cell, including but not limited to cells of species of the genera *Candida*, *Hansenula*, *Saccharomyces*, *Schizosaccharomyces*, *Pichia*, *Kluyveromyces*, or *Yarrowia*. In some embodiments of the present invention, the yeast cells are *Hansenula polymorpha*, *Saccharomyces cerevisiae*, *Saccharomyces carlsbergensis*, *Saccharomyces diastaticus*, *Saccharomyces norbensis*, *Saccharomyces kluyveri*, *Schizosaccharomyces pombe*, *Pichia pastoriensis*, *Pichia finlandica*, *Pichia trehalophila*, *Pichia kodamae*, *Pichia membranaefaciens*, *Pichia opuntiae*, *Pichia thermotolerans*, *Pichia salictaria*, *Pichia quercuum*, *Pichia pijperi*, *Pichia stipitis*, *Pichia methanolica*, *Pichia angusta*, and *Kluyveromyces lactis*. 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 this invention, including, but not limited to, *Agrobacterium*, *Alicyclobacillus*, *Anabaena*, *Anacystis*, *Acinetobacter*, *Acidothermus*, *Arthrobacter*, *Azobacter*, *Bacillus*, *Bifidobacterium*, and *Brevibacterium*. ), Butyrivibrio, Buchnera, Campestris, Campylobacter, Clostridium, Corynebacterium, Chromatium, Coprococcus, Escherichia, Enterococcus, Enterobacter, Erwinia, Fusobacterium bacterium), Faecalibacterium, Francisella, Flavobacterium, Geobacillus, Haemophilus, Helicobacter, Klebsiella, Lactobacillus, Lactococcus, Ilyobacter, Micrococcus, Micrococcus Microbacterium, Mesorhizobium, Methylobacterium, Methylobacterium, Mycobacterium, Neisseria, Pantoea, Pseudomonas, Prochlorococcus, Rhodobacter, Rhodopseudomonas, Rhodopseudomonas, Roseburia,The genera *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 the genus *Agrobacterium*, *Acinetobacter*, *Azotobacter*, *Bacillus*, *Bifidobacterium*, *Buchnerella*, *Geobryophyte*, *Campylobacter*, *Clostridium*, *Corynebacterium*, *Escherichia*, *Enterococcus*, *Erwinia*, *Flavobacterium*, *Lactobacillus*, *Lactococcus*, *Pantospira*, *Pseudomonas*, *Staphylococcus*, *Salmonella*, *Streptococcus*, *Streptomyces*, or *Fermentomonas*. 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 this invention. In some embodiments of the invention, the bacterial host cell is a species of the genus *Agrobacterium* (e.g., *Agrobacterium radiobacter*, *Agrobacterium rhizogenes*, and *Agrobacterium rubi*). In some embodiments of the invention, the bacterial host cell is an *Arthrobacter* species (e.g., *Arthrobacter aurescens*, *Arthrobacter citreus*, *Arthrobacter globiformis*, *Arthrobacter hydrocarboglutamicus*, *Arthrobacter mysorens*, *Arthrobacter nicotianae*, *Arthrobacter paraffineus*, *Arthrobacter protophonniae*, *Arthrobacter roseoparqffinus*, *Arthrobacter sulfureus*, and *Arthrobacter ureafaciens*). In some embodiments of the invention, the bacterial host cell is a *Bacillus* species (e.g., *Bacillus thuringensis*, *Bacillus anthracis*).Bacillus megaterium, Bacillus subtilis, Bacillus lentus, Bacillus circulans, Bacillus pumilus, Bacillus lautus, Bacillus coagulans, Bacillus brevis, Bacillus firmus, Bacillus alkaophius, Bacillus licheniformis, Bacillus clausii, Bacillus stearothermophilus, Bacillus halodurans, and Bacillus amyloliquefaciens. In some embodiments, the host cell is a strain of the genus Bacillus, including but not limited to Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis, Bacillus megaterium, Bacillus clausii, Bacillus stearothermophilus, or Bacillus amyloliquefaciens. In some embodiments, the Bacillus host cells are *Bacillus subtilis*, *Bacillus licheniformis*, *Bacillus megaterium*, *Bacillus stearothermophilus*, and / or *Bacillus amyloliquefaciens*. In some embodiments, the bacterial host cells are *Clostridium* species (e.g., *C. acetobutylicum*, *C. tetani* E88, *C. lituseburense*, *C. saccharobutylicum*, *C. perfringens*, and *C. beijerinckii*). In some embodiments, the bacterial host cells are *Corynebacterium* species (e.g., *C. glutamicum* and *C. acetoacidophilum*). In some embodiments, the bacterial host cells are *Escherichia* species (e.g., *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*, *Erwinia punctata*, and *Erwinia terreus*). In some embodiments, the bacterial host cell is a Pantotheca species (e.g., *P. citrea* and *P. agglomerans*). In some embodiments, the bacterial host cell is a Pseudomonas species (e.g., *P. putida* and *P. aeruginosa*).P. mevalonii and Pseudomonas species D-0l 10 (P. sp. D-0l 10)). In some embodiments, the bacterial host cell is a Streptococcus species (e.g., S. equisimiles, S. pyogenes, and S. uberis). In some embodiments, the bacterial host cell is a Streptomyces species (e.g., S. ambofaciens, S. achromogenes, S. avermitilis, S. coelicolor, S. aureofaciens, S. aureus, S. fungicididicus, S. griseus, and S. lividans)). In some implementations, the bacterial host cell is a species of the genus *Zygomyces* (e.g., *Z. mobilis* and *Z. lipolytica*).

[0182] Many of the prokaryotic and eukaryotic strains that can be used in this invention are readily available to the public from many culture collections, such as the American Type Culture Collection (ATCC), the German Microbiology and Fungi Collection (DSM), the Central Agricultural Research Center (Centraalbureau Voor Schimmelcultures, CBS) in the Netherlands, and the Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

[0183] In some embodiments, host cell is genetically modified to have the feature of improving protein secretion, protein stability and / or protein expression and / or other properties desired for secretion.Genetic modification can be realized by genetic engineering technology and / or typical microbiological technology (for example, chemical or UV mutagenesis and subsequent selection).In fact, in some embodiments, the combination of recombinant modification and typical selection technology is used to produce host cell.Use recombinant technology, nucleic acid molecule can introduce, disappearance, suppress or modify in the mode that causes the output increase of one or more pantothenate kinase variants in host cell and / or substratum.For example, knock out the cell of Alp1 function generation protease defect, and knock out pyr5 function generation and have pyrimidine defect phenotype cell.In a kind of genetic engineering method, homologous recombination is used for inducing the genetic modification of targeting by specific targeting gene in vivo, to suppress the expression of coded protein.In alternative method, siRNA, antisense and / or ribozyme technology can be used for suppressing gene expression.The method for protein expression in various reduction cells known in the art, include but not limited to all or part of the disappearance of the gene of coded protein, and site-specific mutagenesis to destroy 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, eg, 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. Patent No. 9,714,437, incorporated herein by reference) can be used.

[0185] In some embodiments, the engineered host cells of the present invention (i.e., “recombinant host cells”) are cultured in conventional nutrient media that are appropriately modified to activate promoters, select transformants, or amplify pantothenic acid kinase polynucleotides. Culture conditions, such as temperature and pH, are those previously used with the selection of host cells for expression and are well known to those skilled in the art. As noted, numerous standard references and textbooks are available for the culture and production of many cells, including those derived from bacteria, plants, animals (especially mammals), and archaeology.

[0186] In some embodiments, cells expressing the variant pantothenic acid kinase peptide of the present invention are grown under batch or continuous fermentation conditions. A typical “batch fermentation” is a closed system in which the composition of the culture medium is set at the start of fermentation and is unaffected by human intervention during fermentation. A variation of the batch system is “feed-batch fermentation,” which can also be used in this invention. In this variation, the substrate is added incrementally as fermentation proceeds. Feed-batch systems are useful when catabolite repression might inhibit cellular metabolism, and when a limited amount of substrate in the culture medium is desired. Batch fermentation and fed-batch fermentation are common and well-known in the art. “Continuous fermentation” is an open system in which a defined fermentation medium is continuously added to a bioreactor, and an equal amount of conditioned medium is simultaneously removed for treatment. Continuous fermentation typically maintains the culture at a constant high density, where cells are primarily in the logarithmic growth phase. Continuous fermentation systems aim to maintain steady-state growth conditions. Methods for regulating nutrients and growth factors used in continuous fermentation processes, as well as techniques for maximizing product formation rates, are well-known in the field of industrial microbiology.

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

[0188] This invention provides a method for preparing a variant pantothenic acid kinase polypeptide or a bioactive fragment thereof. In some embodiments, the method includes: providing a host cell transformed with a polynucleotide encoding a sequence that is 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%) of the sequence and containing at least one mutant amino acid sequence provided herein; culturing the transformed host cell in a culture medium under conditions in which the host cell expresses the variant pantothenic acid kinase polypeptide; and optionally recovering or isolating the expressed variant pantothenic acid kinase polypeptide, and / or recovering or isolating the culture medium containing the expressed variant pantothenic acid kinase polypeptide. In some embodiments, the method also provides optionally lysing transformed host cells after expression of the encoded pantothenic acid kinase peptide, and optionally recovering and / or isolating the expressed variant pantothenic acid kinase peptide from the cell lysate. The invention also provides a method for preparing a variant pantothenic acid kinase peptide, the method comprising culturing host cells transformed with a variant pantothenic acid kinase polynucleotide under conditions suitable for producing the variant pantothenic acid kinase peptide, and recovering the variant pantothenic acid kinase peptide. Typically, protein recovery techniques well known in the art, including those described herein, are used to recover or isolate the pantothenic acid kinase peptide from host cell culture medium, host cells, or both. In some embodiments, host cells are collected by centrifugation, destroyed by physical or chemical means, and the resulting crude extract is retained for further purification. Microbial cells used 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] Engineered pantothenic acid kinase expressed in host cells can be recovered from cells and / or culture media 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. Suitable solutions for lysing and efficiently extracting proteins from bacteria such as *Escherichia coli* are marketed under the trade name CelLytic B. TM(Sigma-Aldrich) Commercially available. Therefore, in some embodiments, the resulting peptide is recovered / separated and optionally purified by any of a variety of methods known in the art. For example, in some embodiments, the peptide is separated from the nutrient medium by a routine procedure including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation, chromatography (e.g., ion exchange, affinity, hydrophobic interaction, chromatofocusing, and size exclusion), or precipitation. In some embodiments, a protein refolding step is used to complete the construction of the mature protein, if desired. Furthermore, in some embodiments, high-performance liquid chromatography (HPLC) is employed in the final purification step. For example, in some embodiments, methods known in the art can be used in this invention (see, for example, Parry et al., Biochem. J., 353:117

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

[2007] , both incorporated herein by reference). Indeed, any suitable purification method known in the art can be used in this invention.

[0190] Chromatographic techniques used to separate pantothenic acid kinase peptides include, but are not limited to, reversed-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, and molecular shape, which are known to those skilled in the art.

[0191] In some implementations, affinity chromatography can be used to isolate modified pantothenic acid kinase. For affinity chromatography purification, any antibody that specifically binds to the pantothenic acid kinase peptide can be used. To generate antibodies, various host animals, including but not limited to rabbits, mice, rats, etc., can be immunized by injection of pantothenic acid kinase. The pantothenic acid kinase peptide can be attached to a suitable vector, such as BSA, via a side-chain functional group or a linker attached to the side-chain functional group. 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 lysophosphatidylcholine, Pluronic polyols, polyanionic peptides, oil emulsions, keyhole polymers, etc. Keyholelimpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacillus Calmette-Guérin) and Corynebacterium parvum.

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

[0193] In some embodiments, the pantothenic acid kinase peptide is linked 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, polyvinyl fluoride, polyethyleneoxy, and polyacrylamide, as well as copolymers and grafts thereof. Solid supports can also be inorganic, such as glass, silica, controlled-pore glass (CPG), inverted silica, or metals such as gold or platinum. The substrate configuration can be in the form of beads, spheres, particles, granules, gels, membranes, or surfaces. Surfaces can be planar, substantially planar, or non-planar. Solid supports can be porous or non-porous and can have swelling or non-swelling features. Solid supports can be configured as pores, depressions, or other containers, vessels, features, or locations. More than one support can be configured on an array at various locations, which can be addressed by automated reagent delivery or by detection methods and / or instruments.

[0194] In some embodiments, immunological methods are used to purify pantothenic acid kinase variants. In one method, antibodies against variant pantothenic acid kinase peptides (e.g., peptides comprising SEQ ID NO:2, SEQ ID NO:30, SEQ ID NO:60, SEQ ID NO:132, SEQ ID NO:222, SEQ ID NO:230, SEQ ID NO:240 and / or SEQ ID NO:276, and / or their immunogenic fragments) generated using conventional methods are immobilized on beads, mixed with cell culture medium under conditions where variant pantothenic acid kinase is bound, and precipitated. In a related method, immunochromatography may be used.

[0195] In some embodiments, the variant pantothenic acid kinase is expressed as a fusion protein comprising a non-enzymatic portion. In some embodiments, the variant pantothenic acid kinase sequence is fused with a purification-enhancing domain. As used herein, the term "purification-enhancing domain" refers to a domain that mediates the purification of the polypeptide fused thereto. Suitable purification domains include, but are not limited to, metal chelate peptides, histidine-tryptophan modules that allow purification on immobilized metals, glutathione-binding sequences (e.g., GST), hemagglutinin (HA) tags (corresponding to epitopes derived from influenza hemagglutinin proteins; see, for example, 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.), and the like. Consider an expression vector for use in the compositions and methods described herein that provides expression of a fusion protein comprising the polypeptide of the invention fused to a multihistidine region separated by enterokinase cleavage sites. Histidine residues facilitate purification on IMIAC (immobilized metal ion affinity chromatography; see, for example, Porath et al., Prot. Exp. Purif., 3:263-281

[1992] ), while enterokinase cleavage sites provide a means of isolating variant pantothenic acid kinase peptides from fusion proteins. The pGEX vector (Promega) can also be used to express exogenous peptides as fusion proteins with glutathione S-transferase (GST). Generally, such fusion proteins are soluble and can be readily purified from lysed cells by adsorption onto ligand-agarose beads (e.g., glutathione-agarose in the case of GST-fusion proteins) followed by elution in the presence of free ligands.

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

[0197] Suitable culture media and growth conditions for host cells are well known in the art. Any suitable method for introducing polynucleotides for expressing enzyme peptides into cells is considered and may be used in this invention. Suitable techniques include, but are not limited to, electroporation, bio-ejector particle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion.

[0198] Various features and embodiments of the present invention are illustrated in the following representative examples, which are intended to be illustrative and not 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 of the 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 and equipment item.

[0201] In the following experimental disclosures, the following abbreviations will be used: M (mol / L); mM (millimoles / L), uM and μM (micromoles / L); nM (nanomoles / L); mol (moles); gm and g (grams); mg (milligrams); ug and μg (micrograms); L and l (liters); ml and mL (milliliters); cm (centimeters); mm (millimeters); um and μm (micrometers); sec. (seconds); min(s) (minutes); h(s) and hr(s) (hours); U (units); MW (molecular weight); rpm (revolutions / minutes); psi and PSI (pounds per square inch); ℃ (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, Luria broth; TB (terrific broth); SFP (shake flask powder); CDS (coding sequence); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); nt (nucleotide; polynucleotide); aa (amino acid; polypeptide); 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-performance liquid chromatography); HPLC-UV (HPLC-UV-Vis detector); 1H NMR (proton nuclear magnetic resonance spectroscopy); FIOPC (fold improvement relative to positive control); Sigma and Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO); Difco (Difco Laboratories, BD Diagnostic Systems, Detroit, MI); Microfluidics (Microfluidics, Westwood, MA); Life Technologies (Life Technologies, 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] Variants generated

[0204] The parent gene for the evolved pantothenate kinase (EV-PanK) used to generate the variants of the present invention is E. coli pantothenate kinase. This gene was synthesized and cloned into the pET-30a(+) vector. The gene sequence was optimized for expression in the E. coli BL21(DE3) strain.

[0205] BL21(DE3) *E. coli* cells were transformed with the corresponding plasmid containing the PanK encoding gene and plated on Luria broth (LB) agar plates containing 1% glucose and 50 μg / mL kanamycin (KAN), and grown overnight at 37°C. Single colonies were selected and inoculated into 180 μL of LB containing 1% glucose and 50 μg / mL KAN in 96-well shallow microtiter plates. The plates were sealed with an air-permeable seal and the cultures were grown overnight at 30°C, 200 rpm, and 85% relative humidity (RH). Then, 10 μL of each cell culture was transferred to the wells of a 96-well deep-well plate containing 390 μL of TB and 50 μg / mL KAN. The deep-well plates were sealed with an air-permeable seal and incubated at 30°C, 250 rpm, and 85% RH until OD was reached. 600 0.6-0.8. The cell culture was then induced with isopropylthioglycoside (IPTG) to a final concentration of 1 mM and incubated overnight at 30°C, 250 rpm. The cells were then pelleted using centrifugation at 4000 rpm for 10 min. The supernatant was discarded and the pellet was frozen at -80°C prior to lysis.

[0206] The frozen pellet was lysed with 300 μL of lysis buffer containing 50 mM triethanolamine buffer, pH 7.5, 1 mg / mL lysozyme, 0.5 mg / mL PMBS, and 0.05% v / v DNase. The lysis mixture was shaken for 2.5 hours at room temperature (RT). The plate was then centrifuged at 4000 rpm and 4°C for 10 minutes. The supernatant was then used as the clarified lysate for biocatalytic reactions to determine activity levels.

[0207] Example 2

[0208] Activity assay

[0209] The libraries were generated using well-established techniques (e.g., recombination of previously identified beneficial mutations). As described in Example 1, each gene-encoded polypeptide was generated using HTP, producing clear lysates.

[0210] Each 50 μL reaction was performed in a shallow 96-well plate with 10 μL of clarified lysate solution, 5-40 g / L compound 3 (EGA), 50 mM sodium phosphate buffer at pH 7.5. The plate was heat-sealed and incubated at 30° C. and shaken overnight at 600 RPM in a 50 mmthrow Infors shaker maintained at 85% RH for 20 hours.

[0211] To generate chromophore-containing substances and enable simple reaction monitoring, the reaction sample was derivatized using 2,4-dinitrophenylhydrazine (DNPH). DNPH derivatization creates a covalent bond between DNPH and aldehydes (such as glyceraldehydes explored in this invention). After the reaction, 20 μL of the reaction was combined with 200 μL of DNPH (15 mg / mL in DMSO with 2.5% 6N HCl) in a new 96-well plate. The derivatization reaction was incubated at room temperature for 1 hour. The sample was then filtered by centrifugation using a 0.22 μm 96-well filter plate, ready for analysis by UHPLC-MS.

[0212] The activity of each sample was measured using an analytical method that monitored the mass spectrometry (MS) signal (SIM=373) of the desired product, phospho-ethynylglyceraldehyde. The UHPLC-MS method was an isocratic method run at 1 mL / min on a Zorbax Eclipse HD C18 column. Mobile phase A consisted of HO with 0.05% formic acid, and mobile phase B consisted of acetonitrile with 0.05% formic acid, with a ratio of 60% A to 40% B.

[0213] The activity of the variant enzymes was calculated by dividing the MS area counts for each variant by the average MS area counts of eight positive parent enzyme samples contained in the same 96-well plate. The amount of product produced by each variant was calculated using a previously determined standard curve.

[0214] The table below lists the sequences and activities of a series of evolved enzymes with significantly improved enzyme activity and tolerance to increased substrate loads.

[0215]

[0216]

[0217] Example 3

[0218] Engineered peptides produced using pCK110900

[0219] The polynucleotide sequence encoding SEQ ID NO: 30 (ie, SEQ ID NO: 29) was cloned into the pCK110900 vector system (see, e.g., U.S. Patent No. 9,714,437, which is incorporated herein by reference in its entirety), and then expressed in E. coli W3110fhuA under the control of the lac promoter.

[0220] In a 96-well format, single colonies were picked and grown in 180 μL of LB containing 1% glucose and 30 μg / mL CAM at 30°C, 200 rpm, and 85% humidity. After overnight growth, 20 μL of the grown culture was transferred to a deep-well plate containing 380 μL of TB and 30 μg / mL CAM. The culture was grown at 30°C, 250 rpm, and 85% humidity. When the optical density (OD) of the culture reached 0. 600 When the HTP concentration reached 0.6–0.8, pantothenic acid kinase gene expression was induced by adding IPTG to a final concentration of 1 mM. After induction, cells were allowed to continue growth for 18–20 hours. Cells were harvested by centrifugation at 4000 rpm for 10 minutes at 4°C, and the culture medium was discarded. The cell pellet was stored at -80°C until ready for use. Before assay, the cell pellet was resuspended in 250 μL of lysis buffer containing 50 mM potassium phosphate, pH 7.5, 1 g / L lysozyme, and 0.5 g / L PMBS. The plate was shaken at medium speed on a microtiter plate shaker for 2 hours at room temperature. The plate was then centrifuged at 4000 rpm for 20 minutes at 4°C, and the clear supernatant was used in the HTP assay reaction described below.

[0221] The shake-flask procedure can be used to generate engineered pantothenic acid kinase peptide shake-flask powders (SFPs) that can be used for secondary screening assays and / or for use in the biocatalytic methods described herein. Compared to cell lysates used in HTP assays, shake-flask powder (SFP) preparations of the enzyme provide a more purified engineered enzyme preparation (e.g., up to 30% of total protein) and also allow for the use of more concentrated enzyme solutions. To initiate cultivation, a single colony of *E. coli* containing a plasmid encoding the engineered peptide of interest was inoculated into 50 mL of LB containing 30 μg / mL CAM and 1% glucose. The culture was allowed to grow overnight (at least 16 hours) in an incubator at 30°C with shaking at 250 rpm. After overnight growth, the OD of the culture was measured. 600 The grown culture was diluted in 250 mL of TB containing 30 μg / mL CAM in a 1 L shake flask until a final OD value of 0.2 was obtained. 600 Incubate 250 mL of culture at 30°C and 250 rpm until OD reaches 100%. 600Reach 0.6-0.8. Induce pantothenic acid kinase gene expression by adding IPTG to a final concentration of 1 mM and continue growth for another 18-20 hours. Harvest cells by transferring the culture to a pre-weighed centrifuge flask and centrifuging at 4000 rpm for 20 minutes at 4°C. Resuspend the cell pellet and wash with 30 mL of cold 50 mM potassium phosphate, pH 7.5 buffer, and centrifuge again at 4000 rpm for 20 minutes at 4°C. Discard the supernatant and weigh the remaining cell pellet. Keep the cells frozen at -80°C for at least 2 hours before lysis. In some embodiments, the cells are stored at -80°C until ready for use. For lysis, resuspend the cell pellet in 6 mL of cold 50 mM potassium phosphate, pH 7.5 per 1 g of cell pellet. Use 110 L The resuspended cells were lysed using 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.

[0222] Example 4

[0223] For improved activity, evolution and screening of engineered peptides derived from SEQ ID NO:30

[0224] The engineered polynucleotide encoding the polypeptide having the pantothenate kinase activity of SEQ ID NO: 30 (i.e., SEQ ID NO: 29) was used to generate the additional engineered polypeptides of Table 4-1. These polypeptides exhibited improved pantothenate kinase activity (e.g., % conversion of acetylglyceraldehyde to acetylglyceraldehyde phosphate product) compared to the starting polypeptide.

[0225]

[0226]

[0227] The engineered peptides were generated from the “backbone” amino acid sequence of SEQ ID NO:30 using the directed evolution method described above, along with the HTP assay and analysis method described below.

[0228] Directed evolution began with the polynucleotides listed in SEQ ID NO:29. Engineered peptides were then selected as the starting “backbone” gene sequences. Libraries of engineered peptides were generated using various 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 peptides to convert the acetylenylglyceraldehyde substrate into the acetylenylglyceraldehyde phosphate product (compound 6 shown in Scheme 1 is converted into compound 5).

[0229] Enzyme assays were performed in 96-well configurations at a total volume of 100 μL per well, comprising HTP enzyme lysate, 20 g / L (151 mM) acetylacetal, 2 equivalents of acetyl phosphate, 5 g / L (9.8 mM) ATP, 20 g / L WT acetate kinase from *Thermotogamaritima*, 10 mM MgCl2, and 100 mM potassium phosphate, pH 7.8 (final concentration). The reaction was performed by adding the following to each well: (i) 80 μL of a solution containing 25 g / L acetylacetal, 2 equivalents of acetyl phosphate, 6.25 g / L ATP, 25 g / L WT acetate kinase from *Thermophyton floccosum*, 12.5 mM MgCl2, and 125 mM potassium phosphate, adjusting the pH of the mixture to 7.8; and (ii) 20 μL of clear lysate of HTP (prepared as described above), diluted 4-fold in 50 mM potassium phosphate, pH 7.5 buffer. The reaction plate was heat-sealed and incubated at 30 °C with shaking at 600 rpm for 20–22 hours.

[0230] After 20–22 hours, 10 μL of sample was transferred to a separate plate. The sample was then mixed with 20 g / L (S)-(-)-1-amino-2-(methoxymethyl)pyrrolidine in 190 μL of water. The plate was sealed and shaken at 400 rpm for 1 hour at 25 °C. The derivatization reaction was quenched by adding 200 μL of CH3CN. The sample was then shaken in a microtiter plate shaker at room temperature and then centrifuged at 4000 rpm for 10 min at 4 °C. The quenched sample was further diluted 10x in water before UPLC analysis. The UPLC running parameters are described below.

[0231]

[0232]

[0233] Example 5

[0234] Evolution and screening of engineered polypeptides derived from SEQ ID NO: 60 for improved activity

[0235] Engineered polynucleotides encoding the peptide having the pantothenic acid kinase activity of SEQ ID NO:60 (i.e., SEQ ID NO:59) were used to generate additional engineered peptides as shown in Table 5-1. These peptides exhibited improved pantothenic acid kinase activity (e.g., % conversion of acetylenylglyceraldehyde to acetylenylglyceraldehyde phosphate) compared to the starting peptides. Engineered peptides with even-numbered sequence identifiers were generated from the “backbone” amino acid sequence of SEQ ID NO:59 using directed evolution methods as described above, along with HTP assays and analytical methods described below.

[0236] Directed evolution began with the polynucleotides listed in SEQ ID NO:59. Engineered peptides were then selected as the starting “backbone” gene sequences. Libraries of engineered peptides were generated using various 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 peptides to convert acetylenol glyceraldehyde substrates into acetylenol glyceraldehyde phosphate products.

[0237] Enzyme assays were performed in 96-well configurations at a total volume of 100 μL / well, comprising HTP enzyme lysate, 30 g / L (227 mM) acetylglucaldehyde, 2 equivalents of acetyl phosphate, 5 g / L (9.8 mM) ATP, 20 g / L WT acetate kinase from *Thermophyton floccosum*, 10 mM MgCl2, and 100 mM potassium phosphate, pH 7.8 (final concentration). The reaction was performed by adding the following to each well: (i) 86 μL of a solution containing 34.9 g / L acetylglucaldehyde, 528 mM acetyl phosphate, 5.8 g / L ATP, 23.3 g / L WT acetate kinase from *Thermophyton floccosum*, 11.6 mM MgCl2, and 116 mM potassium phosphate, adjusting the pH of the mixture to 7.8; and (ii) 14 μL of clear HTP lysate (prepared as previously described). Heat-seal the reaction plate and shake it at 600 rpm at 30°C for 20-22 hours.

[0238] After 20–22 hours, mix 200 μL of 50 mM potassium phosphate, pH 7.5 with the sample. Transfer 20 μL of the sample to a separate plate and mix with 180 μL of a 6 g / L solution of (S)-(-)-1-amino-2-(methoxymethyl)pyrrolidine in water. Seal the plate and incubate at 25 °C with shaking at 400 rpm for 1 hour. The derivatization reaction is quenched by adding 200 μL of CH3CN. The sample is then incubated at room temperature in a microtiter plate shaker and then centrifuged at 4000 rpm for 10 min at 4 °C. The quenched sample is further diluted 2x in water before UPLC analysis.

[0239]

[0240]

[0241]

[0242] Example 6

[0243] Evolution and screening of engineered peptides derived from SEQ ID NO:132 for improved activity and thermal stability.

[0244] Engineered polynucleotides encoding polypeptides having the pantothenate kinase activity of SEQ ID NO: 132 (i.e., SEQ ID NO: 131) were used to generate additional engineered polypeptides of Table 6-1. These polypeptides exhibited improved pantothenate kinase activity (e.g., % conversion of acetylglyceraldehyde to acetylglyceraldehyde phosphate product) compared to the starting polypeptide. Engineered polypeptides having amino acid sequences with even-numbered sequence identifiers were generated from the "backbone" amino acid sequence of SEQ ID NO: 132 using directed evolution methods as described above in conjunction with HTP assays and analytical methods described below.

[0245] Directed evolution begins with the polynucleotide listed in SEQ ID NO: 131. An engineered polypeptide is then selected as the starting "backbone" gene sequence. Libraries of engineered polypeptides are generated using various well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using an HTP assay and an analytical method that measures the ability of the polypeptide to convert an acetylene glyceraldehyde substrate into an acetylene glyceraldehyde phosphate product.

[0246] Cells were lysed as previously described. The clarified lysate was transferred to a Bio-RAD hard-shell PCR plate and incubated at 34°C for 2 hours. After pre-incubation, the lysate was diluted to 40% by mixing 40 μL of the clarified lysate with 60 μL of 50 mM potassium phosphate, pH 7.5.

[0247] The enzyme assay was performed in a 96-well format with a total volume of 100 μL / well and included HTPase lysate, 30 g / L (227 mM) acetylglyceraldehyde, 2 equivalents of acetyl phosphate, 5 g / L (9.8 mM) ATP, 20 g / L WT acetate kinase from Thermotoga maritima, 10 mM MgCl2, 100 mM potassium phosphate, pH 7.8 (final concentration). The reaction was performed by adding the following to each well: (i) 87.5 μL of a solution containing 34.3 g / L acetylglyceraldehyde, 519 mM acetyl phosphate, 5.7 g / L ATP, 22.9 g / L WT acetate kinase from Thermotoga maritima, 11.4 mM MgCl2, 114 mM potassium phosphate, adjusting the pH of the mixture to 7.8, (ii) 12.5 μL of diluted, preheated lysate (prepared as described above). Heat-seal the reaction plate and shake it at 600 rpm at 30°C for 20-22 hours.

[0248] After 20–22 hours, mix 200 μL of 50 mM potassium phosphate, pH 7.5 with the sample. Transfer 20 μL of the sample to a separate plate and mix with 180 μL of a 6 g / L solution of (S)-(-)-1-amino-2-(methoxymethyl)pyrrolidine in water. Seal the plate and incubate at 25 °C with shaking at 400 rpm for 1 hour. The derivatization reaction is quenched by adding 200 μL of CH3CN. The sample is then incubated at room temperature in a microtiter plate shaker and then centrifuged at 4000 rpm for 10 min at 4 °C. The quenched sample is further diluted 2x in water before UPLC analysis.

[0249]

[0250] In addition to the preheated pyrolytes, many broadly active variants, including those listed in Table 6-2, were regenerated and re-evaluated using unheated pyrolytes. The reaction proceeded as described above, except that the heating step was omitted. Table 6-2 lists the variants improved relative to SEQ ID NO:132 under unheated conditions.

[0251]

[0252] Example 7

[0253] Evolution and screening of engineered peptides derived from SEQ ID NO:222 for improved activity and thermal stability.

[0254] Engineered polynucleotides encoding the peptide having the pantothenic acid kinase activity of SEQ ID NO:222 (i.e., SEQ ID NO:221) were used to generate additional engineered peptides as shown in Table 7-1. These peptides exhibited improved pantothenic acid kinase activity (e.g., % conversion of acetylenylglyceraldehyde to acetylenylglyceraldehyde phosphate) compared to the starting peptides. Engineered peptides with even-numbered sequence identifier amino acid sequences were generated from the “backbone” amino acid sequence of SEQ ID NO:222 using directed evolution methods as described above, along with HTP assays and analytical methods described below.

[0255] Directed evolution began with the polynucleotides listed in SEQ ID NO:221. Engineered peptides were then selected as the starting “backbone” gene sequences. Libraries of engineered peptides were generated using various 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 peptides to convert acetylenol glyceraldehyde substrates into acetylenol glyceraldehyde phosphate products.

[0256] Enzyme assays were performed in 96-well configurations at a total volume of 50 μL / well, comprising HTP enzyme lysate, 18 g / L (136 mM) acetylenol, 2 equivalents of acetyl phosphate, 5 g / L (9.8 mM) ATP, 20 g / L WT acetate kinase from *Thermophyton floccosum*, 10 mM MgCl2, and 100 mM potassium phosphate, pH 7.8 (final concentration). The reaction was performed by adding the following to each well: (i) 43.3 μL of a solution containing 20.8 g / L acetylenol, 315 mM acetyl phosphate, 5.8 g / L ATP, 23.1 g / L WT acetate kinase from *Thermophyton floccosum*, 11.5 mM MgCl2, and 115 mM potassium phosphate, adjusting the pH of the mixture to 7.8; and (ii) 6.7 μL of 1.5% (v / v) diluted PanK HTP lysate. Heat-seal the reaction plate and shake it at 600 rpm at 30°C for 20-22 hours.

[0257] After 20–22 hours, mix 200 μL of 50 mM potassium phosphate, pH 7.5 with the sample. Transfer 20 μL of the sample to a separate plate and mix with 180 μL of a 6 g / L solution of (S)-(-)-1-amino-2-(methoxymethyl)pyrrolidine in water. Seal the plate and incubate at 25 °C with shaking at 400 rpm for 1 hour. The derivatization reaction is quenched by adding 200 μL of CH3CN. The sample is then incubated at room temperature in a microtiter plate shaker and then centrifuged at 4000 rpm for 10 min at 4 °C. The quenched sample is further diluted 2x in water before UPLC analysis.

[0258]

[0259] Example 8

[0260] Evolution and screening of engineered polypeptides derived from SEQ ID NO: 230 for improved activity

[0261] Engineered polynucleotides (i.e., SEQ ID NO: 229) encoding peptides having the pantothenic acid kinase activity of SEQ ID NO: 230 were used to generate additional engineered peptides as shown in Table 8-1. These peptides exhibited improved pantothenic acid kinase activity (e.g., % conversion of acetylenylglyceraldehyde to acetylenylglyceraldehyde phosphate) compared to the starting peptides. Engineered peptides with even-numbered sequence identifier amino acid sequences were generated from the “backbone” amino acid sequence of SEQ ID NO: 230 using directed evolution methods as described above, along with HTP assays and analytical methods described below.

[0262] Directed evolution began with the polynucleotides listed in SEQ ID NO:229. Engineered peptides were then selected as the starting “backbone” gene sequences. Libraries of engineered peptides were generated using various 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 peptides to convert acetylenol glyceraldehyde substrates into acetylenol glyceraldehyde phosphate products.

[0263] Enzyme assays were performed in 96-well plates at a total volume of 50 μL / well, comprising HTP enzyme lysate, 3 g / L ethynylglycerolaldehyde, 2 equivalents of acetyl phosphate, 1 g / L ATP, 10 g / L WT acetate kinase (AcK) from *Thermophyton floccosum*, 10 mM MgCl2, and 100 mM potassium phosphate, pH 6.8 (final concentration). The reaction was performed by adding the following to each well: (i) 30 μL of a solution containing 5 g / L ethynylglycerolaldehyde, 75.7 mM acetyl phosphate, 1.7 g / L ATP, 16.7 g / L WT acetate kinase from *Thermophyton floccosum*, 16.7 mM MgCl2, and 167 mM potassium phosphate, adjusting the pH of the mixture to 6.8; and (ii) 20 μL of 1.125% (v / v) diluted PanK HTP lysate. The reaction plates were heat-sealed and incubated at 30 °C with shaking at 600 rpm for 3 hours.

[0264] After 3 hours, 100 μL of 50 mM potassium phosphate, pH 7.5, was mixed with the sample. In a separate plate, 20 μL of the sample was transferred and mixed with 180 μL of a 5 g / L solution of (S)-(-)-1-amino-2-(methoxymethyl)pyrrolidine in water. The plate was sealed and shaken at 400 rpm for 1 hour at 25 °C. The derivatization reaction was quenched by adding 200 μL of CH3CN. The sample was then shaken in a microtiter plate shaker at room temperature and then centrifuged at 4000 rpm for 10 min at 4 °C. The quenched sample was further diluted 5x in water before UPLC analysis.

[0265] Hit variants from high-throughput screening were grown in 250-mL shake flasks and enzyme powder was produced. The activity of the enzyme powder was evaluated using a similar assay as described above using 0.025-0.5 g / L enzyme powder, 3 g / L ethynylglyceraldehyde, 2 equivalents of acetyl phosphate, 1 g / L ATP, 10 g / L AcK WT, 10 mM MgCl 2 , 100 mM potassium phosphate, pH 6.8, 600 rpm, 30° C., for 3 hours. Polypeptides with improved activity are listed in Table 8-1.

[0266]

[0267] Example 9

[0268] For improved activity, evolution and screening of engineered peptides derived from SEQ ID NO:240.

[0269] Engineered polynucleotides (i.e., SEQ ID NO: 239) encoding peptides having the pantothenic acid kinase activity of SEQ ID NO: 240 were used to generate additional engineered peptides as shown in Table 9-1. These peptides exhibited improved pantothenic acid kinase activity (e.g., % conversion of acetylenylglyceraldehyde to acetylenylglyceraldehyde phosphate) compared to the starting peptides. Engineered peptides with even-numbered sequence identifier amino acid sequences were generated from the “backbone” amino acid sequence of SEQ ID NO: 240 using directed evolution methods as described above, along with HTP assays and analytical methods described below.

[0270] Directed evolution began with the polynucleotide listed in SEQ ID NO:239. An engineered peptide was then selected as the starting “backbone” gene sequence. Libraries of the engineered peptides were generated using various 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 peptide’s ability to convert the acetylenol glyceraldehyde substrate into the acetylenol glyceraldehyde phosphate product.

[0271] The enzyme assay was performed in a 96-well format with a total volume of 50 μL / well containing HTPase lysate, 3 g / L acetylglyceraldehyde, 2 equivalents of acetyl phosphate, 0.125 g / L ATP, 0.5 g / L improved acetate kinase (SEQ ID NO: 272), 10 mM MgCl , 100 mM potassium phosphate, pH 6.8 (final concentration). The reaction was performed by adding the following to each well: (i) 30 μL of a solution containing 5 g / L acetylglyceraldehyde, 75.7 mM acetyl phosphate, 0.21 g / L ATP, 0.83 g / L improved acetate kinase (SEQ ID NO: 272), 16.7 mM MgCl , 167 mM potassium phosphate, the pH of the mixture being adjusted to 6.8, and (ii) 20 μL of a 2.5% (v / v) diluted PanK HTP lysate. The reaction plate was heat-sealed and shaken at 30° C. for 3 hours at 600 rpm.

[0272] After 3 hours, mix 100 μL of 50 mM potassium phosphate, pH 7.5 with the sample. Transfer 20 μL of the sample to a separate plate and mix with 180 μL of o-benzylhydroxylamine in a 10 g / L solution in methanol. Seal the plate and incubate at 25°C with shaking at 400 rpm for 20–30 minutes. Further dilute the sample 4x in methanol before performing UPLC analysis using the method described in Table 9-2 below.

[0273] The targeted variants were grown in 250-mL shake flasks, producing enzyme powder. The activity of the enzyme powder was evaluated using a similar assay as described above with 0.025–0.5 g / L SF powder, 3 g / L acetylenol, 2 equivalents of acetyl phosphate, 0.1 g / L ATP, 0.5 g / L modified acetate kinase (SEQ ID NO: 272), 10 mM MgCl2, 100 mM potassium phosphate, pH 6.8, 600 rpm, 30 °C, for 3 h. Peptides with improved activity are listed in Table 9-1.

[0274]

[0275]

[0276] Example 10

[0277] For improved activity, evolution and screening of engineered peptides derived from SEQ ID NO:252.

[0278] The engineered polynucleotides encoding the polypeptides having the pantothenate kinase activity of SEQ ID NO: 252 (i.e., SEQ ID NO: 251) were used to generate the additional engineered polypeptides of Table 10-1. These polypeptides exhibited improved pantothenate kinase activity (e.g., % conversion of acetylglyceraldehyde to acetylglyceraldehyde phosphate product) compared to the starting polypeptides. Engineered polypeptides having amino acid sequences with even-numbered sequence identifiers were generated from the "backbone" amino acid sequence of SEQ ID NO: 252 using the directed evolution method as described above in conjunction with the HTP assay and analysis methods described below.

[0279] Directed evolution begins with the polynucleotide listed in SEQ ID NO: 251. An engineered polypeptide is then selected as the starting "backbone" gene sequence. Libraries of engineered polypeptides are generated using various well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using an HTP assay and an analytical method that measures the ability of the polypeptide to convert an acetylene glyceraldehyde substrate into an acetylene glyceraldehyde phosphate product.

[0280] Enzyme assays were performed in 96-well plates at a total volume of 50 μL / well, comprising HTP enzyme lysate, 3 g / L ethynylglycerolaldehyde, 2 equivalents of acetyl phosphate, 0.125 g / L ATP, 0.5 g / L modified acetate kinase (SEQ ID NO:274), 10 mM MgCl2, and 100 mM potassium phosphate, pH 6.8 (final concentration). The reaction was performed by adding the following to each well: (i) 30 μL of a solution containing 5 g / L ethynylglycerolaldehyde, 75.7 mM acetyl phosphate, 0.21 g / L ATP, 0.83 g / L modified acetate kinase (SEQ ID NO:274), 16.7 mM MgCl2, and 167 mM potassium phosphate, adjusting the pH of the mixture to 6.8; and (ii) 20 μL of 5% (v / v) diluted PanK HTP lysate. The reaction plates were heat-sealed and incubated at 30 °C with shaking at 600 rpm for 3 hours.

[0281] After 3 hours, mix 100 μL of 50 mM potassium phosphate, pH 7.5 with the sample. Transfer 20 μL of the sample to a separate plate and mix with 180 μL of a 10 g / L solution of o-benzylhydroxylamine in methanol. Seal the plate and incubate at 25°C with shaking at 400 rpm for 20–30 minutes. Further dilute the sample 4x in methanol before performing UPLC analysis using the method described in Table 9.2.

[0282] The hit variant was grown in a 250-mL shake flask to produce an enzyme powder. The activity of the enzyme powder was evaluated using a similar assay as described above with 0.025-0.5 g / L LSF powder, 3 g / L acetylenol, 2 equivalents of acetyl phosphate, 0.1 g / L ATP, 0.5 g / L modified acetate kinase (SEQ ID NO: 274), 10 mM MgCl2, 100 mM potassium phosphate, pH 6.8, 600 rpm, 30 °C, for 3 h. The engineered peptides in this example were expressed with a 10-amino acid histidine tag at the beginning of the amino acid sequence of each engineered peptide, and were screened using the histidine tag of said 10 amino acids.

[0283] Peptides with improved activity are listed in Table 10-1. Amino acid differences are listed relative to SEQ ID NO:276, which is the engineered peptide of SEQ ID NO:252 without a 10-amino acid histidine tag. Using SEQ ID NO:276 as a reference sequence for amino acid differences preserves the consistent numbering of amino acids and the location of residue differences / mutations among the engineered peptides described in these examples.

[0284]

[0285] This invention also provides the following items:

[0286] 1. An engineered pantothenate 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: 30, SEQ ID NO: 60, SEQ ID NO: 132, SEQ ID NO: 222, SEQ ID NO: 230, SEQ ID NO: 240 and / or SEQ ID NO: 276, or a functional fragment thereof, wherein the engineered pantothenate kinase comprises at least one substitution or set of substitutions in the polypeptide sequence, and wherein amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 2, SEQ ID NO: 30, SEQ ID NO: 60, SEQ ID NO: 132, SEQ ID NO: 222, SEQ ID NO: 230, SEQ ID NO: 240 and / or SEQ ID NO: 276.

[0287] 2. The engineered pantothenate kinase according to claim 1, 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: 2, wherein the engineered pantothenate 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.

[0288] 3. The engineered pantothenate kinase of claim 2, wherein the engineered pantothenate kinase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 2, or a functional fragment thereof, and wherein the engineered pantothenate kinase comprises at least one substitution or set of substitutions at one or more positions selected from the group consisting of 277 / 281, 54 / 240 / 277 / 281, 240, 240 / 277, 240 / 277 / 281, 240 / 277 / 281 / 282, 240 / 281 and 240 / 281 / 282, 277, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 2.

[0289] 4. The engineered pantothenic acid kinase according to Project 1, wherein the engineered pantothenic acid 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 with SEQ ID NO:30, wherein the engineered pantothenic acid kinase comprises at least one substitution or set of substitutions in the polypeptide sequence, and wherein the amino acid positions of the polypeptide sequence refer to the number SEQ ID NO:30.

[0290] 5. The engineered pantothenic acid kinase according to item 2, wherein the engineered pantothenic acid 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 with SEQ ID NO: 30, and wherein the engineered pantothenic acid kinase contains at least one substitution or set of substitutions at one or more positions selected from the following: 13, 13 / 14 / 19 / 22, 13 / 14 / 22, 13 / 14 / 22 / 37, 13 / 14 / 22 / 106 / 247, 13 / 14 / 218 / 247 / 305, 13 / 19, 13 / 22, 15 / 26 / 283, 15 / 27, 15 / 27 / 283, 15 / 27 / 283 / 305, 15 / 27 / 305, 15 / 283, 19 / 22 / 26 / 69, 19 / 29, 20, 20 / 24, 20 / 24 / 25 / 69 / 75, 20 / 24 / 30 / 75, 20 / 70 / 75, 22 / 218, 22 / 218 / 271, 24, 24 / 30, 24 / 30 / 75, 24 / 75 / 86 / 134, 26, 26 / 29, 27, 27 / 78, 27 / 78 / 123 / 283, 27 / 283, 29 and 75, wherein the amino acid positions of the polypeptide sequences are referenced to SEQ ID NO:30.

[0291] 6. The engineered pantothenic acid kinase according to Project 1, wherein the engineered pantothenic acid 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 with SEQ ID NO:60, wherein the engineered pantothenic acid 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 referenced to SEQ ID NO:60.

[0292] 7. The engineered pantothenic acid kinase according to item 2, wherein the engineered pantothenic acid 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 with SEQ ID NO: 60, and wherein the engineered pantothenic acid kinase comprises at least one substitution or set of substitutions at one or more positions selected from: 14 / 19 / 41 / 157 / 161 / 261, 19 / 22 / 41 / 44 / 54 / 119 / 157 / 261 / 298 / 308, 19 / 22 / 54 / 157 / 169, 22 / 106 / 218, 41, 41 / 44 / 54 / 119 / 120 / 157 / 169 / 261, 41 / 44 / 54 / 119 / 120 / 157 / 261 / 298 / 308, 41 / 44 / 54 / 119 / 120 / 161 / 169 / 261 / 298, 41 / 44 / 54 / 119 / 298 / 305, 41 / 44 / 161 / 169 / 261, 41 / 44 / 169 / 261 / 298 / 308, 41 / 44 / 169 / 261 / 308, 41 / 54 / 119 / 157 / 169 / 261, 41 / 119 / 161 / 169 / 261 / 308, 41 / 119 / 161 / 308, 44 / 54 / 119 / 120 / 157 / 161 / 169, 44 / 54 / 119 / 120 / 157 / 161 / 261, 44 / 54 / 119 / 120 / 169 / 261, 44 / 54 / 119 / 157 / 161 / 261 / 298, 44 / 54 / 119 / 169, 44 / 76 / 119 / 157 / 161, 44 / 119 / 120 / 261, 44 / 119 / 157 / 161, 44 / 119 / 161 / 261 / 298, 44 / 157 / 161 / 169, 44 / 157 / 298, 44 / 261 / 2 98 / 308, 44 / 261 / 308, 54 / 119 / 157 / 161 / 169, 54 / 157 / 161 / 261 / 308, 119 / 157 / 161, 119 / 157 / 161 / 169 / 261, 119 / 169 / 261, 119 / 261 / 298 / 308, 120 / 157 / 261, 157, 157 / 161 / 169 / 261, 157 / 161 / 308, 157 / 169 / 261 / 298 / 308, 157 / 308, 250, 302 and 310, and wherein the amino acid positions of the polypeptide sequences are referenced to SEQ ID NO:60.

[0293] 8. The engineered pantothenate kinase of claim 1, 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: 132, or a functional fragment thereof, wherein the engineered pantothenate 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: 132.

[0294] 9. The engineered pantothenate kinase of claim 2, wherein the engineered pantothenate 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: 132, or a functional fragment thereof, and wherein the engineered pantothenate kinase comprises at least one substitution or set of substitutions at one or more positions selected from the group consisting of: 24 / 48, 64, 71, 123, 125, 134, and 180, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 132.

[0295] 10. The engineered pantothenate kinase of claim 1, 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: 132, or a functional fragment thereof, wherein the engineered pantothenate kinase comprises at least one substitution or set of substitutions in the polypeptide sequence, and wherein amino acid positions in the polypeptide sequence are numbered with reference to SEQ ID NO: 132.

[0296] 11. The engineered pantothenate kinase of claim 2, wherein the engineered pantothenate 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: 132, or a functional fragment thereof, and wherein the engineered pantothenate kinase comprises at least one substitution or set of substitutions at one or more positions selected from the group consisting of: 92 / 301, 125, and 180, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 132.

[0297] 12. The engineered pantothenic acid kinase according to Project 1, wherein the engineered pantothenic acid 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 with SEQ ID NO:222, wherein the engineered pantothenic acid kinase comprises at least one substitution or set of substitutions in the polypeptide sequence, and wherein the amino acid positions of the polypeptide sequence refer to the number SEQ ID NO:222.

[0298] 13. The engineered pantothenic acid kinase according to item 2, wherein the engineered pantothenic acid 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 with SEQ ID NO:222, and wherein the engineered pantothenic acid kinase contains at least one substitution or set of substitutions at one or more positions selected from the following: 83 / 84 / 305, 143 and 154, and wherein the amino acid positions of the polypeptide sequence refer to the number SEQ ID NO:222.

[0299] 14. The engineered pantothenic acid kinase according to Project 1, wherein the engineered pantothenic acid 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 with SEQ ID NO:230, wherein the engineered pantothenic acid kinase comprises at least one substitution or set of substitutions in the polypeptide sequence, and wherein the amino acid positions of the polypeptide sequence refer to the number SEQ ID NO:230.

[0300] 15. The engineered pantothenic acid kinase according to item 2, wherein the engineered pantothenic acid 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 with SEQ ID NO:230, and wherein the engineered pantothenic acid kinase comprises at least one substitution or set of substitutions at one or more positions selected from the following: 13 / 169 / 213 / 247 / 283 / 288, 16 / 247, 49 / 247, 64 / 104 / 154 / 284, 75 / 104 / 284, 169 / 247 and 247, and wherein the amino acid positions of the polypeptide sequence refer to the numbers in SEQ ID NO:230.

[0301] 16. The engineered pantothenic acid kinase according to Project 1, wherein the engineered pantothenic acid 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 with SEQ ID NO:240, wherein the engineered pantothenic acid kinase comprises at least one substitution or set of substitutions in the polypeptide sequence, and wherein the amino acid positions of the polypeptide sequence refer to the number SEQ ID NO:240.

[0302] 17. The engineered pantothenic acid kinase according to item 2, wherein the engineered pantothenic acid 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 with SEQ ID NO:240, and wherein the engineered pantothenic acid kinase comprises at least one substitution or set of substitutions at one or more positions selected from the following: 8, 8 / 13 / 14 / 83 / 247, 8 / 64 / 213 / 247, 8 / 64 / 247, 8 / 247 / 283, 10 and 264 / 276, and wherein the amino acid positions of the polypeptide sequence refer to the number of SEQ ID NO:240.

[0303] 18. The engineered pantothenic acid kinase according to Project 1, wherein the engineered pantothenic acid 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 with SEQ ID NO:276, wherein the engineered pantothenic acid kinase comprises at least one substitution or set of substitutions in the polypeptide sequence, and wherein the amino acid positions of the polypeptide sequence refer to the number SEQ ID NO:276.

[0304] 19. The engineered pantothenic acid kinase according to item 2, wherein the engineered pantothenic acid 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 with SEQ ID NO:276, and wherein the engineered pantothenic acid kinase comprises at least one substitution or set of substitutions at one or more positions selected from the following: 11 / 13 / 23 / 61 / 85 / 304, 13 / 19 / 61, 23 / 61 and 23 / 61 / 304, and wherein the amino acid positions of the polypeptide sequence refer to the number of SEQ ID NO:276.

[0305] 20. The engineered pantothenic kinase according to Item 1, wherein the engineered pantothenic 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 pantothenic kinase variant listed in Tables 2-1, 4-1, 5-1, 6-1, 6-2, 7-1, 8-1, 9-1 and / or Table 10-1.

[0306] 21. The engineered pantothenic acid kinase according to Project 1, wherein the engineered pantothenic acid kinase comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the same polypeptide sequence as SEQ ID NO:2.

[0307] 22. The engineered pantothenate kinase of item 1, wherein the engineered pantothenate kinase comprises a variant engineered polypeptide listed in SEQ ID NO: 2.

[0308] 23. The engineered pantothenic acid kinase according to Project 1, wherein the engineered pantothenic acid kinase comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the same polypeptide sequence as SEQ ID NO:30.

[0309] 24. The engineered pantothenate kinase according to item 1, wherein the engineered pantothenate kinase comprises a variant engineered polypeptide listed in SEQ ID NO: 30.

[0310] 25. The engineered pantothenic acid kinase according to item 1, wherein the engineered pantothenic acid kinase comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the same polypeptide sequence as SEQ ID NO: 60.

[0311] 26. The engineered pantothenate kinase of item 1, wherein the engineered pantothenate kinase comprises a variant engineered polypeptide set forth in SEQ ID NO: 60.

[0312] 27. The engineered pantothenic acid kinase according to Project 1, wherein the engineered pantothenic acid kinase comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the same polypeptide sequence as SEQ ID NO: 132.

[0313] 28. The engineered pantothenic acid kinase according to item 1, wherein the engineered pantothenic acid kinase comprises a variant engineered peptide listed in SEQ ID NO:132.

[0314] 29. The engineered pantothenic acid kinase according to item 1, wherein the engineered pantothenic acid kinase comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the same polypeptide sequence as SEQ ID NO: 222.

[0315] 30. The engineered pantothenic acid kinase according to item 1, wherein the engineered pantothenic acid kinase comprises a variant engineered peptide listed in SEQ ID NO:222.

[0316] 31. The engineered pantothenic acid kinase according to Project 1, wherein the engineered pantothenic acid kinase comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the same polypeptide sequence as SEQ ID NO:230.

[0317] 32. The engineered pantothenic acid kinase according to item 1, wherein the engineered pantothenic acid kinase comprises a variant engineered peptide listed in SEQ ID NO:230.

[0318] 33. The engineered pantothenic acid kinase according to item 1, wherein the engineered pantothenic acid kinase comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the same polypeptide sequence as SEQ ID NO:240.

[0319] 34. The engineered pantothenic acid kinase according to item 1, wherein the engineered pantothenic acid kinase comprises a variant engineered peptide listed in SEQ ID NO:240.

[0320] 35. The engineered pantothenic acid kinase according to item 1, wherein the engineered pantothenic acid kinase comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the same polypeptide sequence as SEQ ID NO:276.

[0321] 36. The engineered pantothenic acid kinase according to item 1, wherein the engineered pantothenic acid kinase comprises a variant engineered peptide listed in SEQ ID NO:276.

[0322] 37. The engineered pantothenic kinase according to item 1, wherein the engineered pantothenic kinase comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the same polypeptide sequence as at least one engineered pantothenic kinase variant listed in the even-numbered sequences of SEQ ID NO: 2-270.

[0323] 38. The engineered pantothenic acid kinase according to item 1, wherein the engineered pantothenic acid kinase comprises the polypeptide sequence listed in the even-numbered sequences of SEQ ID NO:2-270.

[0324] 39. The engineered pantothenic kinase according to any one of items 1-38, wherein the engineered pantothenic kinase comprises at least one improved property compared with wild-type Escherichia coli (E. coli) pantothenic kinase.

[0325] 40. The engineered pantothenic acid kinase according to item 39, wherein the improved properties include improved substrate activity compared to wild-type pantothenic acid kinase.

[0326] 41. The engineered pantothenic acid kinase according to item 40, wherein the substrate comprises 2,4-dinitrophenylhydrazine and at least one aldehyde.

[0327] 42. The engineered pantothenic acid kinase according to item 39, wherein the improved properties include improved production of phospho-ethynylglyceraldehyde compared to wild-type pantothenic acid kinase.

[0328] 43. The engineered pantothenic acid kinase according to any one of items 1-42, wherein the engineered pantothenic acid kinase is purified.

[0329] 44. A composition comprising at least one engineered pantothenic acid kinase according to any one of items 1-43.

[0330] 45. A polynucleotide sequence encoding at least one engineered pantothenic acid kinase according to any one of items 1-43.

[0331] 46. ​​A polynucleotide sequence encoding at least one engineered pantothenic acid kinase, wherein the polynucleotide sequence comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of sequence identity with SEQ ID NO:1, SEQ ID NO:29, SEQ ID NO:59, SEQ ID NO:131, SEQ ID NO:221, SEQ ID NO:229, SEQ ID NO:239 and / or SEQ ID NO:275, wherein the polynucleotide sequence of the engineered pantothenic acid kinase contains at least one substitution at one or more positions.

[0332] 47. The polynucleotide sequence according to item 46, the polynucleotide sequence encoding at least one engineered pantothenic acid kinase or a functional fragment thereof, the polynucleotide sequence comprising 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.

[0333] 48. The polynucleotide sequence according to item 46, the polynucleotide sequence encoding at least one engineered pantothenic acid kinase or a functional fragment thereof, the polynucleotide sequence comprising 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:29.

[0334] 49. The polynucleotide sequence according to item 46, the polynucleotide sequence encoding at least one engineered pantothenic acid kinase or a functional fragment thereof, the polynucleotide sequence comprising 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:59.

[0335] 50. The polynucleotide sequence according to item 46, the polynucleotide sequence encoding at least one engineered pantothenic acid kinase or a functional fragment thereof, the polynucleotide sequence comprising 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:131.

[0336] 51. The polynucleotide sequence according to item 46, the polynucleotide sequence encoding at least one engineered pantothenic acid kinase or a functional fragment thereof, the polynucleotide sequence comprising 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:221.

[0337] 52. The polynucleotide sequence according to item 46, the polynucleotide sequence encoding at least one engineered pantothenic acid kinase or a functional fragment thereof, the polynucleotide sequence comprising 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:229.

[0338] 53. The polynucleotide sequence according to item 46, the polynucleotide sequence encoding at least one engineered pantothenic acid kinase or a functional fragment thereof, the polynucleotide sequence comprising 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:239.

[0339] 54. The polynucleotide sequence according to item 46, the polynucleotide sequence encoding at least one engineered pantothenic acid kinase or a functional fragment thereof, the polynucleotide sequence comprising 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:275.

[0340] 55. A polynucleotide sequence according to any one of items 45-54, wherein the polynucleotide sequence is operatively linked to a control sequence.

[0341] 56. The polynucleotide sequence according to any one of items 45-54, wherein the polynucleotide sequence is codon-optimized.

[0342] 57. The polynucleotide sequence according to any one of items 45-56, wherein the polynucleotide sequence comprises the sequences numbered odd numbers in SEQ ID NO:1-269.

[0343] 58. An expression vector comprising at least one polynucleotide sequence according to any one of items 45-57.

[0344] 59. A host cell comprising at least one expression vector according to item 58.

[0345] 60. A host cell comprising at least one polynucleotide sequence according to any one of items 45-57.

[0346] 61. A method for producing engineered pantothenic acid kinase in host cells, the method comprising culturing host cells according to items 59 and / or 60 under suitable conditions to produce at least one engineered pantothenic acid kinase.

[0347] 62. The method according to item 61, the method further comprising recovering at least one engineered pantothenic acid kinase from the culture and / or the host cell.

[0348] 63. The method according to items 61 and / or 62, the method further comprising the step of purifying the at least one engineered pantothenic acid kinase.

[0349] For all purposes, all publications, patents, patent applications and other documents cited in this application are incorporated herein by reference in their entirety as if each individual publication, patent, patent application or other document were individually indicated as being incorporated herein by reference for all purposes.

[0350] Although various specific implementation schemes have been described and illustrated, it will be understood that various changes can be made without departing from the spirit and scope of the invention. Sequence Listing <110> Kodxheath <120> Engineered pantothenic acid kinase variant enzyme <130> CX2-177WO2 <140> <141> 2019-07-02 <150> 62 / 695,587 <151> 2018-07-09 <150> 62 / 822,301 <151> 2019-03-22 <160> 276 <210> 1 <211> 951 <212> DNA <213> Artificial Sequence <220> <223>Pantothenate kinase from Escherichia coli <400> 1 atgagcatca aagagcagac cctgatgacc ccgtacctgc agttcgaccg taaccaatgg 60 gcggcgctgc gtgatagcgt tccgatgacc ctgagcgagg acgaaatcgc gcgtctgaag 120 ggtattaacg aggatctgag cctggaggaa gtggcggaaa tctacctgcc gctgagccgt 180 ctgctgaact tctatattag cagcaacctg cgtcgtcagg cggttctgga acaatttctg 240 ggtaccaacg gccagcgtat cccgtatatc attagcattg cgggtagcgt ggcggttggc 300 aaaagcacca ccgcgcgtgt gctgcaggcg ctgctgagcc gttggccgga gcaccgtcgt 360 gttgaactga tcaccaccga cggcttcctg cacccgaacc aagtgctgaa ggagcgtggt 420 ctgatgaaga aaaagggctt cccggaaagc tacgatatgc accgtctggt gaaatttgtt 480 agcgacctga agagcggtgt gccgaacgtt accgcgccgg tgtacagcca cctgatctat 540 gatgttattc cggacggcga taaaaccgtg gttcagccgg acatcctgat tctggagggt 600 ctgaacgtgc tgcaaagcgg catggactat ccgcacgatc cgcaccacgt gtttgttagc 660 gacttcgtgg attttagcat ctacgttgac gcgccggagg atctgctgca gacctggtat 720 attaaccgtt tcctgaaatt tcgtgagggt gcgttcaccg acccggatag ctactttcac 780 aactatgcga aactgaccaa ggaagaggcg atcaaaaccg cgatgaccct gtggaaggag 840 atcaactggc tgaacctgaa gcaaaacatt ctgccgaccc gtgaacgtgc gagcctgatt 900 ctgaccaaaa gcgcgaacca cgcggtggag gaagttcgtc tgcgtaagta a 951 <210> 2 <211> 316 <212> PRT <213> Artificial Sequence <220> <223> Pantothenate kinase from Escherichia coli <400> 2 Met Ser Ile Lys Glu Gln Thr Leu Met Thr Pro Tyr Leu Gln Phe Asp 1 5 10 15 Arg Asn Gln Trp Ala Ala Leu Arg Asp Ser Val Pro Met Thr Leu Ser 20 25 30 Glu Asp Glu Ile Ala Arg Leu Lys Gly Ile Asn Glu Asp Leu Ser Leu 35 40 45 Glu Glu Val Ala Glu Ile Tyr Leu Pro Leu Ser Arg Leu Leu Asn Phe 50 55 60 Tyr Ile Ser Ser Asn Leu Arg Arg Gln Ala Val Leu Glu Gln Phe Leu 65 70 75 80 Gly Thr Asn Gly Gln Arg Ile Pro Tyr Ile Ile Ser Ile Ala Gly Ser 85 90 95 Val Ala Val Gly Lys Ser Thr Thr Ala Arg Val Leu Gln Ala Leu Leu 100 105 110 Ser Arg Trp Pro Glu His Arg Arg Val Glu Leu Ile Thr Thr Asp Gly 115 120 125 Phe Leu His Pro Asn Gln Val Leu Lys Glu Arg Gly Leu Met Lys Lys 130 135 140 Lys Gly Phe Pro Glu Ser Tyr Asp Met His Arg Leu Val Lys Phe Val 145 150 155 160 Ser Asp Leu Lys Ser Gly Val Pro Asn Val Thr Ala Pro Val Tyr Ser 165 170 175 His Leu Ile Tyr Asp Val Ile Pro Asp Gly Asp Lys Thr Val Val Gln 180 185 190 Pro Asp Ile Leu Ile Leu Glu Gly Leu Asn Val Leu Gln Ser Gly Met 195 200 205 Asp Tyr Pro His Asp Pro His His Val Phe Val Ser Asp Phe Val Asp 210 215 220 Phe Ser Ile Tyr Val Asp Ala Pro Glu Asp Leu Leu Gln Thr Trp Tyr 225 230 235 240 Ile Asn Arg Phe Leu Lys Phe Arg Glu Gly Ala Phe Thr Asp Pro Asp 245 250 255 Ser Tyr Phe His Asn Tyr Ala Lys Leu Thr Lys Glu Glu Ala Ile Lys 260 265 270 Thr Ala Met Thr Leu Trp Lys Glu Ile Asn Trp Leu Asn Leu Lys Gln 275 280 285 Asn Ile Leu Pro Thr Arg Glu Arg Ala Ser Leu Ile Leu Thr Lys Ser 290 295 300 Ala Asn His Ala Val Glu Glu Val Arg Leu Arg Lys 305 310 315 <210> 3 <211> 951 <212> DNA <213> Artificial Sequence <220> <223> A variant of pantothenic acid kinase from Escherichia coli <400> 3 atgagcatca aagagcagac cctgatgacc ccgtacctgc agttcgaccg taaccaatgg 60 gcggcgctgc gtgatagcgt tccgatgacc ctgagcgagg acgaaatcgc gcgtctgaag 120 ggtattaacg aggatctgag cctggaggaa gtggcggaaa tctacctgcc gctgagccgt 180 ctgctgaact tctatattag cagcaacctg cgtcgtcagg cggttctgga acaatttctg 240 ggtaccaacg gccagcgtat cccgtatatc attagcattg cgggtagcgt ggcggttggc 300 aaaagcacca ccgcgcgtgt gctgcaggcg ctgctgagcc gttggccgga gcaccgtcgt 360 gttgaactga tcaccaccga cggcttcctg cacccgaacc aagtgctgaa ggagcgtggt 420 ctgatgaaga aaaagggttt cccggaaagc tacgatatgc accgtctggt gaaatttgtt 480 agcgacctga agagcggtgt gccgaacgtt accgcgccgg tgtacagcca cctgatctat 540 gatgttattc cggacggcga taaaaccgtg gttcagccgg acatcctgat tctggagggt 600 ctgaacgtgc tgcaaagcgg catggactat ccgcacgatc cgcaccacgt gtttgttagc 660 gacttcgtgg attttagcat ctacgttgac gcgccggagg atctgctgca gacctggttc 720 attaaccgtt tcctgaaatt tcgtgagggt gcgttcaccg acccggatag ctactttcac 780 aactatgcga aactgaccaa ggaagaggcg atcaaaaccg cgatgaccat gtggaaggag 840 atgatgtggc tgaacctgaa gcaaaacatt ctgccgaccc gtgaacgtgc gagcctgatt 900 ctgaccaaaa gcgcgaacca cgcggtggag gaagttcgtc tgcgtaagta a 951 <210> 4 <211> 316 <212> PRT <213> Artificial Sequence <220> <223> A variant of pantothenic acid kinase from Escherichia coli <400> 4 Met Ser Ile Lys Glu Gln Thr Leu Met Thr Pro Tyr Leu Gln Phe Asp 1 5 10 15 Arg Asn Gln Trp Ala Ala Leu Arg Asp Ser Val Pro Met Thr Leu Ser 20 25 30 Glu Asp Glu Ile Ala Arg Leu Lys Gly Ile Asn Glu Asp Leu Ser Leu 35 40 45 Glu Glu Val Ala Glu Ile Tyr Leu Pro Leu Ser Arg Leu Leu Asn Phe 50 55 60 Tyr Ile Ser Ser Asn Leu Arg Arg Gln Ala Val Leu Glu Gln Phe Leu 65 70 75 80 Gly Thr Asn Gly Gln Arg Ile Pro Tyr Ile Ile Ser Ile Ala Gly Ser 85 90 95 Val Ala Val Gly Lys Ser Thr Thr Ala Arg Val Leu Gln Ala Leu Leu 100 105 110 Ser Arg Trp Pro Glu His Arg Arg Val Glu Leu Ile Thr Thr Asp Gly 115 120 125 Phe Leu His Pro Asn Gln Val Leu Lys Glu Arg Gly Leu Met Lys Lys 130 135 140 Lys Gly Phe Pro Glu Ser Tyr Asp Met His Arg Leu Val Lys Phe Val 145 150 155 160 Ser Asp Leu Lys Ser Gly Val Pro Asn Val Thr Ala Pro Val Tyr Ser 165 170 175 His Leu Ile Tyr Asp Val Ile Pro Asp Gly Asp Lys Thr Val Val Gln 180 185 190 Pro Asp Ile Leu Ile Leu Glu Gly Leu Asn Val Leu Gln Ser Gly Met 195 200 205 Asp Tyr Pro His Asp Pro His His Val Phe Val Ser Asp Phe Val Asp 210 215 220 Phe Ser Ile Tyr Val Asp Ala Pro Glu Asp Leu Leu Gln Thr Trp Phe 225 230 235 240 Ile Asn Arg Phe Leu Lys Phe Arg Glu Gly Ala Phe Thr Asp Pro Asp 245 250 255 Ser Tyr Phe His Asn Tyr Ala Lys Leu Thr Lys Glu Glu Ala Ile Lys 260 265 270 Thr Ala Met Thr Met Trp Lys Glu Met Met Trp Leu Asn Leu Lys Gln 275 280 285 Asn Ile Leu Pro Thr Arg Glu Arg Ala Ser Ile Leu Thr Lys Ser 290 295 300 Ala Asn His Ala Val Glu Glu Val Arg Leu Arg Lys 305 310 315 <210> 5 <211> 951 <212> DNA <213> Artificial Sequence <220> <223> Recombinant strains of yeast strains (Escherichia coli). <400> 5 atgagcatca aagagcagac cctgatgacc ccgtacctgc agttcgaccg taaccaatgg gcggcgctgc gtgatagcgt tccgatgacc ctgagcgagg acgaaatcgc gcgtctgaag ggtattaacg aggatctgag cctggagga gtggcggaa tctacctgcc gctgagccgt 240. ctgctgaact tctatattag cagcaacctg cgtcgtcagg cggttctgga acaatttctg ggtaccaacg gccagcgtat cccgtatatc attagcattg cgggtagcgt ggcggttggc 300 aaaagcacca ccgcgcgtgt gctgcaggcg ctgctgagcc gttggccgga gcaccgtcgt 360 gttgaactga tcaccaccga cggcttcctg cacccgaacc aagtgctgaa ggagcgtggt 420 ctgatgaaga aaaagggttt cccggaaagc tacgatatgc accgtctggt gaaatttgtt 480 agcgacctga agagcggtgt gccgaacgtt accgcgccgg tgtacagcca cctgatctat 540 gatgttattc cggacggcga taaaaccgtg gttcagccgg acatcctgat tctggagggt 600 ctgaacgtgc tgcaaagcgg catggactat ccgcacgatc cgcaccacgt gtttgttagc 660 gacttcgtgg attttagcat ctacgttgac gcgccggagg atctgctgca gacctggttc 720 attaaccgtt tcctgaaatt tcgtgagggt gcgttcaccg acccggatag ctactttcac 780 aactatgcga aactgaccaa ggaagaggcg atcaaaaccg cgatgaccat ttggaaggag 840 attaactggc tgaacctgaa gcaaaacatt ctgccgaccc gtgaacgtgc gagcctgatt 900 ctgaccaaaa gcgcgaacca cgcggtggag gaagttcgtc tgcgtaagta a 951 <210> 6 <211> 316 <212> PRT <213> Artificial Sequence <220> <223> Variant of pantothenate kinase from Escherichia coli <400> 6 Met Ser Ile Lys Glu Gln Thr Leu Met Thr Pro Tyr Leu Gln Phe Asp 1 5 10 15 Arg Asn Gln Trp Ala Ala Leu Arg Asp Ser Val Pro Met Thr Leu Ser 20 25 30 Glu Asp Glu Ile Ala Arg Leu Lys Gly Ile Asn Glu Asp Leu Ser Leu 35 40 45 Glu Glu Val Ala Glu Ile Tyr Leu Pro Leu Ser Arg Leu Leu Asn Phe 50 55 60 Tyr Ile Ser Ser Asn Leu Arg Arg Gln Ala Val Leu Glu Gln Phe Leu 65 70 75 80 Gly Thr Asn Gly Gln Arg Ile Pro Tyr Ile Ile Ser Ile Ala Gly Ser 85 90 95 Val Ala Val Gly Lys Ser Thr Thr Ala Arg Val Leu Gln Ala Leu Leu 100 105 110 Ser Arg Trp Pro Glu His Arg Arg Val Glu Leu Ile Thr Thr Asp Gly 115 120 125 Phe Leu His Pro Asn Gln Val Leu Lys Glu Arg Gly Leu Met Lys Lys 130 135 140 Lys Gly Phe Pro Glu Ser Tyr Asp Met His Arg Leu Val Lys Phe Val 145 150 155 160 Ser Asp Leu Lys Ser Gly Val Pro Asn Val Thr Ala Pro Val Tyr Ser 165 170 175 His Leu Ile Tyr Asp Val Ile Pro Asp Gly Asp Lys Thr Val Val Gln 180 185 190 Pro Asp Ile Leu Ile Leu Glu Gly Leu Asn Val Leu Gln Ser Gly Met 195 200 205 Asp Tyr Pro His Asp Pro His His Val Phe Val Ser Asp Phe Val Asp 210 215 220 Phe Ser Ile Tyr Val Asp Ala Pro Glu Asp Leu Leu Gln Thr Trp Phe 225 230 235 240 Ile Asn Arg Phe Leu Lys Phe Arg Glu Gly Ala Phe Thr Asp Pro Asp 245 250 255 Ser Tyr Phe His Asn Tyr Ala Lys Leu Thr Lys Glu Glu Ala Ile Lys 260 265 270 Thr Ala Met Thr Ile Trp Lys Glu Ile Asn Trp Leu Asn Leu Lys Gln 275 280 285 Asn Ile Leu Pro Thr Arg Glu Arg Ala Ser Leu Ile Leu Thr Lys Ser 290 295 300 Ala Asn His Ala Val Glu Glu Val Arg Leu Arg Lys 305 310 315 <210> 7 <211> 951 <212> DNA <213> Artificial Sequence <220> <223> Recombinant strains of yeast strains (Escherichia coli). <400> 7 atgagcatca aagagcagac cctgatgacc ccgtacctgc agttcgaccg taaccaatgg gcggcgctgc gtgatagcgt tccgatgacc ctgagcgagg acgaaatcgc gcgtctgaag ggtattaacg aggatctgag cctggagga gtggcggaa tctacctgcc gctgagccgt 240. ctgctgaact tctatattag cagcaacctg cgtcgtcagg cggttctgga acaatttctg ggtaccaacg gccagcgtat cccgtatatc attagcattg cgggtagcgt ggcggttggc 300 aaaagcacca ccgcgcgtgt gctgcaggcg ctgctgagcc gttggccgga gcaccgtcgt 360 gttgaactga tcaccaccga cggcttcctg cacccgaacc aagtgctga ggagcgtggt ctgatgaaga aaaagggctt cccggaaagc tacgatatgc accgtctggt gaaatttgtt agcgacctga agagcggtgt gccgaacgtt accgcgccgg tgtacagcca cctgatctat 540 gatgttattc cggacggcga taaaaccgtg gttcagccgg acatcctgat tctggagggt 600 ctgaacgtgc tgcaaagcgg catggactat ccgcacgatc cgcaccacgt gtttgttagc 660 gacttcgtgg attttagcat ctacgttgac gcgccggagg atctgctgca gacctggtac 720 attaaccgtt tcctgaaatt tcgtgagggt gcgttcaccg acccggatag ctactttcac 780 aactatgcga aactgaccaa ggaagaggcg atcaaaaccg cgatgaccat gtggaaggag 840 attaactggc tgaacctgaa gcaaaacatt ctgccgaccc gtgaacgtgc gagcctgatt 900 ctgaccaaaa gcgcgaacca cgcggtggag gaagttcgtc tgcgtaagta a 951 <210> 8 <211> 316 <212> PRT <213> Artificial Sequence <220> [[ID=2​​​​​​​Arg Asn Gln Trp Ala Ala Leu Arg Asp Ser Val Pro Met Thr Leu Ser 20 25 30 Glu Asp Glu Ile Ala Arg Leu Lys Gly Ile Asn Glu Asp Leu Ser Leu 35 40 45 Glu Glu Val Ala Glu Ile Tyr Leu Pro Leu Ser Arg Leu Leu Asn Phe 50 55 60 Tyr Ile Ser Ser Asn Leu Arg Arg Gln Ala Val Leu Glu Gln Phe Leu 65 70 75 80 Gly Thr Asn Gly Gln Arg Ile Pro Tyr Ile Ile Ser Ile Ala Gly Ser 85 90 95 Val Ala Val Gly Lys Ser Thr Thr Ala Arg Val Leu Gln Ala Leu Leu 100 105 110 Ser Arg Trp Pro Glu His Arg Arg Val Glu Leu Ile Thr Thr Asp Gly 115 120 125 Phe Leu His Pro Asn Gln Val Leu Lys Glu Arg Gly Leu Met Lys Lys 130 135 140 Lys Gly Phe Pro Glu Ser Tyr Asp Met His Arg Leu Val Lys Phe Val 145 150 155 160 Ser Asp Leu Lys Ser Gly Val Pro Asn Val Thr Ala Pro Val Tyr Ser 165 170 175 His Leu Ile Tyr Asp Val Ile Pro Asp Gly Asp Lys Thr Val Val Gln 180 185 190 Pro Asp Ile Leu Ile Leu Glu Gly Leu Asn Val Leu Gln Ser Gly Met 195 200 205 Asp Tyr Pro His Asp Pro His His Val Phe Val Ser Asp Phe Val Asp 210 215 220 Phe Ser Ile Tyr Val Asp Ala Pro Glu Asp Leu Leu Gln Thr Trp Tyr 225 230 235 240 Ile Asn Arg Phe Leu Lys Phe Arg Glu Gly Ala Phe Thr Asp Pro Asp 245 250 255 Ser Tyr Phe His Asn Tyr Ala Lys Leu Thr Lys Glu Glu Ala Ile Lys 260 265 270 Thr Ala Met Thr Met Trp Lys Glu Ile Asn Trp Leu Asn Leu Lys Gln 275 280 285 Asn Ile Leu Pro Thr Arg Glu Arg Ala Ser Leu Ile Leu Thr Lys Ser 290 295 300 Ala Asn His Ala Val Glu Glu Val Arg Leu Arg Lys 305 310 315 <210> 9 <211> 951 <212> DNA <213> Artificial Sequence <220> <223> Variant of pantothenate kinase from Escherichia coli <400> 9 atgagcatca aagagcagac cctgatgacc ccgtacctgc agttcgaccg taaccaatgg 60 gcggcgctgc gtgatagcgt tccgatgacc ctgagcgagg acgaaatcgc gcgtctgaag 120 ggtattaacg aggatctgag cctggaggaa gtggcggaaa gctacctgcc gctgagccgt 180 ctgctgaact tctatattag cagcaacctg cgtcgtcagg cggttctgga acaatttctg 240 ggtaccaacg gccagcgtat cccgtatatc attagcattg cgggtagcgt ggcggttggc 300 aaaagcacca ccgcgcgtgt gctgcaggcg ctgctgagcc gttggccgga gcaccgtcgt 360 gttgaactga tcaccaccga cggcttcctg cacccgaacc aagtgctgaa ggagcgtggt 420 ctgatgaaga aaaagggttt cccggaaagc tacgatatgc accgtctggt gaaatttgtt 480 agcgacctga agagcggtgt gccgaacgtt accgcgccgg tgtacagcca cctgatctat 540 gatgttattc cggacggcga taaaaccgtg gttcagccgg acatcctgat tctggagggt 600 ctgaacgtgc tgcaaagcgg catggactat ccgcacgatc cgcaccacgt gtttgttagc 660 gacttcgtgg attttagcat ctacgttgac gcgccggagg atctgctgca gacctggtgg 720 attaaccgtt tcctgaaatt tcgtgagggt gccttcaccg acccggatag ctactttcac 780 aactatgcga aactgaccaa ggaagaggcg atcaaaaccg cgatgaccat gtggaaggag 840 atgaactggc tgaacctgaa gcaaaacatt ctgccgaccc gtgaacgtgc gagcctgatt 900 ctgaccaaaa gcgcgaacca cgcggtggag gaagttcgtc tgcgtaagta a 951 <210> 10 <211> 316 <212> PRT <213> artificial sequence <220> <223> It comes from Escherichia coli <400> 10 Met Ser Ile Lys Glu Gln Thr Leu Met Thr Pro Tyr Leu Gln Phe Asp 1 5 10 15 Arg Asn Gln Trp Ala Ala Leu Arg Asp Ser Val Pro Met Thr Leu Ser 20 25 30 Glu Asp Glu Ile Ala Arg Leu Lys Gly Ile Asn Glu Asp Leu Ser Leu 35 40 45 Glu Glu Val Ala Glu Ser Tyr Leu Pro Leu Ser Arg Leu Leu Asn Phe 50 55 60 Tyr Ile Ser Ser Asn Leu Arg Arg Gln Ala Val Leu Glu Gln Phe Leu 65 70 75 80 Gly Thr Asn Gly Gln Arg Ile Pro Tyr Ile Ile Ser Ile Ala Gly Ser 85 90 95 Val Ala Val Gly Lys Ser Thr Thr Ala Arg Val Leu Gln Ala Leu Leu 100 105 110 Ser Arg Trp Pro Glu His Arg Arg Val Glu Leu Ile Thr Thr Asp Gly 115 120 125 Phe Leu His Pro Asn Gln Val Leu Lys Glu Arg Gly Leu Met Lys Lys 130 135 140 Lys Gly Phe Pro Glu Ser Tyr Asp Met His Arg Leu Val Lys Phe Val 145 150 155 160 Ser Asp Leu Lys Ser Gly Val Pro Asn Val Thr Ala Pro Val Tyr Ser 165 170 175 His Leu Ile Tyr Asp Val Ile Pro Asp Gly Asp Lys Thr Val Val Gln 180 185 190 Pro Asp Ile Leu Ile Leu Glu Gly Leu Asn Val Leu Gln Ser Gly Met 195 200 205 Asp Tyr Pro His Asp Pro His His Val Phe Val Ser Asp Phe Val Asp 210 215 220 Phe Ser Ile Tyr Val Asp Ala Pro Glu Asp Leu Leu Gln Thr Trp Trp 225 230 235 240 Ile Asn Arg Phe Leu Lys Phe Arg Glu Gly Ala Phe Thr Asp Pro Asp 245 250 255 Ser Tyr Phe His Asn Tyr Ala Lys Leu Thr Lys Glu Glu Ala Ile Lys 260 265 270 Thr Ala Met Thr Met Trp Lys Glu Met Asn Trp Leu Asn Leu Lys Gln 275 280 285 Asn Ile Leu Pro Thr Arg Glu Arg Ala Ser Leu Ile Leu Thr Lys Ser 290 295 300 Ala Asn His Ala Val Glu Glu Val Arg Leu Arg Lys 305 310 315 <210> 11 <211> 951 <212> DNA <213> Artificial Sequence <220> <223> Variants of pantothenate kinase from Escherichia coli <400> 11 atgagcatca aagagcagac cctgatgacc ccgtacctgc agttcgaccg taaccaatgg 60 gcggcgctgc gtgatagcgt tccgatgacc ctgagcgagg acgaaatcgc gcgtctgaag 120 ggtattaacg aggatctgag cctggaggaa gtggcggaaa tctacctgcc gctgagccgt 180 ctgctgaact tctatattag cagcaacctg cgtcgtcagg cggttctgga acaatttctg 240 ggtaccaacg gccagcgtat cccgtatatc attagcattg cgggtagcgt ggcggttggc 300 aaaagcacca ccgcgcgtgt gctgcaggcg ctgctgagcc gttggccgga gcaccgtcgt 360 gttgaactga tcaccaccga cggcttcctg cacccgaacc aagtgctgaa ggagcgtggt 420 ctgatgaaga aaaagggctt cccggaaagc tacgatatgc accgtctggt gaaatttgtt 480 agcgacctga agagcggtgt gccgaacgtt accgcgccgg tgtacagcca cctgatctat 540 gatgttattc cggacggcga taaaaccgtg gttcagccgg acatcctgat tctggagggt 600 ctgaacgtgc tgcaaagcgg catggactat ccgcacgatc cgcaccacgt gtttgttagc 660 gacttcgtgg attttagcat ctacgttgac gcgccggagg atctgctgca gacctggttc 720 attaaccgtt tcctgaaatt tcgtgagggt gcgttcaccg acccggatag ctactttcac 780 aactatgcga aactgaccaa ggaagaggcg atcaaaaccg cgatgaccat gtggaaggag 840 atgaactggc tgaacctgaa gcaaaacatt ctgccgaccc gtgaacgtgc gagcctgatt 900 ctgaccaaaa gcgcgaacca cgcggtggag gaagttcgtc tgcgtaagta a 951 <210> 12 <211> 316 <212> PRT <213> Artificial Sequence <220> <223> Variants of pantothenate kinase from Escherichia coli <400> 12 Met Ser Ile Lys Glu Gln Thr Leu Met Thr Pro Tyr Leu Gln Phe Asp 1 5 10 15 Arg Asn Gln Trp Ala Ala Leu Arg Asp Ser Val Pro Met Thr Leu Ser 20 25 30 Glu Asp Glu Ile Ala Arg Leu Lys Gly Ile Asn Glu Asp Leu Ser Leu 35 40 45 Glu Glu Val Ala Glu Ile Tyr Leu Pro Leu Ser Arg Leu Leu Asn Phe 50 55 60 Tyr Ile Ser Ser Asn Leu Arg Arg Gln Ala Val Leu Glu Gln Phe Leu 65 70 75 80 Gly Thr Asn Gly Gln Arg Ile Pro Tyr Ile Ile Ser Ile Ala Gly Ser 85 90 95 Val Ala Val Gly Lys Ser Thr Thr Ala Arg Val Leu Gln Ala Leu Leu 100 105 110 Ser Arg Trp Pro Glu His Arg Arg Val Glu Leu Ile Thr Thr Asp Gly 115 120 125 Phe Leu His Pro Asn Gln Val Leu Lys Glu Arg Gly Leu Met Lys Lys 130 135 140 Lys Gly Phe Pro Glu Ser Tyr Asp Met His Arg Leu Val Lys Phe Val 145 150 155 160 Ser Asp Leu Lys Ser Gly Val Pro Asn Val Thr Ala Pro Val Tyr Ser 165 170 175 His Leu Ile Tyr Asp Val Ile Pro Asp Gly Asp Lys Thr Val Val Gln 180 185 190 Pro Asp Ile Leu Ile Leu Glu Gly Leu Asn Val Leu Gln Ser Gly Met 195 200 205 Asp Tyr Pro His Asp Pro His His Val Phe Val Ser Asp Phe Val Asp 210 215 220 Phe Ser Ile Tyr Val Asp Ala Pro Glu Asp Leu Leu Gln Thr Trp Phe 225 230 235 240 Ile Asn Arg Phe Leu Lys Phe Arg Glu Gly Ala Phe Thr Asp Pro Asp 245 250 255 Ser Tyr Phe His Asn Tyr Ala Lys Leu Thr Lys Glu Glu Ala Ile Lys 260 265 270 Thr Ala Met Thr Met Trp Lys Glu Met Asn Trp Leu Asn Leu Lys Gln 275 280 285 Asn Ile Leu Pro Thr Arg Glu Arg Ala Ser Ile Leu Thr Lys Ser 290 295 300 Ala Asn His Ala Val Glu Glu Val Arg Leu Arg Lys 305 310 315 <210> 13 <211> 951 <212> DNA <213> Artificial Sequence <220> <223> Recombinant strains of yeast strains (Escherichia coli). <400> 13 atgagcatca aagagcagac cctgatgacc ccgtacctgc agttcgaccg taaccaatgg gcggcgctgc gtgatagcgt tccgatgacc ctgagcgagg acgaaatcgc gcgtctgaag ggtattaacg aggatctgag cctggagga gtggcggaa tctacctgcc gctgagccgt 240. ctgctgaact tctatattag cagcaacctg cgtcgtcagg cggttctgga acaatttctg ggtaccaacg gccagcgtat cccgtatatc attagcattg cgggtagcgt ggcggttggc 300 aaaagcacca ccgcgcgtgt gctgcaggcg ctgctgagcc gttggccgga gcaccgtcgt 360 gttgaactga tcaccaccga cggcttcctg cacccgaacc aagtgctgaa ggagcgtggt 420 ctgatgaaga aaaagggctt cccggaaagc tacgatatgc accgtctggt gaaatttgtt 480 agcgacctga agagcggtgt gccgaacgtt accgcgccgg tgtacagcca cctgatctat 540 gatgttattc cggacggcga taaaaccgtg gttcagccgg acatcctgat tctggagggt 600 ctgaacgtgc tgcaaagcgg catggactat ccgcacgatc cgcaccacgt gtttgttagc 660 gacttcgtgg attttagcat ctacgttgac gcgccggagg atctgctgca gacctggtac 720 attaaccgtt tcctgaaatt tcgtgagggt gcgttcaccg acccggatag ctactttcac 780 aactatgcga aactgaccaa ggaagaggcg atcaaaaccg cgatgaccat gtggaaggag 840 atgaactggc tgaacctgaa gcaaaacatt ctgccgaccc gtgaacgtgc gagcctgatt 900 ctgaccaaaa gcgcgaacca cgcggtggag gaagttcgtc tgcgtaagta a 951 <210> 14 <211> 316 <212> PRT <213> Artificial Sequence <220> <223> Variant of pantothenate kinase from Escherichia coli <400> 14 Met Ser Ile Lys Glu Gln Thr Leu Met Thr Pro Tyr Leu Gln Phe Asp 1 5 10 15 Arg Asn Gln Trp Ala Ala Leu Arg Asp Ser Val Pro Met Thr Leu Ser 20 25 30 Glu Asp Glu Ile Ala Arg Leu Lys Gly Ile Asn Glu Asp Leu Ser Leu 35 40 45 Glu Glu Val Ala Glu Ile Tyr Leu Pro Leu Ser Arg Leu Leu Asn Phe 50 55 60 Tyr Ile Ser Ser Asn Leu Arg Arg Gln Ala Val Leu Glu Gln Phe Leu 65 70 75 80 Gly Thr Asn Gly Gln Arg Ile Pro Tyr Ile Ile Ser Ile Ala Gly Ser 85 90 95 Val Ala Val Gly Lys Ser Thr Thr Ala Arg Val Leu Gln Ala Leu Leu 100 105 110 Ser Arg Trp Pro Glu His Arg Arg Val Glu Leu Ile Thr Thr Asp Gly 115 120 125 Phe Leu His Pro Asn Gln Val Leu Lys Glu Arg Gly Leu Met Lys Lys 130 135 140 Lys Gly Phe Pro Glu Ser Tyr Asp Met His Arg Leu Val Lys Phe Val 145 150 155 160 Ser Asp Leu Lys Ser Gly Val Pro Asn Val Thr Ala Pro Val Tyr Ser 165 170 175 His Leu Ile Tyr Asp Val Ile Pro Asp Gly Asp Lys Thr Val Val Gln 180 185 190 Pro Asp Ile Leu Ile Leu Glu Gly Leu Asn Val Leu Gln Ser Gly Met 195 200 205 Asp Tyr Pro His Asp Pro His His Val Phe Val Ser Asp Phe Val Asp 210 215 220 Phe Ser Ile Tyr Val Asp Ala Pro Glu Asp Leu Leu Gln Thr Trp Tyr 225 230 235 240 Ile Asn Arg Phe Leu Lys Phe Arg Glu Gly Ala Phe Thr Asp Pro Asp 245 250 255 Ser Tyr Phe His Asn Tyr Ala Lys Leu Thr Lys Glu Glu Ala Ile Lys 260 265 270 Thr Ala Met Thr Met Trp Lys Glu Met Asn Trp Leu Asn Leu Lys Gln 275 280 285 Asn Ile Leu Pro Thr Arg Glu Arg Ala Ser Leu Ile Leu Thr Lys Ser 290 295 300 Ala Asn His Ala Val Glu Glu Val Arg Leu Arg Lys 305 310 315 <210> 15 <211> 951 <212> DNA <213> Artificial Sequence <220> <223> Recombinant strains of yeast strains (Escherichia coli). <400> 15 atgagcatca aagagcagac cctgatgacc ccgtacctgc agttcgaccg taaccaatgg gcggcgctgc gtgatagcgt tccgatgacc ctgagcgagg acgaaatcgc gcgtctgaag ggtattaacg aggatctgag cctggagga gtggcggaa tctacctgcc gctgagccgt 240. ctgctgaact tctatattag cagcaacctg cgtcgtcagg cggttctgga acaatttctg ggtaccaacg gccagcgtat cccgtatatc attagcattg cgggtagcgt ggcggttggc 300 aaaagcacca ccgcgcgtgt gctgcaggcg ctgctgagcc gttggccgga gcaccgtcgt 360 gttgaactga tcaccaccga cggcttcctg cacccgaacc aagtgctga ggagcgtggt ctgatgaaga aaaagggttt cccggaaagc tacgatatgc accgtctggt gaaatttgtt agcgacctga agagcggtgt gccgaacgtt accgcgccgg tgtacagcca cctgatctat 540 gatgttattc cggacggcga taaaaccgtg gttcagccgg acatcctgat tctggagggt 600 ctgaacgtgc tgcaaagcgg catggactat ccgcacgatc cgcaccacgt gtttgttagc 660 gacttcgtgg attttagcat ctacgttgac gcgccggagg atctgctgca gacctggttc 720 attaaccgtt tcctgaaatt tcgtgagggt gcgttcaccg acccggatag ctactttcac 780 aactatgcga aactgaccaa ggaagaggcg atcaaaaccg cgatgacctt gtggaaggag 840 atgaactggc tgaacctgaa gcaaaacatt ctgccgaccc gtgaacgtgc gagcctgatt 900 ctgaccaaaa gcgcgaacca cgcggtggag gaagttcgtc tgcgtaagta a 951 <210> 16 <211> 316 <212> PRT <213> Artificial Sequence <220> <223> Variant of pantothenate kinase from Escherichia coli <400> 16 Met Ser Ile Lys Glu Gln Thr Leu Met Thr Pro Tyr Leu Gln Phe Asp 1 5 10 15 Arg Asn Gln Trp Ala Ala Leu Arg Asp Ser Val Pro Met Thr Leu Ser 20 25 30 Glu Asp Glu Ile Ala Arg Leu Lys Gly Ile Asn Glu Asp Leu Ser Leu 35 40 45 Glu Glu Val Ala Glu Ile Tyr Leu Pro Leu Ser Arg Leu Leu Asn Phe 50 55 60 Tyr Ile Ser Ser Asn Leu Arg Arg Gln Ala Val Leu Glu Gln Phe Leu 65 70 75 80 Gly Thr Asn Gly Gln Arg Ile Pro Tyr Ile Ile Ser Ile Ala Gly Ser 85 90 95 Val Ala Val Gly Lys Ser Thr Thr Ala Arg Val Leu Gln Ala Leu Leu 100 105 110 Ser Arg Trp Pro Glu His Arg Arg Val Glu Leu Ile Thr Thr Asp Gly 115 120 125 Phe Leu His Pro Asn Gln Val Leu Lys Glu Arg Gly Leu Met Lys Lys 130 135 140 Lys Gly Phe Pro Glu Ser Tyr Asp Met His Arg Leu Val Lys Phe Val 145 150 155 160 Ser Asp Leu Lys Ser Gly Val Pro Asn Val Thr Ala Pro Val Tyr Ser 165 170 175 His Leu Ile Tyr Asp Val Ile Pro Asp Gly Asp Lys Thr Val Val Gln 180 185 190 Pro Asp Ile Leu Ile Leu Glu Gly Leu Asn Val Leu Gln Ser Gly Met 195 200 205 Asp Tyr Pro His Asp Pro His His Val Phe Val Ser Asp Phe Val Asp 210 215 220 Phe Ser Ile Tyr Val Asp Ala Pro Glu Asp Leu Leu Gln Thr Trp Phe 225 230 235 240 Ile Asn Arg Phe Leu Lys Phe Arg Glu Gly Ala Phe Thr Asp Pro Asp 245 250 255 Ser Tyr Phe His Asn Tyr Ala Lys Leu Thr Lys Glu Glu Ala Ile Lys 260 265 270 Thr Ala Met Thr Leu Trp Lys Glu Met Asn Trp Leu Asn Leu Lys Gln 275 280 285 Asn Ile Leu Pro Thr Arg Glu Arg Ala Ser Leu Ile Leu Thr Lys Ser 290 295 300 Ala Asn His Ala Val Glu Glu Val Arg Leu Arg Lys 305 310 315 <210> 17 <211> 951 <212> DNA <213> Artificial Sequence <220> <223> Variant of pantothenate kinase from Escherichia coli <400> 17 atgagcatca aagagcagac cctgatgacc ccgtacctgc agttcgaccg taaccaatgg 60 gcggcgctgc gtgatagcgt tccgatgacc ctgagcgagg acgaaatcgc gcgtctgaag 120 ggtattaacg aggatctgag cctggaggaa gtggcggaaa tctacctgcc gctgagccgt 180 ctgctgaact tctatattag cagcaacctg cgtcgtcagg cggttctgga acaatttctg 240 ggtaccaacg gccagcgtat cccgtatatc attagcattg cgggtagcgt ggcggttggc 300 aaaagcacca ccgcgcgtgt gctgcaggcg ctgctgagcc gttggccgga gcaccgtcgt 360 gttgaactga tcaccaccga cggcttcctg cacccgaacc aagtgctgaa ggagcgtggt 420 ctgatgaaga aaaagggttt cccggaaagc tacgatatgc accgtctggt gaaatttgtt 480 agcgacctga agagcggtgt gccgaacgtt accgcgccgg tgtacagcca cctgatctat 540 gatgttattc cggacggcga taaaaccgtg gttcagccgg acatcctgat tctggagggt 600 ctgaacgtgc tgcaaagcgg catggactat ccgcacgatc cgcaccacgt gtttgttagc 660 gacttcgtgg attttagcat ctacgttgac gcgccggagg atctgctgca gacctggtat 720 attaaccgtt tcctgaaatt tcgtgagggt gccttcaccg acccggatag ctactttcac 780 aactatgcga aactgaccaa ggaagaggcg atcaaaaccg cgatgaccat ttggaaggag 840 ttgaactggc tgaacctgaa gcaaaacatt ctgccgaccc gtgaacgtgc gagcctgatt 900 ctgaccaaaa gcgcgaacca cgcggtggag gaagttcgtc tgcgtaagta a 951 <210> 18 <211> 316 <212> PRT <213> artificial sequence <220> <223> It comes from Escherichia coli <400> 18 Met Ser Ile Lys Glu Gln Thr Leu Met Thr Pro Tyr Leu Gln Phe Asp 1 5 10 15 Arg Asn Gln Trp Ala Ala Leu Arg Asp Ser Val Pro Met Thr Leu Ser 20 25 30 Glu Asp Glu Ile Ala Arg Leu Lys Gly Ile Asn Glu Asp Leu Ser Leu 35 40 45 Glu Glu Val Ala Glu Ile Tyr Leu Pro Leu Ser Arg Leu Leu Asn Phe 50 55 60 Tyr Ile Ser Ser Asn Leu Arg Arg Gln Ala Val Leu Glu Gln Phe Leu 65 70 75 80 Gly Thr Asn Gly Gln Arg Ile Pro Tyr Ile Ile Ser Ile Ala Gly Ser 85 90 95 Val Ala Val Gly Lys Ser Thr Thr Ala Arg Val Leu Gln Ala Leu Leu 100 105 110 Ser Arg Trp Pro Glu His Arg Arg Val Glu Leu Ile Thr Thr Asp Gly 115 120 125 Phe Leu His Pro Asn Gln Val Leu Lys Glu Arg Gly Leu Met Lys Lys 130 135 140 Lys Gly Phe Pro Glu Ser Tyr Asp Met His Arg Leu Val Lys Phe Val 145 150 155 160 Ser Asp Leu Lys Ser Gly Val Pro Asn Val Thr Ala Pro Val Tyr Ser 165 170 175 His Leu Ile Tyr Asp Val Ile Pro Asp Gly Asp Lys Thr Val Val Gln 180 185 190 Pro Asp Ile Leu Ile Leu Glu Gly Leu Asn Val Leu Gln Ser Gly Met 195 200 205 Asp Tyr Pro His Asp Pro His His Val Phe Val Ser Asp Phe Val Asp 210 215 220 Phe Ser Ile Tyr Val Asp Ala Pro Glu Asp Leu Leu Gln Thr Trp Tyr 225 230 235 240 Ile Asn Arg Phe Leu Lys Phe Arg Glu Gly Ala Phe Thr Asp Pro Asp 245 250 255 Ser Tyr Phe His Asn Tyr Ala Lys Leu Thr Lys Glu Glu Ala Ile Lys 260 265 270 Thr Ala Met Thr Ile Trp Lys Glu Leu Asn Trp Leu Asn Leu Lys Gln 275 280 285 Asn Ile Leu Pro Thr Arg Glu Arg Ala Ser Leu Ile Leu Thr Lys Ser 290 295 300 Ala Asn His Ala Val Glu Glu Val Arg Leu Arg Lys 305 310 315 <210> 19 <211> 951 <212> DNA <213> Artificial Sequence <220> <223> A variant of pantothenic acid kinase from Escherichia coli <400> 19 atgagcatca aagagcagac cctgatgacc ccgtacctgc agttcgaccg taaccaatgg 60 gcggcgctgc gtgatagcgt tccgatgacc ctgagcgagg acgaaatcgc gcgtctgaag 120 ggtattaacg aggatctgag cctggaggaa gtggcggaaa tctacctgcc gctgagccgt 180 ctgctgaact tctatattag cagcaacctg cgtcgtcagg cggttctgga acaatttctg 240 ggtaccaacg gccagcgtat cccgtatatc attagcattg cgggtagcgt ggcggttggc 300 aaaagcacca ccgcgcgtgt gctgcaggcg ctgctgagcc gttggccgga gcaccgtcgt 360 gttgaactga tcaccaccga cggcttcctg cacccgaacc aagtgctgaa ggagcgtggt 420 ctgatgaaga aaaagggctt cccggaaagc tacgatatgc accgtctggt gaaatttgtt 480 agcgacctga agagcggtgt gccgaacgtt accgcgccgg tgtacagcca cctgatctat 540 gatgttattc cggacggcga taaaaccgtg gttcagccgg acatcctgat tctggagggt 600 ctgaacgtgc tgcaaagcgg catggactat ccgcacgatc cgcaccacgt gtttgttagc 660 gacttcgtgg attttagcat ctacgttgac gcgccggagg atctgctgca gacctggttc 720 attaaccgtt tcctgaaatt tcgtgagggt gcgttcaccg acccggatag ctactttcac 780 aactatgcga aactgaccaa ggaagaggcg atcaaaaccg cgatgaccat gtggaaggag 840 atgaactggc tgaacctgaa gcaaaacatt ctgccgaccc gtgaacgtgc gagcctgatt 900 ctgaccaaaa gcgcgaacca cgcggtggag gaagttcgtc tgcgtaagta a 951 <210> 20 <211> 316 <212> PRT <213> Artificial Sequence <220> <223> A variant of pantothenic acid kinase from Escherichia coli <400> 20 Met Ser Ile Lys Glu Gln Thr Leu Met Thr Pro Tyr Leu Gln Phe Asp 1 5 10 15 Arg Asn Gln Trp Ala Ala Leu Arg Asp Ser Val Pro Met Thr Leu Ser 20 25 30 Glu Asp Glu Ile Ala Arg Leu Lys Gly Ile Asn Glu Asp Leu Ser Leu 35 40 45 Glu Glu Val Ala Glu Ile Tyr Leu Pro Leu Ser Arg Leu Leu Asn Phe 50 55 60 Tyr Ile Ser Ser Asn Leu Arg Arg Gln Ala Val Leu Glu Gln Phe Leu 65 70 75 80 Gly Thr Asn Gly Gln Arg Ile Pro Tyr Ile Ile Ser Ile Ala Gly Ser 85 90 95 Val Ala Val Gly Lys Ser Thr Thr Ala Arg Val Leu Gln Ala Leu Leu 100 105 110 Ser Arg Trp Pro Glu His Arg Arg Val Glu Leu Ile Thr Thr Asp Gly 115 120 125 Phe Leu His Pro Asn Gln Val Leu Lys Glu Arg Gly Leu Met Lys Lys 130 135 140 Lys Gly Phe Pro Glu Ser Tyr Asp Met His Arg Leu Val Lys Phe Val 145 150 155 160 Ser Asp Leu Lys Ser Gly Val Pro Asn Val Thr Ala Pro Val Tyr Ser 165 170 175 His Leu Ile Tyr Asp Val Ile Pro Asp Gly Asp Lys Thr Val Val Gln 180 185 190 Pro Asp Ile Leu Ile Leu Glu Gly Leu Asn Val Leu Gln Ser Gly Met 195 200 205 Asp Tyr Pro His Asp Pro His His Val Phe Val Ser Asp Phe Val Asp 210 215 220 Phe Ser Ile Tyr Val Asp Ala Pro Glu Asp Leu Leu Gln Thr Trp Phe 225 230 235 240 Ile Asn Arg Phe Leu Lys Phe Arg Glu Gly Ala Phe Thr Asp Pro Asp 245 250 255 Ser Tyr Phe His Asn Tyr Ala Lys Leu Thr Lys Glu Glu Ala Ile Lys 260 265 270 Thr Ala Met Thr Met Trp Lys Glu Met Asn Trp Leu Asn Leu Lys Gln 275 280 285 Asn Ile Leu Pro Thr Arg Glu Arg Ala Ser Ile Leu Thr Lys Ser 290 295 300 Ala Asn His Ala Val Glu Glu Val Arg Leu Arg Lys 305 310 315 <210> 21 <211> 951 <212> DNA <213> Artificial Sequence <220> <223> Recombinant strains of yeast strains (Escherichia coli). <400> 21 atgagcatca aagagcagac cctgatgacc ccgtacctgc agttcgaccg taaccaatgg gcggcgctgc gtgatagcgt tccgatgacc ctgagcgagg acgaaatcgc gcgtctgaag ggtattaacg aggatctgag cctggagga gtggcggaa tctacctgcc gctgagccgt 240. ctgctgaact tctatattag cagcaacctg cgtcgtcagg cggttctgga acaatttctg ggtaccaacg gccagcgtat cccgtatatc attagcattg cgggtagcgt ggcggttggc 300 aaaagcacca ccgcgcgtgt gctgcaggcg ctgctgagcc gttggccgga gcaccgtcgt 360 gttgaactga tcaccaccga cggcttcctg cacccgaacc aagtgctgaa ggagcgtggt 420 ctgatgaaga aaaagggttt cccggaaagc tacgatatgc accgtctggt gaaatttgtt 480 agcgacctga agagcggtgt gccgaacgtt accgcgccgg tgtacagcca cctgatctat 540 gatgttattc cggacggcga taaaaccgtg gttcagccgg acatcctgat tctggagggt 600 ctgaacgtgc tgcaaagcgg catggactat ccgcacgatc cgcaccacgt gtttgttagc 660 gacttcgtgg attttagcat ctacgttgac gcgccggagg atctgctgca gacctggtgg 720 attaaccgtt tcctgaaatt tcgtgagggt gcgttcaccg acccggatag ctactttcac 780 aactatgcga aactgaccaa ggaagaggcg atcaaaaccg cgatgaccat ttggaaggag 840 atgaactggc tgaacctgaa gcaaaacatt ctgccgaccc gtgaacgtgc gagcctgatt 900 ctgaccaaaa gcgcgaacca cgcggtggag gaagttcgtc tgcgtaagta a 951 <210> 22 <211> 316 <212> PRT <213> Artificial Sequence <220> <223> Variant of pantothenate kinase from Escherichia coli <400> 22 Met Ser Ile Lys Glu Gln Thr Leu Met Thr Pro Tyr Leu Gln Phe Asp 1 5 10 15 Arg Asn Gln Trp Ala Ala Leu Arg Asp Ser Val Pro Met Thr Leu Ser 20 25 30 Glu Asp Glu Ile Ala Arg Leu Lys Gly Ile Asn Glu Asp Leu Ser Leu 35 40 45 Glu Glu Val Ala Glu Ile Tyr Leu Pro Leu Ser Arg Leu Leu Asn Phe 50 55 60 Tyr Ile Ser Ser Asn Leu Arg Arg Gln Ala Val Leu Glu Gln Phe Leu 65 70 75 80 Gly Thr Asn Gly Gln Arg Ile Pro Tyr Ile Ile Ser Ile Ala Gly Ser[[ID=2�]] 85 90 95 Val Ala Val Gly Lys Ser Thr Thr Ala Arg Val Leu Gln Ala Leu Leu 100 105 110 Ser Arg Trp Pro Glu His Arg Arg Val Glu Leu Ile Thr Thr Asp Gly 115 120 125 Phe Leu His Pro Asn Gln Val Leu Lys Glu Arg Gly Leu Met Lys Lys 130 135 140 Lys Gly Phe Pro Glu Ser Tyr Asp Met His Arg Leu Val Lys Phe Val 145 150 155 160 Ser Asp Leu Lys Ser Gly Val Pro Asn Val Thr Ala Pro Val Tyr Ser 165 170 175 His Leu Ile Tyr Asp Val Ile Pro Asp Gly Asp Lys Thr Val Val Gln 180 185 190 Pro Asp Ile Leu Ile Leu Glu Gly Leu Asn Val Leu Gln Ser Gly Met 195 200 205 Asp Tyr Pro His Asp Pro His His Val Phe Val Ser Asp Phe Val Asp 210 215 220 Phe Ser Ile Tyr Val Asp Ala Pro Glu Asp Leu Leu Gln Thr Trp Trp 225 230 235 240 Ile Asn Arg Phe Leu Lys Phe Arg Glu Gly Ala Phe Thr Asp Pro Asp 245 250 255 Ser Tyr Phe His Asn Tyr Ala Lys Leu Thr Lys Glu Glu Ala Ile Lys 260 265 270 Thr Ala Met Thr Ile Trp Lys Glu Met Asn Trp Leu Asn Leu Lys Gln 275 280 285 Asn Ile Leu Pro Thr Arg Glu Arg Ala Ser Leu Ile Leu Thr Lys Ser 290 295 300 Ala Asn His Ala Val Glu Glu Val Arg Leu Arg Lys 305 310 315 <210> 23 <211> 951 <212> DNA <213> Artificial Sequence <220> <223> Recombinant strains of yeast strains (Escherichia coli). <400> 23 atgagcatca aagagcagac cctgatgacc ccgtacctgc agttcgaccg taaccaatgg gcggcgctgc gtgatagcgt tccgatgacc ctgagcgagg acgaaatcgc gcgtctgaag ggtattaacg aggatctgag cctggagga gtggcggaa tctacctgcc gctgagccgt 240. ctgctgaact tctatattag cagcaacctg cgtcgtcagg cggttctgga acaatttctg ggtaccaacg gccagcgtat cccgtatatc attagcattg cgggtagcgt ggcggttggc 300 aaaagcacca ccgcgcgtgt gctgcaggcg ctgctgagcc gttggccgga gcaccgtcgt 360 gttgaactga tcaccaccga cggcttcctg cacccgaacc aagtgctga ggagcgtggt ctgatgaaga aaaagggttt cccggaaagc tacgatatgc accgtctggt gaaatttgtt agcgacctga agagcggtgt gccgaacgtt accgcgccgg tgtacagcca cctgatctat 540 gatgttattc cggacggcga taaaaccgtg gttcagccgg acatcctgat tctggagggt 600 ctgaacgtgc tgcaaagcgg catggactat ccgcacgatc cgcaccacgt gtttgttagc 660 gacttcgtgg attttagcat ctacgttgac gcgccggagg atctgctgca gacctggtgg 720 attaaccgtt tcctgaaatt tcgtgagggt gcgttcaccg acccggatag ctactttcac 780 aactatgcga aactgaccaa ggaagaggcg atcaaaaccg cgatgaccct gtggaaggag 840 atgaactggc tgaacctgaa gcaaaacatt ctgccgaccc gtgaacgtgc gagcctgatt 900 ctgaccaaaa gcgcgaacca cgcggtggag gaagttcgtc tgcgtaagta a 951 <210> 24 <211> 316 <212> PRT <213> Artificial Sequence <220> <223> Variant of pantothenate kinase from Escherichia coli <400> 24 Met Ser Ile Lys Glu Gln Thr Leu Met Thr Pro Tyr Leu Gln Phe Asp 1 5 10 15 Arg Asn Gln Trp Ala Ala Leu Arg Asp Ser Val Pro Met Thr Leu Ser 20 25 30 Glu Asp Glu Ile Ala Arg Leu Lys Gly Ile Asn Glu Asp Leu Ser Leu 35 40 45 Glu Glu Val Ala Glu Ile Tyr Leu Pro Leu Ser Arg Leu Leu Asn Phe 50 55 60 Tyr Ile Ser Ser Asn Leu Arg Arg Gln Ala Val Leu Glu Gln Phe Leu 65 70 75 80 Gly Thr Asn Gly Gln Arg Ile Pro Tyr Ile Ile Ser Ile Ala Gly Ser 85 90 95 Val Ala Val Gly Lys Ser Thr Thr Ala Arg Val Leu Gln Ala Leu Leu 100 105 110 Ser Arg Trp Pro Glu His Arg Arg Val Glu Leu Ile Thr Thr Asp Gly 115 120 125 Phe Leu His Pro Asn Gln Val Leu Lys Glu Arg Gly Leu Met Lys Lys 130 135 140 Lys Gly Phe Pro Glu Ser Tyr Asp Met His Arg Leu Val Lys Phe Val 145 150 155 160 Ser Asp Leu Lys Ser Gly Val Pro Asn Val Thr Ala Pro Val Tyr Ser 165 170 175 His Leu Ile Tyr Asp Val Ile Pro Asp Gly Asp Lys Thr Val Val Gln 180 185 190 Pro Asp Ile Leu Ile Leu Glu Gly Leu Asn Val Leu Gln Ser Gly Met 195 200 205 Asp Tyr Pro His Asp Pro His His Val Phe Val Ser Asp Phe Val Asp 210 215 220 Phe Ser Ile Tyr Val Asp Ala Pro Glu Asp Leu Leu Gln Thr Trp Trp 225 230 235 240 Ile Asn Arg Phe Leu Lys Phe Arg Glu Gly Ala Phe Thr Asp Pro Asp 245 250 255 Ser Tyr Phe His Asn Tyr Ala Lys Leu Thr Lys Glu Glu Ala Ile Lys 260 265 270 Thr Ala Met Thr Leu Trp Lys Glu Met Asn Trp Leu Asn Leu Lys Gln 275 280 285 Asn Ile Leu Pro Thr Arg Glu Arg Ala Ser Leu Ile Leu Thr Lys Ser 290 295 300 Ala Asn His Ala Val Glu Glu Val Arg Leu Arg Lys 305 310 315 <210> 25 <211> 951 <212> DNA <213> Artificial Sequence <220> <223> Variant of pantothenate kinase from Escherichia coli <400> 25 atgagcatca aagagcagac cctgatgacc ccgtacctgc agttcgaccg taaccaatgg 60 gcggcgctgc gtgatagcgt tccgatgacc ctgagcgagg acgaaatcgc gcgtctgaag 120 ggtattaacg aggatctgag cctggaggaa gtggcggaaa tctacctgcc gctgagccgt 180 ctgctgaact tctatattag cagcaacctg cgtcgtcagg cggttctgga acaatttctg 240 ggtaccaacg gccagcgtat cccgtatatc attagcattg cgggtagcgt ggcggttggc 300 aaaagcacca ccgcgcgtgt gctgcaggcg ctgctgagcc gttggccgga gcaccgtcgt 360 gttgaactga tcaccaccga cggcttcctg cacccgaacc aagtgctgaa ggagcgtggt 420 ctgatgaaga aaaagggctt cccggaaagc tacgatatgc accgtctggt gaaatttgtt 480 agcgacctga agagcggtgt gccgaacgtt accgcgccgg tgtacagcca cctgatctat 540 gatgttattc cggacggcga taaaaccgtg gttcagccgg acatcctgat tctggagggt 600 ctgaacgtgc tgcaaagcgg catggactat ccgcacgatc cgcaccacgt gtttgttagc 660 gacttcgtgg attttagcat ctacgttgac gcgccggagg atctgctgca gacctggttc 720 attaaccgtt tcctgaaatt tcgtgagggt gccttcaccg acccggatag ctactttcac 780 aactatgcga aactgaccaa ggaagaggcg atcaaaaccg cgatgaccat gtggaaggag 840 attaactggc tgaacctgaa gcaaaacatt ctgccgaccc gtgaacgtgc gagcctgatt 900 ctgaccaaaa gcgcgaacca cgcggtggag gaagttcgtc tgcgtaagta a 951 <210> 26 <211> 316 <212> PRT <213> artificial sequence <220> <223> It comes from Escherichia coli <400> 26 Met Ser Ile Lys Glu Gln Thr Leu Met Thr Pro Tyr Leu Gln Phe Asp 1 5 10 15 Arg Asn Gln Trp Ala Ala Leu Arg Asp Ser Val Pro Met Thr Leu Ser 20 25 30 Glu Asp Glu Ile Ala Arg Leu Lys Gly Ile Asn Glu Asp Leu Ser Leu 35 40 45 Glu Glu Val Ala Glu Ile Tyr Leu Pro Leu Ser Arg Leu Leu Asn Phe 50 55 60 Tyr Ile Ser Ser Asn Leu Arg Arg Gln Ala Val Leu Glu Gln Phe Leu 65 70 75 80 Gly Thr Asn Gly Gln Arg Ile Pro Tyr Ile Ile Ser Ile Ala Gly Ser 85 90 95 Val Ala Val Gly Lys Ser Thr Thr Ala Arg Val Leu Gln Ala Leu Leu 100 105 110 Ser Arg Trp Pro Glu His Arg Arg Val Glu Leu Ile Thr Thr Asp Gly 115 120 125 Phe Leu His Pro Asn Gln Val Leu Lys Glu Arg Gly Leu Met Lys Lys 130 135 140 Lys Gly Phe Pro Glu Ser Tyr Asp Met His Arg Leu Val Lys Phe Val 145 150 155 160 Ser Asp Leu Lys Ser Gly Val Pro Asn Val Thr Ala Pro Val Tyr Ser 165 170 175 His Leu Ile Tyr Asp Val Ile Pro Asp Gly Asp Lys Thr Val Val Gln 180 185 190 Pro Asp Ile Leu Ile Leu Glu Gly Leu Asn Val Leu Gln Ser Gly Met 195 200 205 Asp Tyr Pro His Asp Pro His His Val Phe Val Ser Asp Phe Val Asp 210 215 220 Phe Ser Ile Tyr Val Asp Ala Pro Glu Asp Leu Leu Gln Thr Trp Phe 225 230 235 240 Ile Asn Arg Phe Leu Lys Phe Arg Glu Gly Ala Phe Thr Asp Pro Asp 245 250 255 Ser Tyr Phe His Asn Tyr Ala Lys Leu Thr Lys Glu Glu Ala Ile Lys 260 265 270 Thr Ala Met Thr Met Trp Lys Glu Ile Asn Trp Leu Asn Leu Lys Gln 275 280 285 Asn Ile Leu Pro Thr Arg Glu Arg Ala Ser Leu Ile Leu Thr Lys Ser 290 295 300 Ala Asn His Ala Val Glu Glu Val Arg Leu Arg Lys 305 310 315 <210> 27 <211> 951 <212> DNA <213> Artificial Sequence <220> <223> A variant of pantothenic acid kinase from Escherichia coli <400> 27 atgagcatca aagagcagac cctgatgacc ccgtacctgc agttcgaccg taaccaatgg 60 gcggcgctgc gtgatagcgt tccgatgacc ctgagcgagg acgaaatcgc gcgtctgaag 120 ggtattaacg aggatctgag cctggaggaa gtggcggaaa tctacctgcc gctgagccgt 180 ctgctgaact tctatattag cagcaacctg cgtcgtcagg cggttctgga acaatttctg 240 ggtaccaacg gccagcgtat cccgtatatc attagcattg cgggtagcgt ggcggttggc 300 aaaagcacca ccgcgcgtgt gctgcaggcg ctgctgagcc gttggccgga gcaccgtcgt 360 gttgaactga tcaccaccga cggcttcctg cacccgaacc aagtgctgaa ggagcgtggt 420 ctgatgaaga aaaagggttt cccggaaagc tacgatatgc accgtctggt gaaatttgtt 480 agcgacctga agagcggtgt gccgaacgtt accgcgccgg tgtacagcca cctgatctat 540 gatgttattc cggacggcga taaaaccgtg gttcagccgg acatcctgat tctggagggt 600 ctgaacgtgc tgcaaagcgg catggactat ccgcacgatc cgcaccacgt gtttgttagc 660 gacttcgtgg attttagcat ctacgttgac gcgccggagg atctgctgca gacctggttc 720 attaaccgtt tcctgaaatt tcgtgagggt gcgttcaccg acccggatag ctactttcac 780 aactatgcga aactgaccaa ggaagaggcg atcaaaaccg cgatgaccat gtggaaggag 840 attaactggc tgaacctgaa gcaaaacatt ctgccgaccc gtgaacgtgc gagcctgatt 900 ctgaccaaaa gcgcgaacca cgcggtggag gaagttcgtc tgcgtaagta a 951 <210> 28 <211> 316 <212> PRT <213> Artificial Sequence <220> <223> A variant of pantothenic acid kinase from Escherichia coli <400> 28 Met Ser Ile Lys Glu Gln Thr Leu Met Thr Pro Tyr Leu Gln Phe Asp 1 5 10 15 Arg Asn Gln Trp Ala Ala Leu Arg Asp Ser Val Pro Met Thr Leu Ser 20 25 30 Glu Asp Glu Ile Ala Arg Leu Lys Gly Ile Asn Glu Asp Leu Ser Leu 35 40 45 Glu Glu Val Ala Glu Ile Tyr Leu Pro Leu Ser Arg Leu Leu Asn Phe 50 55 60 Tyr Ile Ser Ser Asn Leu Arg Arg Gln Ala Val Leu Glu Gln Phe Leu 65 70 75 80 Gly Thr Asn Gly Gln Arg Ile Pro Tyr Ile Ile Ser Ile Ala Gly Ser 85 90 95 Val Ala Val Gly Lys Ser Thr Thr Ala Arg Val Leu Gln Ala Leu Leu 100 105 110 Ser Arg Trp Pro Glu His Arg Arg Val Glu Leu Ile Thr Thr Asp Gly 115 120 125 Phe Leu His Pro Asn Gln Val Leu Lys Glu Arg Gly Leu Met Lys Lys 130 135 140 Lys Gly Phe Pro Glu Ser Tyr Asp Met His Arg Leu Val Lys Phe Val 145 150 155 160 Ser Asp Leu Lys Ser Gly Val Pro Asn Val Thr Ala Pro Val Tyr Ser 165 170 175 His Leu Ile Tyr Asp Val Ile Pro Asp Gly Asp Lys Thr Val Val Gln 180 185 190 Pro Asp Ile Leu Ile Leu Glu Gly Leu Asn Val Leu Gln Ser Gly Met 195 200 205 Asp Tyr Pro His Asp Pro His His Val Phe Val Ser Asp Phe Val Asp 210 215 220 Phe Ser Ile Tyr Val Asp Ala Pro Glu Asp Leu Leu Gln Thr Trp Phe 225 230 235 240 Ile Asn Arg Phe Leu Lys Phe Arg Glu Gly Ala Phe Thr Asp Pro Asp 245 250 255 Ser Tyr Phe His Asn Tyr Ala Lys Leu Thr Lys Glu Glu Ala Ile Lys 260 265 270 Thr Ala Met Thr Met Trp Lys Glu Ile Asn Trp Leu Asn Leu Lys Gln 275 280 285 Asn Ile Leu Pro Thr Arg Glu Arg Ala Ser Leu Ile Leu Thr Lys Ser 290 295 300 Ala Asn His Ala Val Glu Glu Val Arg Leu Arg Lys 305 310 315 <210> 29 <211> 951 <212> DNA <213> Artificial Sequence <220> <223> Variant of pantothenate kinase from Escherichia coli <400> 29 atgagcatca aagagcagac cctgatgacc ccgtacctgc agttcgaccg taaccaatgg 60 gcggcgctgc gtgatagcgt tccgatgacc ctgagcgagg acgaaatcgc gcgtctgaag 120 ggtattaacg aggatctgag cctggaggaa gtggcggaaa tctacctgcc gctgagccgt 180 ctgctgaact tctatattag cagcaacctg cgtcgtcagg cggttctgga acaatttctg 240 ggtaccaacg gccagcgtat cccgtatatc attagcattg cgggtagcgt ggcggttggc 300 aaaagcacca ccgcgcgtgt gctgcaggcg ctgctgagcc gttggccgga gcaccgtcgt 360 gttgaactga tcaccaccga cggcttcctg cacccgaacc aagtgctgaa ggagcgtggt 420 ctgatgaaga aaaagggttt cccggaaagc tacgatatgc accgtctggt gaaatttgtt 480 agcgacctga agagcggtgt gccgaacgtt accgcgccgg tgtacagcca cctgatctat 540 gatgttattc cggacggcga taaaaccgtg gttcagccgg acatcctgat tctggagggt 600 ctgaacgtgc tgcaaagcgg catggactat ccgcacgatc cgcaccacgt gtttgttagc 660 gacttcgtgg attttagcat ctacgttgac gcgccggagg atctgctgca gacctggtac 720 attaaccgtt tcctgaaatt tcgtgagggt gcgttcaccg acccggatag ctactttcac 780 aactatgcga aactgaccaa ggaagaggcg atcaaaaccg cgatgaccat ttggaaggag 840 atgaactggc tgaacctgaa gcaaaacatt ctgccgaccc gtgaacgtgc gagcctgatt 900 ctgaccaaaa gcgcgaacca cgcggtggag gaagttcgtc tgcgtaagta a 951 <210> 30 <211> 316 <212> PRT <213> Artificial Sequence <220> <223> Variant of pantothenate kinase from Escherichia coli <400> 30 Met Ser Ile Lys Glu Gln Thr Leu Met Thr Pro Tyr Leu Gln Phe Asp 1 5 10 15 Arg Asn Gln Trp Ala Ala Leu Arg Asp Ser Val Pro Met Thr Leu Ser 20 25 30 Glu Asp Glu Ile Ala Arg Leu Lys Gly Ile Asn Glu Asp Leu Ser Leu 35 40 45 Glu Glu Val Ala Glu Ile Tyr Leu Pro Leu Ser Arg Leu Leu Asn Phe 50 55 60 Tyr Ile Ser Ser Asn Leu Arg Arg Gln Ala Val Leu Glu Gln Phe Leu 65 70 75 80 Gly Thr Asn Gly Gln Arg Ile Pro Tyr Ile Ile Ser Ile Ala Gly Ser 85 90 95 Val Ala Val Gly Lys Ser Thr Thr Ala Arg Val Leu Gln Ala Leu Leu 100 105 110 Ser Arg Trp Pro Glu His Arg Arg Val Glu Leu Ile Thr Thr Asp Gly 115 120 125 Phe Leu His Pro Asn Gln Val Leu Lys Glu Arg Gly Leu Met Lys Lys 130 135 14<strong>[0]] Lys Gly Phe Pro Glu Ser Tyr Asp Met His Arg Leu Val Lys Phe Val 145 150 155 160 Ser Asp Leu Lys Ser Gly Val Pro Asn Val Thr Ala Pro Val Tyr Ser 165 170 175 His Leu Ile Tyr Asp Val Ile Pro Asp Gly Asp Lys Thr Val Val Gln 180 185 190 Pro Asp Ile Leu Ile Leu Glu Gly Leu Asn Val Leu Gln Ser Gly Met 195 200 205 Asp Tyr Pro His Asp Pro His His Val Phe Val Ser Asp Phe Val Asp 210 215 220 Phe Ser Ile Tyr Val Asp Ala Pro Glu Asp Leu Leu Gln Thr Trp Tyr 225 230 235 240 Ile Asn Arg Phe Leu Lys Phe Arg Glu Gly Ala Phe Thr Asp Pro Asp 245 250 255 Ser Tyr Phe His Asn Tyr Ala Lys Leu Thr Lys Glu Glu Ala Ile Lys 260 265 270 Thr Ala Met Thr Ile Trp Lys Glu Met Asn Trp Leu Asn Leu Lys Gln 275 280 285 Asn Ile Leu Pro Thr Arg Glu Arg Ala Ser Leu Ile Leu Thr Lys Ser 290 295 300 Ala Asn His Ala Val Glu Glu Val Arg Leu Arg Lys 305 310 315 <210> 31 <211> 951 <212> DNA <213> Artificial Sequence <220> <223> Recombinant strains of yeast strains (Escherichia coli). <400> 31 atgagcatca aagagcagac cctgatgacc ccgtacctgc agttcgaccg taaccaatgg gcggcgctgc gtgatagcgt tccgatgacc ctgagcgagg acgaaatcgc gcgtctgaag ggtattaacg aggatctgag cctggagga gtggcggaa tctacctgcc gctgagccgt 240. ctgctgaact tctatattag cagcaacctg cgtcgtcagg cggttctgga acaatttctg ggtaccaacg gccagcgtat cccgtatatc attagcattg cgggtagcgt ggcggttggc 300 aaaagcacca ccgcgcgtgt gctgcaggcg ctgctgagcc gttggccgga gcaccgtcgt 360 gttgaactga tcaccaccga cggcttcctg cacccgaacc aagtgctga ggagcgtggt ctgatgaaga aaaagggttt cccggaaagc tacgatatgc accgtctggt gaaatttgtt agcgacctga agagcggtgt gccgaacgtt accgcgccgg tgtacagcca cctgatctat 540 gatgttattc cggacggcga taaaaccgtg gttcagccgg acatcctgat tctggagggt 600 ctgaacgtgc tgcaaagcgg catggactat ccgcacgatc cgcaccacgt gtttgttagc 660 gacttcgtgg attttagcat ctacgttgac gcgccggagg atctgctgca gacctggtgg 720 attaaccgtt tcctgaaatt tcgtgagggt gcgttcaccg acccggatag ctactttcac 780 aactatgcga aactgaccaa ggaagaggcg atcaaaaccg cgatgaccat ttggaaggag 840 atgaactggc tgaacctgaa gcaaaacatt ctgccgaccc gtgaacgtgc gagcctgatt 900 ctgaccaaaa gcgcgaacca cgcggtggag gaagttcgtc tgcgtaagta a 951 <210> 32 <211> 316 <212> PRT <213> Artificial Sequence <220> <223> Variant of pantothenate kinase from Escherichia coli <400> 32 Met Ser Ile Lys Glu Gln Thr Leu Met Thr Pro Tyr Leu Gln Phe Asp 1 5 10 15 Arg Asn Gln Trp Ala Ala Leu Arg Asp Ser Val Pro Met Thr Leu Ser 20 25 30 Glu Asp Glu Ile Ala Arg Leu Lys Gly Ile Asn Glu Asp Leu Ser Leu 35 40 45 Glu Glu Val Ala Glu Ile Tyr Leu Pro Leu Ser Arg Leu Leu Asn Phe 50 55 60 Tyr Ile Ser Ser Asn Leu Arg Arg Gln Ala Val Leu Glu Gln Phe Leu 65 70 75 80 Gly Thr Asn Gly Gln Arg Ile Pro Tyr Ile Ile Ser Ile Ala Gly Ser 85 90 95 Val Ala Val Gly Lys Ser Thr Thr Ala Arg Val Leu Gln Ala Leu Leu 100 105 110 Ser Arg Trp Pro Glu His Arg Arg Val Glu Leu Ile Thr Thr Asp Gly 115 120 125 Phe Leu His Pro Asn Gln Val Leu Lys Glu Arg Gly Leu Met Lys Lys 130 135 140 Lys Gly Phe Pro Glu Ser Tyr Asp Met His Arg Leu Val Lys Phe Val 145 150 155 160 Ser Asp Leu Lys Ser Gly Val Pro Asn Val Thr Ala Pro Val Tyr Ser 165 170 175 His Leu Ile Tyr Asp Val Ile Pro Asp Gly Asp Lys Thr Val Val Gln 180 185 190 Pro Asp Ile Leu Ile Leu Glu Gly Leu Asn Val Leu Gln Ser Gly Met 195 200 205 Asp Tyr Pro His Asp Pro His His Val Phe Val Ser Asp Phe Val Asp 210 215 220 Phe Ser Ile Tyr Val Asp Ala Pro Glu Asp Leu Leu Gln Thr Trp Trp 225 230 235 240 Ile Asn Arg Phe Leu Lys Phe Arg Glu Gly Ala Phe Thr Asp Pro Asp 245 250 255 Ser Tyr Phe His Asn Tyr Ala Lys Leu Thr Lys Glu Glu Ala Ile Lys 260 265 270 Thr Ala Met Thr Ile Trp Lys Glu Met Asn Trp Leu Asn Leu Lys Gln 275 280 285 Asn Ile Leu Pro Thr Arg Glu Arg Ala Ser Leu Ile Leu Thr Lys Ser 290 295 300 Ala Asn His Ala Val Glu Glu Val Arg Leu Arg Lys 305 310 315 <210> 33 <211> 951 <212> DNA <213> Artificial Sequence <220> <223> Variant of pantothenate kinase from Escherichia coli <400> 33 atgagcatca aagagcagac cctgatgacc ccgtacctgc agttcgaccg taaccaatgg 60 gcggcgctgc gtgatagcgt tccgatgacc ctgagcgagg acgaaatcgc gcgtctgaag 120 ggtattaacg aggatctgag cctggaggaa gtggcggaaa tctacctgcc gctgagccgt 180 ctgctgaact tctatattag cagcaacctg cgtcgtcagg cggttctgga acaatttctg 240 ggtaccaacg gccagcgtat cccgtatatc attagcattg cgggtagcgt ggcggttggc 300 aaaagcacca ccgcgcgtgt gctgcaggcg ctgctgagcc gttggccgga gcaccgtcgt 360 gttgaactga tcaccaccga cggcttcctg cacccgaacc aagtgctgaa ggagcgtggt 420 ctgatgaaga aaaagggttt cccggaaagc tacgatatgc accgtctggt gaaatttgtt 480 agcgacctga agagcggtgt gccgaacgtt accgcgccgg tgtacagcca cctgatctat 540 gatgttattc cggacggcga taaaaccgtg gttcagccgg acatcctgat tctggagggt 600 ctgaacgtgc tgcaaagcgg catggactat ccgcacgatc cgcaccacgt gtttgttagc 660 gacttcgtgg attttagcat ctacgttgac gcgccggagg atctgctgca gacctggttc 720 attaaccgtt tcctgaaatt tcgtgagggt gccttcaccg acccggatag ctactttcac 780 aactatgcga aactgaccaa ggaagaggcg atcaaaaccg cgatgacctt gtggaaggag 840 atgatgtggc tgaacctgaa gcaaaacatt ctgccgaccc gtgaacgtgc gagcctgatt 900 ctgaccaaaa gcgcgaacca cgcggtggag gaagttcgtc tgcgtaagta a 951 <210> 34 <211> 316 <212> PRT <213> artificial sequence <220> <223> It comes from Escherichia coli <400> 34 Met Ser Ile Lys Glu Gln Thr Leu Met Thr Pro Tyr Leu Gln Phe Asp 1 5 10 15 Arg Asn Gln Trp Ala Ala Leu Arg Asp Ser Val Pro Met Thr Leu Ser 20 25 30 Glu Asp Glu Ile Ala Arg Leu Lys Gly Ile Asn Glu Asp Leu Ser Leu 35 40 45 Glu Glu Val Ala Glu Ile Tyr Leu Pro Leu Ser Arg Leu Leu Asn Phe 50 55 60 Tyr Ile Ser Ser Asn Leu Arg Arg Gln Ala Val Leu Glu Gln Phe Leu 65 70 75 80 Gly Thr Asn Gly Gln Arg Ile Pro Tyr Ile Ile Ser Ile Ala Gly Ser 85 90 95 Val Ala Val Gly Lys Ser Thr Thr Ala Arg Val Leu Gln Ala Leu Leu 100 105 110 Ser Arg Trp Pro Glu His Arg Arg Val Glu Leu Ile Thr Thr Asp Gly 115 120 125 Phe Leu His Pro Asn Gln Val Leu Lys Glu Arg Gly Leu Met Lys Lys 130 135 140 Lys Gly Phe Pro Glu Ser Tyr Asp Met His Arg Leu Val Lys Phe Val 145 150 155 160 Ser Asp Leu Lys Ser Gly Val Pro Asn Val Thr Ala Pro Val Tyr Ser 165 170 175 His Leu Ile Tyr Asp Val Ile Pro Asp Gly Asp Lys Thr Val Val Gln 180 185 190 Pro Asp Ile Leu Ile Leu Glu Gly Leu Asn Val Leu Gln Ser Gly Met 195 200 205 Asp Tyr Pro His Asp Pro His His Val Phe Val Ser Asp Phe Val Asp 210 215 220 Phe Ser Ile Tyr Val Asp Ala Pro Glu Asp Leu Leu Gln Thr Trp Phe 225 230 235 240 Ile Asn Arg Phe Leu Lys Phe Arg Glu Gly Ala Phe Thr Asp Pro Asp 245 250 255 Ser Tyr Phe His Asn Tyr Ala Lys Leu Thr Lys Glu Glu Ala Ile Lys 260 265 270 Thr Ala Met Thr Leu Trp Lys Glu Met Met Trp Leu Asn Leu Lys Gln 275 280 285 Asn Ile Leu Pro Thr Arg Glu Arg Ala Ser Leu Ile Leu Thr Lys Ser 290 295 300 Ala Asn His Ala Val Glu Glu Val Arg Leu Arg Lys 305 310 315 <210> 35 <211> 951 <212> DNA <213> Artificial Sequence <220> <223> A variant of pantothenic acid kinase from Escherichia coli <400> 35 atgagcatca aagagcagac cctgatgacc ccgtacctgc agttcgaccg taaccaatgg 60 gcggcgctgc gtgatagcgt tccgatgacc ctgagcgagg acgaaatcgc gcgtctgaag 120 ggtattaacg aggatctgag cctggaggaa gtggcggaaa tctacctgcc gctgagccgt 180 ctgctgaact tctatattag cagcaacctg cgtcgtcagg cggttctgga acaatttctg 240 ggtaccaacg gccagcgtat cccgtatatc attagcattg cgggtagcgt ggcggttggc 300 aaaagcacca ccgcgcgtgt gctgcaggcg ctgctgagcc gttggccgga gcaccgtcgt 360 gttgaactga tcaccaccga cggcttcctg cacccgaacc aagtgctgaa ggagcgtggt 420 ctgatgaaga aaaagggttt cccggaaagc tacgatatgc accgtctggt gaaatttgtt 480 agcgacctga agagcggtgt gccgaacgtt accgcgccgg tgtacagcca cctgatctat 540 gatgttattc cggacggcga taaaaccgtg gttcagccag acatcctgat tctggagggt 600 ctgaacgtgc tgcaaagcgg catggactat ccgcacgatc cgcaccacgt gtttgttagc 660 gacttcgtgg attttagcat ctacgttgac gcgccggagg atctgctgca gacctggttc 720 attaaccgtt tcctgaaatt tcgtgagggt gcgttcaccg acccggatag ctactttcac 780 aactatgcga aactgaccaa ggaagaggcg atcaaaaccg cgatgaccat ttggaaggag 840 ttgaactggc tgaacctgaa gcaaaacatt ctgccgaccc gtgaacgtgc gagcctgatt 900 ctgaccaaaa gcgcgaacca cgcggtggag gaagttcgtc tgcgtaagta a 951 <210> 36 <211> 316 <212> PRT <213> Artificial Sequence <220> <223> A variant of pantothenic acid kinase from Escherichia coli <400> 36 Met Ser Ile Lys Glu Gln Thr Leu Met Thr Pro Tyr Leu Gln Phe Asp 1 5 10 15 Arg Asn Gln Trp Ala Ala Leu Arg Asp Ser Val Pro Met Thr Leu Ser 20 25 30 Glu Asp Glu Ile Ala Arg Leu Lys Gly Ile Asn Glu Asp Leu Ser Leu 35 40 45 Glu Glu Val Ala Glu Ile Tyr Leu Pro Leu Ser Arg Leu Leu Asn Phe 50 55 60 Tyr Ile Ser Ser Asn Leu Arg Arg Gln Ala Val Leu Glu Gln Phe Leu 65 70 75 80 Gly Thr Asn Gly Gln Arg Ile Pro Tyr Ile Ile Ser Ile Ala Gly Ser 85 90 95 Val Ala Val Gly Lys Ser Thr Thr Ala Arg Val Leu Gln Ala Leu Leu 100 105 110 Ser Arg Trp Pro Glu His Arg Arg Val Glu Leu Ile Thr Thr Asp Gly 115 120 125 Phe Leu His Pro Asn Gln Val Leu Lys Glu Arg Gly Leu Met Lys Lys 130 135 140 Lys Gly Phe Pro Glu Ser Tyr Asp Met His Arg Leu Val Lys Phe Val 145 150 155 160 Ser Asp Leu Lys Ser Gly Val Pro Asn Val Thr Ala Pro Val Tyr Ser 165 170 175 His Leu Ile Tyr Asp Val Ile Pro Asp Gly Asp Lys Thr Val Val Gln 180 185 190 Pro Asp Ile Leu Ile Leu Glu Gly Leu Asn Val Leu Gln Ser Gly Met 195 200 205 Asp Tyr Pro His Asp Pro His His Val Phe Val Ser Asp Phe Val Asp 210 215 220 Phe Ser Ile Tyr Val Asp Ala Pro Glu Asp Leu Leu Gln Thr Trp Phe 225 230 235 240 Ile Asn Arg Phe Leu Lys Phe Arg Glu Gly Ala Phe Thr Asp Pro Asp 245 250 255 Ser Tyr Phe His Asn Tyr Ala Lys Leu Thr Lys Glu Glu Ala Ile Lys 260 265 270 Thr Ala Met Thr Ile Trp Lys Glu Leu Asn Trp Leu Asn Leu Lys Gln 275 280 285 Asn Ile Leu Pro Thr Arg Glu Arg Ala Ser Leu Ile Leu Thr Lys Ser 290 295 300 Ala Asn His Ala Val Glu Glu Val Arg Leu Arg Lys 305 310 315 <210> 37 <211> 951 <212> DNA <213> Artificial Sequence <220> <223> Variant of pantothenate kinase from Escherichia coli <400> 37 atgagcatca aagagcagac cctgatgacc ccgtacctgc agttcgaccg taaccaatgg 60 gcggcgctgc gtgatagcgt tccgatgacc ctgagcgagg acgaaatcgc gcgtctgaag 120 ggtattaacg aggatctgag cctggaggaa gtggcggaaa tctacctgcc gctgagccgt 180 ctgctgaact tctatattag cagcaacctg cgtcgtcagg cggttctgga acaatttctg 240 ggtaccaacg gccagcgtat cccgtatatc attagcattg cgggtagcgt ggcggttggc 300 aaaagcacca ccgcgcgtgt gctgcaggcg ctgctgagcc gttggccgga gcaccgtcgt 360 gttgaactga tcaccaccga cggcttcctg cacccgaacc aagtgctgaa ggagcgtggt 420 ctgatgaaga aaaagggttt cccggaaagc tacgatatgc accgtctggt gaaatttgtt 480 agcgacctga agagcggtgt gccgaacgtt accgcgccgg tgtacagcca cctgatctat 540 gatgttattc cggacggcga taaaaccgtg gttcagccgg acatcctgat tctggagggt 600 ctgaacgtgc tgcaaagcgg catggactat ccgcacgatc cgcaccacgt gtttgttagc 660 gacttcgtgg attttagcat ctacgttgac gcgccggagg atctgctgca gacctggtac 720 attaaccgtt tcctgaaatt tcgtgagggt gcgttcaccg acccggatag ctactttcac 780 aactatgcga aactgaccaa ggaagaggcg atcaaaaccg cgatgaccat ttggaaggag 840 ttgaactggc tgaacctgaa gcaaaacatt ctgccgaccc gtgaacgtgc gagcctgatt 900 ctgaccaaaa gcgcgaacca cgcggtggag gaagttcgtc tgcgtaagta a 951 <210> 38 <211> 316 <212> PRT <213> Artificial Sequence <220> <223> Variant of pantothenate kinase from Escherichia coli <400> 38 Met Ser Ile Lys Glu Gln Thr Leu Met Thr Pro Tyr Leu Gln Phe Asp 1 5 10 15 Arg Asn Gln Trp Ala Ala Leu Arg Asp Ser Val Pro Met Thr Leu Ser 20 25 30 Glu Asp Glu Ile Ala Arg Leu Lys Gly Ile Asn Glu Asp Leu Ser Leu 35 40 45 Glu Glu Val Ala Glu Ile Tyr Leu Pro Leu Ser Arg Leu Leu Asn Phe 50 55 60 Tyr Ile Ser Ser Asn Leu Arg Arg Gln Ala Val Leu Glu Gln Phe Leu 65 70 75 80 Gly Thr Asn Gly Gln Arg Ile Pro Tyr Ile Ile Ser Ile Ala Gly Ser 85 90 95 Val Ala Val Gly Lys Ser Thr Thr Ala Arg Val Leu Gln Ala Leu Leu 100 105 110 Ser Arg Trp Pro Glu His Arg Arg Val Glu Leu Ile Thr Thr Asp Gly 115 120 125 Phe Leu His Pro Asn Gln Val Leu Lys Glu Arg Gly Leu Met Lys Lys 130 135 140 Lys Gly Phe Pro Glu Ser Tyr Asp Met His Arg Leu Val Lys Phe Val 145 150 155 160 Ser Asp Leu Lys Ser Gly Val Pro Asn Val Thr Ala Pro Val Tyr Ser 165 170 175 His Leu Ile Tyr Asp Val Ile Pro Asp Gly Asp Lys Thr Val Val Gln 180 185 190 Pro Asp Ile Leu Ile Leu Glu Gly Leu Asn Val Leu Gln Ser Gly Met 195 200 205 Asp Tyr Pro His Asp Pro His His Val Phe Val Ser Asp Phe Val Asp 210 215 220 Phe Ser Ile Tyr Val Asp Ala Pro Glu Asp Leu Leu Gln Thr Trp Tyr 225 230 235 240 Ile Asn Arg Phe Leu Lys Phe Arg Glu Gly Ala Phe Thr Asp Pro Asp 245 250 255 Ser Tyr Phe His Asn Tyr Ala Lys Leu Thr Lys Glu Glu Ala Ile Lys 260 265 270 Thr Ala Met Thr Ile Trp Lys Glu Leu Asn Trp Leu Asn Leu Lys Gln 275 280 285 Asn Ile Leu Pro Thr Arg Glu Arg Ala Ser Leu Ile Leu Thr Lys Ser 290 295 300 Ala Asn His Ala Val Glu Glu Val Arg Leu Arg Lys 305 310 315 <210> 39 <211> 951 <212> DNA <213> Artificial Sequence <220> <223> Recombinant strains of yeast strains (Escherichia coli). <400> 39 atgagcatca aagagcagac cctgatgacc ccgtacctgc agttcgaccg taaccaatgg gcggcgctgc gtgatagcgt tccgatgacc ctgagcgagg acgaaatcgc gcgtctgaag ggtattaacg aggatctgag cctggagga gtggcggaa tctacctgcc gctgagccgt 240. ctgctgaact tctatattag cagcaacctg cgtcgtcagg cggttctgga acaatttctg ggtaccaacg gccagcgtat cccgtatatc attagcattg cgggtagcgt ggcggttggc 300 aaaagcacca ccgcgcgtgt gctgcaggcg ctgctgagcc gttggccgga gcaccgtcgt 360 gttgaactga tcaccaccga cggcttcctg cacccgaacc aagtgctga ggagcgtggt ctgatgaaga aaaagggttt cccggaaagc tacgatatgc accgtctggt gaaatttgtt agcgacctga agagcggtgt gccgaacgtt accgcgccgg tgtacagcca cctgatctat 540 gatgttattc cggacggcga taaaaccgtg gttcagccgg acatcctgat tctggagggt 600 ctgaacgtgc tgcaaagcgg catggactat ccgcacgatc cgcaccacgt gtttgttagc 660 gacttcgtgg attttagcat ctacgttgac gcgccggagg atctgctgca gacctggttc 720 attaaccgtt tcctgaaatt tcgtgagggt gcgttcaccg acccggatag ctactttcac 780 aactatgcga aactgaccaa ggaagaggcg atcaaaaccg cgatgacctt gtggaaggag 840 attaactggc tgaacctgaa gcaaaacatt ctgccgaccc gtgaacgtgc gagcctgatt 900 ctgaccaaaa gcgcgaacca cgcggtggag gaagttcgtc tgcgtaagta a 951 <210> 40 <211> 316 <212> PRT <213> Artificial Sequence <220> <223> Variant of pantothenate kinase from Escherichia coli <400> 40 Met Ser Ile Lys Glu Gln Thr Leu Met Thr Pro Tyr Leu Gln Phe Asp 1 5 10 15 Arg Asn Gln Trp Ala Ala Leu Arg Asp Ser Val Pro Met Thr Leu Ser 20 25 30 Glu Asp Glu Ile Ala Arg Leu Lys Gly Ile Asn Glu Asp Leu Ser Leu 35 40 45 Glu Glu Val Ala Glu Ile Tyr Leu Pro Leu Ser Arg Leu Leu Asn Phe 50 55 60 Tyr Ile Ser Ser Asn Leu Arg Arg Gln Ala Val Leu Glu Gln Phe Leu 65 70 75 80 Gly Thr Asn Gly Gln Arg Ile Pro Tyr Ile Ile Ser Ile Ala Gly Ser 85 90 95 Val Ala Val Gly Lys Ser Thr Thr Ala Arg Val Leu Gln Ala Leu Leu 100 105 110 Ser Arg Trp Pro Glu His Arg Arg Val Glu Leu Ile Thr Thr Asp Gly 115 120 125 Phe Leu His Pro Asn Gln Val Leu Lys Glu Arg Gly Leu Met Lys Lys 130 135 140 Lys Gly Phe Pro Glu Ser Tyr Asp Met His Arg Leu Val Lys Phe Val 145 150 155 160 Ser Asp Leu Lys Ser Gly Val Pro Asn Val Thr Ala Pro Val Tyr Ser 165 170 175 His Leu Ile Tyr Asp Val Ile Pro Asp Gly Asp Lys Thr Val Val Gln 180 185 190 Pro Asp Ile Leu Ile Leu Glu Gly Leu Asn Val Leu Gln Ser Gly Met 195 200 205 Asp Tyr Pro His Asp Pro His His Val Phe Val Ser Asp Phe Val Asp 210 215 220 Phe Ser Ile Tyr Val Asp Ala Pro Glu Asp Leu Leu Gln Thr Trp Phe 225 230 235 240 Ile Asn Arg Phe Leu Lys Phe Arg Glu Gly Ala Phe Thr Asp Pro Asp 245 250 255 Ser Tyr Phe His Asn Tyr Ala Lys Leu Thr Lys Glu Glu Ala Ile Lys 260 265 270 Thr Ala Met Thr Leu Trp Lys Glu Ile Asn Trp Leu Asn Leu Lys Gln 275 280 285 Asn Ile Leu Pro Thr Arg Glu Arg Ala Ser Leu Ile Leu Thr Lys Ser 290 295 300 Ala Asn His Ala Val Glu Glu Val Arg Leu Arg Lys 305 310 315 <210> 41 <211> 951 <212> DNA <213> Artificial Sequence <220> <223> Variant of pantothenate kinase from Escherichia coli <400> 41 atgagcatca aagagcagac cctgatgacc ccgtacctgc agttcgaccg taaccaatgg 60 gcggcgctgc gtgatagcgt tccgatgacc ctgagcgagg acgaaatcgc gcgtctgaag 120 ggtattaacg aggatctgag cctggaggaa gtggcggaaa tctacctgcc gctgagccgt 180 ctgctgaact tctatattag cagcaacctg cgtcgtcagg cggttctgga acaatttctg 240 ggtaccaacg gccagcgtat cccgtatatc attagcattg cgggtagcgt ggcggttggc 300 aaaagcacca ccgcgcgtgt gctgcaggcg ctgctgagcc gttggccgga gcaccgtcgt 360 gttgaactga tcaccaccga cggcttcctg cacccgaacc aagtgctgaa ggagcgtggt 420 ctgatgaaga aaaagggttt cccggaaagc tacgatatgc accgtctggt gaaatttgtt 480 agcgacctga agagcggtgt gccgaacgtt accgcgccgg tgtacagcca cctgatctat 540 gatgttattc cggacggcga taaaaccgtg gttcagccgg acatcctgat tctggagggt 600 ctgaacgtgc tgcaaagcgg catggactat ccgcacgatc cgcaccacgt gtttgttagc 660 gacttcgtgg attttagcat ctacgttgac gcgccggagg atctgctgca gacctggtac 720 attaaccgtt tcctgaaatt tcgtgagggt gccttcaccg acccggatag ctactttcac 780 aactatgcga aactgaccaa ggaagaggcg atcaaaaccg cgatgaccat gtggaaggag 840 atgaactggc tgaacctgaa gcaaaacatt ctgccgaccc gtgaacgtgc gagcctgatt 900 ctgaccaaaa gcgcgaacca cgcggtggag gaagttcgtc tgcgtaagta a 951 <210> 42 <211> 316 <212> PRT <213> artificial sequence <220> <223> It comes from Escherichia coli <400> 42 Met Ser Ile Lys Glu Gln Thr Leu Met Thr Pro Tyr Leu Gln Phe Asp 1 5 10 15 Arg Asn Gln Trp Ala Ala Leu Arg Asp Ser Val Pro Met Thr Leu Ser 20 25 30 Glu Asp Glu Ile Ala Arg Leu Lys Gly Ile Asn Glu Asp Leu Ser Leu 35 40 45 Glu Glu Val Ala Glu Ile Tyr Leu Pro Leu Ser Arg Leu Leu Asn Phe 50 55 60 Tyr Ile Ser Ser Asn Leu Arg Arg Gln Ala Val Leu Glu Gln Phe Leu 65 70 75 80 Gly Thr Asn Gly Gln Arg Ile Pro Tyr Ile Ile Ser Ile Ala Gly Ser 85 90 95 Val Ala Val Gly Lys Ser Thr Thr Ala Arg Val Leu Gln Ala Leu Leu 100 105 110 Ser Arg Trp Pro Glu His Arg Arg Val Glu Leu Ile Thr Thr Asp Gly 115 120 125 Phe Leu His Pro Asn Gln Val Leu Lys Glu Arg Gly Leu Met Lys Lys 130 135 140 Lys Gly Phe Pro Glu Ser Tyr Asp Met His Arg Leu Val Lys Phe Val 145 150 155 160 Ser Asp Leu Lys Ser Gly Val Pro Asn Val Thr Ala Pro Val Tyr Ser 165 170 175 His Leu Ile Tyr Asp Val Ile Pro Asp Gly Asp Lys Thr Val Val Gln 180 185 190 Pro Asp Ile Leu Ile Leu Glu Gly Leu Asn Val Leu Gln Ser Gly Met 195 200 205 Asp Tyr Pro His Asp Pro His His Val Phe Val Ser Asp Phe Val Asp 210 215 220 Phe Ser Ile Tyr Val Asp Ala Pro Glu Asp Leu Leu Gln Thr Trp Tyr 225 230 235 240 Ile Asn Arg Phe Leu Lys Phe Arg Glu Gly Ala Phe Thr Asp Pro Asp 245 250 255 Ser Tyr Phe His Asn Tyr Ala Lys Leu Thr Lys Glu Glu Ala Ile Lys 260 265 270 Thr Ala Met Thr Met Trp Lys Glu Met Asn Trp Leu Asn Leu Lys Gln 275 280 285 Asn Ile Leu Pro Thr Arg Glu Arg Ala Ser Leu Ile Leu Thr Lys Ser 290 295 300 Ala Asn His Ala Val Glu Glu Val Arg Leu Arg Lys 305 310 315 <210> 43 <211> 951 <212> DNA <213> Artificial Sequence <220> <223> A variant of pantothenic acid kinase from Escherichia coli <400> 43 atgagcatca aagagcagac cctgatgacc ccgtacctgc agttcgaccg taaccaatgg 60 gcggcgctgc gtgatagcgt tccgatgacc ctgagcgagg acgaaatcgc gcgtctgaag 120 ggtattaacg aggatctgag cctggaggaa gtggcggaaa tctacctgcc gctgagccgt 180 ctgctgaact tctatattag cagcaacctg cgtcgtcagg cggttctgga acaatttctg 240 ggtaccaacg gccagcgtat cccgtatatc attagcattg cgggtagcgt ggcggttggc 300 aaaagcacca ccgcgcgtgt gctgcaggcg ctgctgagcc gttggccgga gcaccgtcgt 360 gttgaactga tcaccaccga cggcttcctg cacccgaacc aagtgctgaa ggagcgtggt 420 ctgatgaaga aaaagggttt cccggaaagc tacgatatgc accgtctggt gaaatttgtt 480 agcgacctga agagcggtgt gccgaacgtt accgcgccgg tgtacagcca cctgatctat 540 gatgttattc cggacggcga taaaaccgtg gttcagccgg acatcctgat tctggagggt 600 ctgaacgtgc tgcaaagcgg catggactat ccgcacgatc cgcaccacgt gtttgttagc 660 gacttcgtgg attttagcat ctacgttgac gcgccggagg atctgctgca gacctggttc 720 attaaccgtt tcctgaaatt tcgtgagggt gcgttcaccg acccggatag ctactttcac 780 aactatgcga aactgaccaa ggaagaggcg atcaaaaccg cgatgaccct gtggaaggag 840 atcaactggc tgaacctgaa gcaaaacatt ctgccgaccc gtgaacgtgc gagcctgatt 900 ctgaccaaaa gcgcgaacca cgcggtggag gaagttcgtc tgcgtaagta a 951 <210> 44 <211> 316 <212> PRT <213> Artificial Sequence <220> <223> A variant of pantothenic acid kinase from Escherichia coli <400> 44 Met Ser Ile Lys Glu Gln Thr Leu Met Thr Pro Tyr Leu Gln Phe Asp 1 5 10 15 Arg Asn Gln Trp Ala Ala Leu Arg Asp Ser Val Pro Met Thr Leu Ser 20 25 30 Glu Asp Glu Ile Ala Arg Leu Lys Gly Ile Asn Glu Asp Leu Ser Leu 35 40 45 Glu Glu Val Ala Glu Ile Tyr Leu Pro Leu Ser Arg Leu Leu Asn Phe 50 55 60 Tyr Ile Ser Ser Asn Leu Arg Arg Gln Ala Val Leu Glu Gln Phe Leu 65 70 75 80 Gly Thr Asn Gly Gln Arg Ile Pro Tyr Ile Ile Ser Ile Ala Gly Ser 85 90 95 Val Ala Val Gly Lys Ser Thr Thr Ala Arg Val Leu Gln Ala Leu Leu 100 105 110 Ser Arg Trp Pro Glu His Arg Arg Val Glu Leu Ile Thr Thr Asp Gly 115 120 125 Phe Leu His Pro Asn Gln Val Leu Lys Glu Arg Gly Leu Met Lys Lys 130 135 140 Lys Gly Phe Pro Glu Ser Tyr Asp Met His Arg Leu Val Lys Phe Val 145 150 155 160 Ser Asp Leu Lys Ser Gly Val Pro Asn Val Thr Ala Pro Val Tyr Ser 165 170 175 His Leu Ile Tyr Asp Val Ile Pro Asp Gly Asp Lys Thr Val Val Gln 180 185 190 Pro Asp Ile Leu Ile Leu Glu Gly Leu Asn Val Leu Gln Ser Gly Met 195 200 205 Asp Tyr Pro His Asp Pro His His Val Phe Val Ser Asp Phe Val Asp 210 215 220 Phe Ser Ile Tyr Val Asp Ala Pro Glu Asp Leu Leu Gln Thr Trp Phe 225 230 235 240 Ile Asn Arg Phe Leu Lys Phe Arg Glu Gly Ala Phe Thr Asp Pro Asp 245 250 255 Ser Tyr Phe His Asn Tyr Ala Lys Leu Thr Lys Glu Glu Ala Ile Lys 260 265 270 Thr Ala Met Thr Leu Trp Lys Glu Ile Asn Trp Leu Asn Leu Lys Gln 275 280 285 Asn Ile Leu Pro Thr Arg Glu Arg Ala Ser Ile Leu Thr Lys Ser 290 295 300 Ala Asn His Ala Val Glu Glu Val Arg Leu Arg Lys 305 310 315 <210> 45 <211> 948 <212> DNA <213> Artificial Sequence <220> <223> Recombinant strains of yeast strains (Escherichia coli). <400> 45 atgagcatca aagagcagac cctgatgacc ccgtacgatg aattcgaccg taacacttgg gcgggcctgc gtgatagcgt tccgatgacc ctgagcgagg acgaaatcgc acgtctgaag 120 ggtattaacg aggatctgag cctggagga gtggcggaa tctacctgcc gctgagccgt 240. ctgctgaact tctatattag cagcaacctg cgtcgtcagg cggttctgga acaatttctg ggtaccaacg gccagcgtat cccgtatatc attagcattg cgggtagcgt ggcggttggc 300 aaaagcacca ccgcgcgtgt gctgcaggcg ctgctgagcc gttggccgga gcaccgtcgt 360 gttgaactga tcaccaccga cggcttcctg cacccgaacc aagtgctgaa ggagcgtggt 420 ctgatgaaga aaaagggttt cccggaaagc tacgatatgc accgtctggt gaaatttgtt 480 agcgacctga agagcggtgt gccgaacgtt accgcgccgg tgtacagcca cctgatctat 540 gatgttattc cggacggcga taaaaccgtg gttcagccgg acatcctgat tctggagggt 600 ctgaacgtgc tgcaaagcgg catggactat ccgcacgatc cgcaccacgt gttcgttagc 660 gacttcgtgg attttagcat ctacgttgac gcgccggagg atctgctgca gacctggtac 720 attaaccgtt tcctgaaatt tcgtgagggt gcgttcaccg acccggatag ctactttcac 780 aactatgcga aactgaccaa ggaagaggcg atcaaaaccg cgatgaccat ttggaaggag 840 atgaactggc tgaacctgaa gcaaaacatt ctgccgaccc gtgaacgtgc gagcctgatt 900 ctgaccaaaa gcgcgaacca cgcggtggag gaagttcgtc tgcgtaag 948 <210> 46 <211> 316 <212> PRT <213> Artificial Sequence <220> <223> Variant of pantothenate kinase from Escherichia coli <400> 46 Met Ser Ile Lys Glu Gln Thr Leu Met Thr Pro Tyr Asp Glu Phe Asp 1 5 10 15 Arg Asn Thr Trp Ala Gly Leu Arg Asp Ser Val Pro Met Thr Leu Ser 20 25 30 Glu Asp Glu Ile Ala Arg Leu Lys Gly Ile Asn Glu Asp Leu Ser Leu 35 40 45 Glu Glu Val Ala Glu Ile Tyr Leu Pro Leu Ser Arg Leu Leu Asn Phe 50 55 60 Tyr Ile Ser Ser Asn Leu Arg Arg Gln Ala Val Leu Glu Gln Phe Leu 65 70 75 80 Gly Thr Asn Gly Gln Arg Ile Pro Tyr Ile Ile Ser Ile Ala Gly Ser 85 90 95 Val Ala Val Gly Lys Ser Thr Thr Ala Arg Val Leu Gln Ala Leu Leu 100 105 110 Ser Arg Trp Pro Glu His Arg Arg Val Glu Leu Ile Thr Thr Asp Gly 115 120 125 Phe Leu His Pro Asn Gln Val Leu Lys Glu Arg Gly Leu Met Lys Lys 130 135 140 Lys Gly Phe Pro Glu Ser Tyr Asp Met His Arg Leu Val Lys Phe Val 145 150 155 160 Ser Asp Leu Lys Ser Gly Val Pro Asn Val Thr Ala Pro Val Tyr Ser 165 170 175 His Leu Ile Tyr Asp Val Ile Pro Asp Gly Asp Lys Thr Val Val Gln 180 185 190 Pro Asp Ile Leu Ile Leu Glu Gly Leu Asn Val Leu Gln Ser Gly Met 195 200 205 Asp Tyr Pro His Asp Pro His His Val Phe Val Ser Asp Phe Val Asp 210 215 220 Phe Ser Ile Tyr Val Asp Ala Pro Glu Asp Leu Leu Gln Thr Trp Tyr 225 230 235 240 Ile Asn Arg Phe Leu Lys Phe Arg Glu Gly Ala Phe Thr Asp Pro Asp 245 250 255 Ser Tyr Phe His Asn Tyr Ala Lys Leu Thr Lys Glu Glu Ala Ile Lys 260 265 270 Thr Ala Met Thr Ile Trp Lys Glu Met Asn Trp Leu Asn Leu Lys Gln 275 280 285 Asn Ile Leu Pro Thr Arg Glu Arg Ala Ser Leu Ile Leu Thr Lys Ser 290 295 300 Ala Asn His Ala Val Glu Glu Val Arg Leu Arg Lys 305 310 315 <210> 47 <211> 948 <212> DNA <213> Artificial Sequence <220> <223> Recombinant strains of yeast strains (Escherichia coli). <400> 47 atgagcatca aagagcagac cctgatgacc ccgtacgatg aattcgaccg taaccaatgg gcgggcctgc gtgatagcgt tccgatgacc ctgagcgagg acgaaatcgc gcgtctgaag 120 ggtattaacg aggatctgag cctggagga gtggcggaa tctacctgcc gctgagccgt 240. ctgctgaact tctatattag cagcaacctg cgtcgtcagg cggttctgga acaatttctg ggtaccaacg gccagcgtat cccgtatatc attagcattg cgggtagcgt ggcggttggc 300 aaaagcacca ccgcgcgtgt gctgcaggcg ctgctgagcc gttggccgga gcaccgtcgt 360 gttgaactga tcaccaccga cggcttcctg cacccgaacc aagtgctga ggagcgtggt ctgatgaaga aaaagggttt cccggaaagc tacgatatgc accgtctggt gaaatttgtt agcgacctga agagcggtgt gccgaacgtt accgcgccgg tgtacagcca cctgatctat 540 gatgttattc cggacggcga taaaaccgtg gttcagccgg acatcctgat tctggagggt 600 ctgaacgtgc tgcaaagcgg catggactat ccgcacgatc cgcaccacgt gtttgttagc 660 gacttcgtgg attttagcat ctacgttgac gcgccggagg atctgctgca gacctggtac 720 attaaccgtt tcctgaaatt tcgtgagggt gcgttcaccg acccggatag ctactttcac 780 aactatgcga aactgaccaa ggaagaggcg atcaaaaccg cgatgaccat ttggaaggag 840 atgaactggc tgaacctgaa gcaaaacatt ctgccgaccc gtgaacgtgc gagcctgatt 900 ctgaccaaaa gcgcgaacca cgcggtggag gaagttcgtc tgcgtaag 948 <210> 48 <211> 316 <212> PRT <213> Artificial Sequence <220> <223> Variant of pantothenate kinase from Escherichia coli <400> 48 Met Ser Ile Lys Glu Gln Thr Leu Met Thr Pro Tyr Asp Glu Phe Asp 1 5 10 15 Arg Asn Gln Trp Ala Gly Leu Arg Asp Ser Val Pro Met Thr Leu Ser 20 25 30 Glu Asp Glu Ile Ala Arg Leu Lys Gly Ile Asn Glu Asp Leu Ser Leu 35 40 45 Glu Glu Val Ala Glu Ile Tyr Leu Pro Leu Ser Arg Leu Leu Asn Phe 50 55 60 Tyr Ile Ser Ser Asn Leu Arg Arg Gln Ala Val Leu Glu Gln Phe Leu 65 70 75 80 Gly Thr Asn Gly Gln Arg Ile Pro Tyr Ile Ile Ser Ile Ala Gly Ser 85 90 95 Val Ala Val Gly Lys Ser Thr Thr Ala Arg Val Leu Gln Ala Leu Leu 100 105 110 Ser Arg Trp Pro Glu His Arg Arg Val Glu Leu Ile Thr Thr Asp Gly 115 120 125 Phe Leu His Pro Asn Gln Val Leu Lys Glu Arg Gly Leu Met Lys Lys 130 135 140 Lys Gly Phe Pro Glu Ser Tyr Asp Met His Arg Leu Val Lys Phe Val 145 150 155 160 Ser Asp Leu Lys Ser Gly Val Pro Asn Val Thr Ala Pro Val Tyr Ser 165 170 175 His Leu Ile Tyr Asp Val Ile Pro Asp Gly Asp Lys Thr Val Val Gln 180 185 190 Pro Asp Ile Leu Ile Leu Glu Gly Leu Asn Val Leu Gln Ser Gly Met 195 200 205 Asp Tyr Pro His Asp Pro His His Val Phe Val Ser Asp Phe Val Asp 210 215 220 Phe Ser Ile Tyr Val Asp Ala Pro Glu Asp Leu Leu Gln Thr Trp Tyr 225 230 235 24'target='_blank'> Ile Asn Arg Phe Leu Lys Phe Arg Glu Gly Ala Phe Thr Asp Pro Asp 245 250 255 Ser Tyr Phe His Asn Tyr Ala Lys Leu Thr Lys Glu Glu Ala Ile Lys 260 265 270 Thr Ala Met Thr Ile Trp Lys Glu Met Asn Trp Leu Asn Leu Lys Gln<> 275 280 285 Asn Ile Leu Pro Thr Arg Glu Arg Ala Ser Leu Ile Leu Thr Lys Ser 290 295 300 Ala Asn His Ala Val Glu Glu Val Arg Leu Arg Lys 305 310 315 <210> 49 <211> 948 <212> DNA <213> Artificial Sequence" <220> <223> Variant of pantothenate kinase from Escherichia coli <400> 49 atgagcatca aagagcagac cctgatgacc ccgtacctgc aattcgaccg taaccaatgg 60 gcgggcctgc gtgatagcgt tccgatgacc ctgagcgagg acgaaatcgc gcgtctgaag 120 ggtattaacg aggatctgag cctggaggaa gtggcggaaa tctacctgcc gctgagccgt 180 ctgctgaact tctatattag cagcaacctg cgtcgtcagg cggttctgga acaatttctg 240 ggtaccaacg gccagcgtat cccgtatatc attagcattg cgggtagcgt ggcggttggc 300 aaaagcacca ccgcgcgtgt gctgcaggcg ctgctgagcc gttggccgga gcaccgtcgt 360 gttgaactga tcaccaccga cggcttcctg cacccgaacc aagtgctgaa ggagcgtggt 420 ctgatgaaga aaaagggttt cccggaaagc tacgatatgc accgtctggt gaaatttgtt 480 agcgacctga agagcggtgt gccgaacgtt accgcgccgg tgtacagcca cctgatctat 540 gatgttattc cggacggcga taaaaccgtg gttcagccgg acatcctgat tctggagggt 600 ctgaacgtgc tgcaaagcgg catggactat ccgcacgatc cgcaccacgt ggaggttagc 660 gacttcgtgg attttagcat ctacgttgac gcgccggagg atctgctgca gacctggtac 720 attaaccgtt tcctgaaatt tcgtgagggt gccttcaccg acccggatag ctactttcac 780 aactatgcga aactgaccaa ggaagaggcg atcaaaaccg cgatgaccat ctggaaggag 840 atgaactggc tgaacctgaa gcaaaacatt ctgccgaccc gtgaacgtgc gagcctgatt 900 ctgaccaaaa gcgcgaacca cgcggtggag gaagttcgtc tgcgtaag 948 <210> 50 <211> 316 <212> PRT <213> artificial sequence <220> <223> It comes from Escherichia coli <400> 50 Met Ser Ile Lys Glu Gln Thr Leu Met Thr Pro Tyr Leu Gln Phe Asp 1 5 10 15 Arg Asn Gln Trp Ala Gly Leu Arg Asp Ser Val Pro Met Thr Leu Ser 20 25 30 Glu Asp Glu Ile Ala Arg Leu Lys Gly Ile Asn Glu Asp Leu Ser Leu 35 40 45 Glu Glu Val Ala Glu Ile Tyr Leu Pro Leu Ser Arg Leu Leu Asn Phe 50 55 60 Tyr Ile Ser Ser Asn Leu Arg Arg Gln Ala Val Leu Glu Gln Phe Leu 65 70 75 80 Gly Thr Asn Gly Gln Arg Ile Pro Tyr Ile Ile Ser Ile Ala Gly Ser 85 90 95 Val Ala Val Gly Lys Ser Thr Thr Ala Arg Val Leu Gln Ala Leu Leu 100 105 110 Ser Arg Trp Pro Glu His Arg Arg Val Glu Leu Ile Thr Thr Asp Gly 115 120 125 Phe Leu His Pro Asn Gln Val Leu Lys Glu Arg Gly Leu Met Lys Lys 130 135 140 Lys Gly Phe Pro Glu Ser Tyr Asp Met His Arg Leu Val Lys Phe Val 145 150 155 160 Ser Asp Leu Lys Ser Gly Val Pro Asn Val Thr Ala Pro Val Tyr Ser 165 170 175 His Leu Ile Tyr Asp Val Ile Pro Asp Gly Asp Lys Thr Val Val Gln 180 185 190 Pro Asp Ile Leu Ile Leu Glu Gly Leu Asn Val Leu Gln Ser Gly Met 195 200 205 Asp Tyr Pro His Asp Pro His His Val Glu Val Ser Asp Phe Val Asp 210 215 220 Phe Ser Ile Tyr Val Asp Ala Pro Glu Asp Leu Leu Gln Thr Trp Tyr 225 230 235 240 Ile Asn Arg Phe Leu Lys Phe Arg Glu Gly Ala Phe Thr Asp Pro Asp 245 250 255 Ser Tyr Phe His Asn Tyr Ala Lys Leu Thr Lys Glu Glu Ala Ile Lys 260 265 270 Thr Ala Met Thr Ile Trp Lys Glu Met Asn Trp Leu Asn Leu Lys Gln 275 280 285 Asn Ile Leu Pro Thr Arg Glu Arg Ala Ser Leu Ile Leu Thr Lys Ser 290 295 300 Ala Asn His Ala Val Glu Glu Val Arg Leu Arg Lys 305 310 315 <210> 51 <211> 948 <212> DNA <213> Artificial Sequence <220> <223> A variant of pantothenic acid kinase from Escherichia coli <400> 51 atgagcatca aagagcagac cctgatgacc ccgtacctgc agtttgaccg taaccaatgg 60 gcggcgctgc gtgatagtaa cccgatgacc ctgagcgagg acgaaatcgc gcgtctgaag 120 ggtattaacg aggatctgag cctggaggaa gtggcggaaa tctacctgcc gctgagccgt 180 ctgctgaact tctatattag cagcaacctg cgtcgtcagg cggttctgga acaatttctg 240 ggtaccaacg gccagcgtat cccgtatatc attagcattg cgggtagcgt ggcggttggc 300 aaaagcacca ccgcgcgtgt gctgcaggcg ctgctgagcc gttggccgga gcaccgtcgt 360 gttgaactga tcaccaccga cggcttcctg cacccgaacc aagtgctgaa ggagcgtggt 420 ctgatgaaga aaaagggttt cccggaaagc tacgatatgc accgtctggt gaaatttgtt 480 agcgacctga agagcggtgt gccgaacgtt accgcgccgg tgtacagcca cctgatctat 540 gatgttattc cggacggcga taaaaccgtg gttcagccgg acatcctgat tctggagggt 600 ctgaacgtgc tgcaaagcgg catggactat ccgcacgatc cgcaccacgt gtttgttagc 660 gacttcgtgg attttagcat ctacgttgac gcgccggagg atctgctgca gacctggtac 720 attaaccgtt tcctgaaatt tcgtgagggt gcgttcaccg acccggatag ctactttcac 780 aactatgcga aactgaccaa ggaagaggcg atcaaaaccg cgatgaccat ttggaaggag 840 atgaaccacc tgaacctgaa gcaaaacatt ctgccgaccc gtgaacgtgc gagcctgatt 900 ctgaccaaaa gcgcgaacca cgcggtggag gaagttcgtc tgcgtaag 948 <210> 52 <211> 316 <212> PRT <213> Artificial Sequence <220> <223> A variant of pantothenic acid kinase from Escherichia coli <400> 52 Met Ser Ile Lys Glu Gln Thr Leu Met Thr Pro Tyr Leu Gln Phe Asp 1 5 10 15 Arg Asn Gln Trp Ala Ala Leu Arg Asp Ser Asn Pro Met Thr Leu Ser 20 25 30 Glu Asp Glu Ile Ala Arg Leu Lys Gly Ile Asn Glu Asp Leu Ser Leu 35 40 45 Glu Glu Val Ala Glu Ile Tyr Leu Pro Leu Ser Arg Leu Leu Asn Phe 50 55 60 Tyr Ile Ser Ser Asn Leu Arg Arg Gln Ala Val Leu Glu Gln Phe Leu 65 70 75 80 Gly Thr Asn Gly Gln Arg Ile Pro Tyr Ile Ile Ser Ile Ala Gly Ser 85 90 95 Val Ala Val Gly Lys Ser Thr Thr Ala Arg Val Leu Gln Ala Leu Leu 100 105 110 Ser Arg Trp Pro Glu His Arg Arg Val Glu Leu Ile Thr Thr Asp Gly 115 120 125 Phe Leu His Pro Asn Gln Val Leu Lys Glu Arg Gly Leu Met Lys Lys 130 135 140 Lys Gly Phe Pro Glu Ser Tyr Asp Met His Arg Leu Val Lys Phe Val 145 150 155 160 Ser Asp Leu Lys Ser Gly Val Pro Asn Val Thr Ala Pro Val Tyr Ser 165 170 175 His Leu Ile Tyr Asp Val Ile Pro Asp Gly Asp Lys Thr Val Val Gln 180 185 190 Pro Asp Ile Leu Ile Leu Glu Gly Leu Asn Val Leu Gln Ser Gly Met 195 200 205 Asp Tyr Pro His Asp Pro His His Val Phe Val Ser Asp Phe Val Asp 210 215 220 Phe Ser Ile Tyr Val Asp Ala Pro Glu Asp Leu Leu Gln Thr Trp Tyr 225 230 235 240 Ile Asn Arg Phe Leu Lys Phe Arg Glu Gly Ala Phe Thr Asp Pro Asp 245 250 255 Ser Tyr Phe His Asn Tyr Ala Lys Leu Thr Lys Glu Glu Ala Ile Lys 260 265 270 Thr Ala Met Thr Ile Trp Lys Glu Met Asn His Leu Asn Leu Lys Gln 275 280 285 Asn Ile Leu Pro Thr Arg Glu Arg Ala Ser Leu Ile Leu Thr Lys Ser 290 295 300 Ala Asn His Ala Val Glu Glu Val Arg Leu Arg Lys 305 310 315 <210> 53 <211> 948 <212> DNA <213> Artificial Sequence <220> <223> Variant of pantothenate kinase from Escherichia coli <400> 53 atgagcatca aagagcagac cctgatgacc ccgtacgatg aattcgaccg taaccaatgg 6 gcgggcctgc gtgatagcgt tccgatgacc ctgagcgagg acgaaatcct gcgtctgaag 120 ggtattaacg aggatctgag cctggaggaa gtggcggaaa tctacctgcc gctgagccgt 180 ctgctgaact tctatattag cagcaacctg cgtcgtcagg cggttctgga acaatttctg 240 ggtaccaacg gccagcgtat cccgtatatc attagcattg cgggtagcgt ggcggttggc 300 aaaagcacca ccgcgcgtgt gctgcaggcg ctgctgagcc gttggccgga gcaccgtcgt 360 gttgaactga tcaccaccga cggcttcctg cacccgaacc aagtgctgaa ggagcgtggt 420 ctgatgaaga aaaagggttt cccggaaagc tacgatatgc accgtctggt gaaatttgtt 480 agcgacctga agagcggtgt gccgaacgtt accgcgccgg tgtacagcca cctgatctat 540 gatgttattc cggacggcga taaaaccgtg gttcagccgg acatcctgat tctggagggt 600 ctgaacgtgc tgcaaagcgg catggactat ccgcacgatc cgcaccacgt gtttgttagc 660 gacttcgtgg attttagcat ctacgttgac gcgccggagg atctgctgca gacctggtac 720 attaaccgtt tcctgaaatt tcgtgagggt gcgttcaccg acccggatag ctactttcac 780 aactatgcga aactgaccaa ggaagaggcg atcaaaaccg cgatgaccat ttggaaggag 840 atgaactggc tgaacctgaa gcaaaacatt ctgccgaccc gtgaacgtgc gagcctgatt 读900 ctgaccaaaa gcgcgaacca cgcggtggag gaagttcgtc tgcgtaag 948 <210> 54 <211> 316 <212> PRT <213> Artificial Sequence It should be noted that there may be an error in the "读900" in line 18 which is likely a misrepresentation in the original text. It is translated as "读900" as it is not clear what the correct content should be.<220> <223> Variant of pantothenate kinase from Escherichia coli <400> 54 Met Ser Ile Lys Glu Gln Thr Leu Met Thr Pro Tyr Asp Glu Phe Asp 1 5 10 15 Arg Asn Gln Trp Ala Gly Leu Arg Asp Ser Val Pro Met Thr Leu Ser 20 25 30 Glu Asp Glu Ile Leu Arg Leu Lys Gly Ile Asn Glu Asp Leu Ser Leu 35 40 45 Glu Glu Val Ala Glu Ile Tyr Leu Pro Leu Ser Arg Leu Leu Asn Phe 50 55 60 Tyr Ile Ser Ser Asn Leu Arg Arg Gln Ala Val Leu Glu Gln Phe Leu 65 70 75 80 Gly Thr Asn Gly Gln Arg Ile Pro Tyr Ile Ile Ser Ile Ala Gly Ser 85 90 95 Val Ala Val Gly Lys Ser Thr Thr Ala Arg Val Leu Gln Ala Leu Leu 100 105 110 Ser Arg Trp Pro Glu His Arg Arg Val Glu Leu Ile Thr Thr Asp Gly 115 120 125 Phe Leu His Pro Asn Gln Val Leu Lys Glu Arg Gly Leu Met Lys Lys 130 135 140 Lys Gly Phe Pro Glu Ser Tyr Asp Met His Arg Leu Val Lys Phe Val 145 150 155 160 Ser Asp Leu Lys Ser Gly Val Pro Asn Val Thr Ala Pro Val Tyr Ser 165 170 175 His Leu Ile Tyr Asp Val Ile Pro Asp Gly Asp Lys Thr Val Val Gln 180 185 190 Pro Asp Ile Leu Ile Leu Glu Gly Leu Asn Val Leu Gln Ser Gly Met 195 200 205 Asp Tyr Pro His Asp Pro His His Val Phe Val Ser Asp Phe Val Asp 210 215 220 Phe Ser Ile Tyr Val Asp Ala Pro Glu Asp Leu Leu Gln Thr Trp Tyr 225 230 235 240 Ile Asn Arg Phe Leu Lys Phe Arg Glu Gly Ala Phe Thr Asp Pro Asp 245 250 255 Ser Tyr Phe His Asn Tyr Ala Lys Leu Thr Lys Glu Glu Ala Ile Lys 260 265 270 Thr Ala Met Thr Ile Trp Lys Glu Met Asn Trp Leu Asn Leu Lys Gln 275 280 285 Asn Ile Leu Pro Thr Arg Glu Arg Ala Ser Leu Ile Leu Thr Lys Ser 290 295 300 Ala Asn His Ala Val Glu Glu Val Arg Leu Arg Lys 305 310 315 <210> 55 <211> 948 <212> DNA <213> Artificial Sequence <220> <223> Recombinant strains of yeast strains (Escherichia coli). <400> 55 atgagcatca aagagcagac cctgatgacc ccgtacctgc agttcgaccg taaccaaatg gcggcgctgc gcgatagcgt tccgatgacg ctgagcgagg acgaaatcgc gcgtctgaag ggtattaacg aggatctgag cctggagga gtggcggaa tctacctgcc gctgagccgt 240. ctgctgaact tctatattag cagcaacctg cgtcgtcagg cggttctgga acaatttctg ggtaccaacg gccagcgtat cccgtatatc attagcattg cgggtagcgt ggcggttggc 300 aaaagcacca ccgcgcgtgt gctgcaggcg ctgctgagcc gttggccgga gcaccgtcgt 360 gttgaactga tcaccaccga cggcttcctg cacccgaacc aagtgctga ggagcgtggt 480. ctgatgaaga aaaagggttt cccggaaagc tacgatatgc accgtctggt gaaatttgtt agcgacctga agagcggtgt gccgaacgtt accgcgccgg tgtacagcca cctgatctat 540 gatgttattc cggacggcga taaaaccgtg gttcagccgg acatcctgat tctggagggt 600 ctgaacgtgc tgcaaagcgg catggactat ccgcacgatc cgcaccacgt gtttgttagc 660 gacttcgtgg attttagcat ctacgttgac gcgccggagg atctgctgca gacctggtac 720 attaaccgtt tcctgaaatt tcgtgagggt gcgttcaccg acccggatag ctactttcac 780 aactatgcga aactgaccaa ggaagaggcg atcaaaaccg cgatgaccat ttggaaggag 840 atgaactggc tgaacctgaa gcaaaacatt ctgccgaccc gtgaacgtgc gagcctgatt 900 ctgaccaaaa gcgcgaacca cgcggtggag gaagttcgtc tgcgtaag 948 <210> 56 <211> 316 <212> PRT <213> Artificial Sequence <220> <223> Variant of pantothenate kinase from Escherichia coli <400> 56 Met Ser Ile Lys Glu Gln Thr Leu Met Thr Pro Tyr Leu Gln Phe Asp 1 5 10 15 Arg Asn Gln Met Ala Ala Leu Arg Asp Ser Val Pro Met Thr Leu Ser 20 25 30 Glu Asp Glu Ile Ala Arg Leu Lys Gly Ile Asn Glu Asp Leu Ser Leu 35 40 45 Glu Glu Val Ala Glu Ile Tyr Leu Pro Leu Ser Arg Leu Leu Asn Phe 50 55 60 Tyr Ile Ser Ser Asn Leu Arg Arg Gln Ala Val Leu Glu Gln Phe Leu 65 70 75 80 Gly Thr Asn Gly Gln Arg Ile Pro Tyr Ile Ile Ser Ile Ala Gly Ser 85 90 95 Val Ala Val Gly Lys Ser Thr Thr Ala Arg Val Leu Gln Ala Leu Leu 100 105 110 Ser Arg Trp Pro Glu His Arg Arg Val Glu Leu Ile Thr Thr Asp Gly 115 120 125 Phe Leu His Pro Asn Gln Val Leu Lys Glu Arg Gly Leu Met Lys Lys 130 135 140 Lys Gly Phe Pro Glu Ser Tyr Asp Met His Arg Leu Val Lys Phe Val 145 150 155 160 Ser Asp Leu Lys Ser Gly Val Pro Asn Val Thr Ala Pro Val Tyr Ser 165 170 175 His Leu Ile Tyr Asp Val Ile Pro Asp Gly Asp Lys Thr Val Val Gln 180 185 190 Pro Asp Ile Leu Ile Leu Glu Gly Leu Asn Val Leu Gln Ser Gly Met 195 200 205 Asp Tyr Pro His Asp Pro His His Val Phe Val Ser Asp Phe Val Asp 210 215 220 Phe Ser Ile Tyr Val Asp Ala Pro Glu Asp Leu Leu Gln Thr Trp Tyr 225 230 235 240 Ile Asn Arg Phe Leu Lys Phe Arg Glu Gly Ala Phe Thr Asp Pro Asp 245 250 255 Ser Tyr Phe His Asn Tyr Ala Lys Leu Thr Lys Glu Glu Ala Ile Lys 260 265 270 Thr Ala Met Thr Ile Trp Lys Glu Met Asn Trp Leu Asn Leu Lys Gln 275 280 285 Asn Ile Leu Pro Thr Arg Glu Arg Ala Ser Leu Ile Leu Thr Lys Ser 290 295 300 Ala Asn His Ala Val Glu Glu Val Arg Leu Arg Lys 305 310 315 <210> 57 <211> 948 <212> DNA <213> Artificial Sequence <220> <223> Variant of pantothenate kinase from Escherichia coli <400> 57 atgagcatca aagagcagac cctgatgacc ccgtacctgc agttcgaccg taaccaatgg 60 gcggctctgc gtgatccggt tccgatgacc ctgagcgagg acgaaatcgc gcgtctgaag 120 ggtattaacg aggatctgag cctggaggaa gtggcggaaa tctacctgcc gctgagccgt 180 ctgctgaact tctatattag cagcaacctg cgtcgtcagg cggttctgga acaatttctg 240 ggtaccaacg gccagcgtat cccgtatatc attagcattg cgggtagcgt ggcggttggc 300 aaaagcacca ccgcgcgtgt gctgcaggcg ctgctgagcc gttggccgga gcaccgtcgt 360 gttgaactga tcaccaccga cggcttcctg cacccgaacc aagtgctgaa ggagcgtggt 420 ctgatgaaga aaaagggttt cccggaaagc tacgatatgc accgtctggt gaaatttgtt 480 agcgacctga agagcggtgt gccgaacgtt accgcgccgg tgtacagcca cctgatctat 540 gatgttattc cggacggcga taaaaccgtg gttcagccgg acatcctgat tctggagggt 600 ctgaacgtgc tgcaaagcgg catggactat ccgcacgatc cgcaccacgt gtttgttagc 660 gacttcgtgg attttagcat ctacgttgac gcgccggagg atctgctgca gacctggtac 720 attaaccgtt tcctgaaatt tcgtgagggt gccttcaccg acccggatag ctactttcac 780 aactatgcga aactgaccaa ggaagaggcg atcaaaaccg cgatgaccat ttggaaggag 840 atgaactggc tgaacctgaa gcaaaacatt ct...

Claims

1. An engineered pantothenate kinase, wherein the amino acid residue differences between the polypeptide sequence of the engineered pantothenate kinase and SEQ ID NO: 2 are L277M / I and I281M / L, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:

2.

2. An engineered pantothenate kinase, wherein the amino acid residue differences between the polypeptide sequence of the engineered pantothenate kinase and SEQ ID NO: 2 are a combination of L277M / I, I281M / L, and 1-2 amino acid residue differences selected from Y240F / W, N282M, and I54S, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 2, and wherein the engineered pantothenate kinase consists of a polypeptide sequence that exhibits an increased ability to convert acetylene glyceraldehyde to acetylene glyceraldehyde phosphate compared to a wild-type polypeptide having the amino acid sequence of SEQ ID NO:

2.

3. An engineered pantothenate kinase, wherein the engineered pantothenate kinase has a polypeptide sequence that differs from SEQ ID NO: 30 by 1 to 5 amino acid residues selected from the group consisting of L13D, Q14E, Q19T / R, A22G / T / D, F218E, A37L, W20M, F15L, V27N, W283H / L, Q78A, L123W, R24A / S, T30R, V75E, L70D, S26P / L, M29S, A305I / T, D25T, N69T / Y, R106A, F247M, R86H, Q134L, and I271S, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 30, and wherein the engineered pantothenate kinase is derived from a polypeptide having an amino acid sequence of SEQ ID NO:

30. The polypeptide sequence composition shows an increased ability to convert ethynylglyceraldehyde into ethynylglyceraldehyde phosphate compared to the polypeptide of NO:

30.

4. An engineered pantothenate kinase, wherein the amino acid residue difference between the polypeptide sequence of the engineered pantothenate kinase and SEQ ID NO: 60 is 1-10 amino acid residue differences selected from the group consisting of R119P / A / K / Q, V157M, S161K / R, G41C / R, N169Q, N261G / P, A308V / I, E44P / Q, L76Q, S298R, I54W / V, Q19A / T, A22G, R120K, Q14E, R106A, F218E, E310S, A305T, T302A and G250R, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 60, and wherein the engineered pantothenate kinase consists of a polypeptide sequence that exhibits an increased ability to convert acetyleneglyceraldehyde to acetyleneglyceraldehyde phosphate compared to a polypeptide having the amino acid sequence of SEQ ID NO:

60.

5. An engineered pantothenate kinase, wherein the amino acid residue difference between the polypeptide sequence of the engineered pantothenate kinase and SEQ ID NO: 132 is 1-2 amino acid residue differences selected from F64L, Y180E, L123W / H, Q134R, T125S / G, R71Q, R24A and L48M, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 132, and wherein the engineered pantothenate kinase consists of a polypeptide sequence that exhibits an increased ability to convert acetyleneglyceraldehyde to acetyleneglyceraldehyde phosphate compared to a polypeptide having the amino acid sequence of SEQ ID NO:

132.

6. An engineered pantothenate kinase, wherein the amino acid residue difference between the polypeptide sequence of the engineered pantothenate kinase and SEQ ID NO: 132 is 1-2 amino acid residue differences selected from the group consisting of Y180E, T125S / G, S92G and L301M, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 132, and wherein the engineered pantothenate kinase consists of a polypeptide sequence that exhibits an increased ability to convert acetyleneglyceraldehyde to acetyleneglyceraldehyde phosphate compared to a polypeptide having the amino acid sequence of SEQ ID NO:

132.

7. An engineered pantothenate kinase, wherein the amino acid residue difference between the polypeptide sequence of the engineered pantothenate kinase and SEQ ID NO: 222 is 1-3 amino acid residue differences selected from K143G, H154P, N83P, G84H and A305T, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 222, and wherein the engineered pantothenate kinase consists of a polypeptide sequence that exhibits an increased ability to convert acetyleneglyceraldehyde to acetyleneglyceraldehyde phosphate compared to a polypeptide having the amino acid sequence of SEQ ID NO:

222.

8. An engineered pantothenate kinase, wherein the amino acid residue difference between the polypeptide sequence of the engineered pantothenate kinase and SEQ ID NO: 230 is 1-6 amino acid residue differences selected from the group consisting of L13H, N169S, D213E, F247L, H283S, Q288T, E49D, V75Q, T104F, L284V, F64I, H154P and D16E, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 230, and wherein the engineered pantothenate kinase consists of a polypeptide sequence that exhibits an increased ability to convert acetyleneglyceraldehyde to acetyleneglyceraldehyde phosphate compared to a polypeptide having the amino acid sequence of SEQ ID NO:

230.

9. An engineered pantothenate kinase, wherein the amino acid residue difference between the polypeptide sequence of the engineered pantothenate kinase and SEQ ID NO: 240 is 1-5 amino acid residue differences selected from the group consisting of L8E / W, F64I, F247L, H283S, L13H, Q14A, N83P, D213E, K264R, T276S and T10S, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 240, and wherein the engineered pantothenate kinase consists of a polypeptide sequence that exhibits an increased ability to convert acetyleneglyceraldehyde to acetyleneglyceraldehyde phosphate compared to a polypeptide having the amino acid sequence of SEQ ID NO:

240.

10. An engineered pantothenate kinase, wherein the amino acid residue difference between the polypeptide sequence of the engineered pantothenate kinase and SEQ ID NO: 276 is 1-6 amino acid residue differences selected from the group consisting of L23V, L61I / V, S304G, P11Q, L13R, Q85V and Q19T, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 276, and wherein the engineered pantothenate kinase consists of a polypeptide sequence that exhibits an increased ability to convert acetyleneglyceraldehyde to acetyleneglyceraldehyde phosphate compared to a polypeptide having the amino acid sequence of SEQ ID NO:

252.

11. The engineered pantothenate kinase of claim 1, wherein the engineered pantothenate kinase is SEQ ID NO:

30.

12. The engineered pantothenate kinase of claim 3, wherein the engineered pantothenate kinase is SEQ ID NO:

60.

13. The engineered pantothenate kinase of claim 4, wherein the engineered pantothenate kinase is SEQ ID NO:

132.

14. The engineered pantothenate kinase of claim 5, wherein the engineered pantothenate kinase is SEQ ID NO:

222.

15. The engineered pantothenate kinase of claim 7, wherein the engineered pantothenate kinase is SEQ ID NO:

230.

16. The engineered pantothenate kinase of claim 8, wherein the engineered pantothenate kinase is SEQ ID NO:

240.

17. An engineered pantothenate kinase, wherein the engineered pantothenate kinase is an even-numbered sequence of SEQ ID NO: 4, 10, 12, 14, 18, 22, 30, 32, 36 or 46-248.

18. The engineered pantothenate kinase of any one of claims 1-17, wherein the engineered pantothenate kinase is purified.

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

20. A polynucleotide encoding at least one engineered pantothenate kinase according to any one of claims 1-17.

21. The polynucleotide of claim 20, encoding at least one engineered pantothenate kinase, wherein the polynucleotide is SEQ ID NO:

29.

22. The polynucleotide of claim 20, encoding at least one engineered pantothenate kinase, wherein the polynucleotide is SEQ ID NO:

59.

23. The polynucleotide of claim 20, encoding at least one engineered pantothenate kinase, wherein the polynucleotide is SEQ ID NO:

131.

24. The polynucleotide of claim 20, encoding at least one engineered pantothenate kinase, wherein the polynucleotide is SEQ ID NO:

221.

25. The polynucleotide of claim 20, encoding at least one engineered pantothenate kinase, wherein the polynucleotide is SEQ ID NO:

229.

26. The polynucleotide of claim 20, encoding at least one engineered pantothenate kinase, wherein the polynucleotide is SEQ ID NO:

239.

27. The polynucleotide of any one of claims 20-26, wherein the polynucleotide is operably linked to a control sequence.

28. The polynucleotide of any one of claims 20-26, wherein the polynucleotide is codon optimized.

29. The polynucleotide of claim 20, wherein the polynucleotide is an odd-numbered sequence of SEQ ID NO: 3, 9, 11, 13, 17, 21, 29, 31, 35, or 45-247.

30. An expression vector comprising at least one polynucleotide according to any one of claims 20-29.

31. A host cell comprising at least one expression vector according to claim 30.

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

33. A method for producing an engineered pantothenate kinase in a host cell, the method comprising culturing the host cell of claim 31 or 32 under suitable conditions, thereby producing at least one engineered pantothenate kinase.

34. The method of claim 33, further comprising recovering at least one engineered pantothenate kinase from the culture and / or the host cell.

35. The method of claim 33 or 34, further comprising the step of purifying the at least one engineered pantothenate kinase.

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