Engineered glycosyltransferases and steviol glycoside glucosylation methods

Abstract

To provide engineered glycosyltransferases and steviol glycoside glucosylation methods.SOLUTION: The present invention provides engineered glycosyltransferase (GT) enzymes, polypeptides having GT activity, and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. The present invention provides engineered sucrose synthase (SuS) enzymes, polypeptides having SuS activity, and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. The present invention also provides compositions comprising the GT enzymes and methods of using the engineered GT enzymes to make products with β-glucose linkages.SELECTED DRAWING: None

Description

[Technical field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 712,199, filed July 30, 2018, and U.S. Provisional Patent Application No. 62 / 712,327, filed July 31, 2018, both of which are incorporated by reference in their entireties for all purposes.

[0002] The present invention provides engineered glycosyltransferase (GT) enzymes, polypeptides having GT activity, and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. The present invention provides engineered sucrose synthase (SuS) enzymes, polypeptides having SuS activity, and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. The present invention further provides compositions comprising GT enzymes, and methods of using engineered GT enzymes to generate products with β-glucose linkages. The present invention further provides compositions and methods for the production of rebaudioside (e.g., rebaudioside M, rebaudioside A, rebaudioside I, and rebaudioside D). The present invention also provides compositions comprising SuS enzymes, and methods of using them. Methods of producing GT and SuS enzymes are also provided.

[0003] Reference to a sequence listing, table or computer program An official copy of the Sequence Listing has been submitted contemporaneously herewith via EFS-Web as an ASCII text file with the filename "CX8-180USP1A_ST25.txt", a creation date of July 31, 2018, and a size of 8,929 kilobytes. This Sequence Listing filed via EFS-Web is a part of the present specification and is incorporated herein by reference in its entirety. [Background technology]

[0004] Glucosyltransferases (GTs) are enzymes that post-translationally transfer glycosyl residues from activated nucleoside sugars to monomeric and polymeric acceptor molecules (e.g., other sugars, proteins, lipids, and other organic substrates). Thus, these enzymes utilize activated donor sugar substrates that contain a substituted phosphate leaving group. The donor sugar substrates (i.e., "glycosyl donors") are typically activated as nucleoside diphosphate sugars. However, other sugars such as nucleoside monophosphate sugars, lipid phosphates, and unsubstituted phosphates are also used (see, e.g., Lairson et al., Ann. Rev. Biochem., 77:25.1-25.35

[2008] ). GTs are classified as either retaining or inverting enzymes based on the stereochemistry of the substrates and reaction products. In reactions in which the stereochemistry of the donor anomeric bond is retained (e.g., alpha to alpha), GTs are retaining enzymes. In reactions where the stereochemistry is inverted (e.g., alpha to beta), GT is an inverting enzyme. These glycosylated products are involved in various metabolic pathways and processes. Indeed, the biosynthesis of a great many di-, oligo- and polysaccharides requires the action of various glycosyltransferases. The transfer of glucosyl moieties can modify the bioactivity, solubility, and intracellular transport properties of the acceptor. GT has been used for the targeted synthesis of specific compounds (e.g., glycoconjugates and glycosides) and for the production of differentially glycosylated drugs, biological probes, or natural product libraries. In some methods, the large-scale use of GT for glycoconjugate synthesis requires large amounts of glycosyl donors, thus increasing the cost of such procedures. Nucleotide recycling systems have been developed to allow the resynthesis of glycosyl donors from the released nucleotides. These recycling systems also reduce the amount of nucleotide by-products formed during the reaction, thereby reducing the inhibition caused by GT. Nevertheless, there remains a need for improved methods suitable for the large-scale production of glycoconjugates by GT. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Lairson et al., Ann. Rev. Biochem. (2008) 77:25.1-25.35 Summary of the Invention [Means for solving the problem]

[0006] The present invention provides engineered glycosyltransferase (GT) enzymes, polypeptides having GT activity, and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. The present invention provides engineered sucrose synthase (SuS) enzymes, polypeptides having SuS activity, and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. The present invention further provides compositions comprising GT enzymes, and methods of using engineered GT enzymes to generate products with β-glucose linkages. The present invention further provides compositions and methods for the production of rebaudioside (e.g., rebaudioside M, rebaudioside A, rebaudioside I, and rebaudioside D). The present invention also provides compositions comprising SuS enzymes, and methods of using them. Methods of producing GT and SuS enzymes are also provided. The present invention provides engineered glycosyltransferase variants comprising a polypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 2. In some embodiments, the engineered glycosyltransferase comprises a polypeptide that is at least 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:20. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence is 41 / 72 / 233 / 338, 41 / 72 / 338, 41 / 144 / 233, 41 / 233, 41 / 233 / 338, 61, 61 / 87 / 91 / 107, 61 / 87 / 91 / 259, 61 / 91 / 431, 61 / 107, 61 / 259 / 428, 61 / 407 / 428, 61 / 411, 72, 72 / 76, 72 / 76 / 163 / 197, 72 / 76 / 195 / 233, 72 / 76 / 197 / 204, 72 / 76 / 207 / 233, 72 / 76 / 207 / 338, 72 / 81, 72 / 81 / 195 / 233, 72 / 139 / 195 / 204, 72 / 144 / 338, 72 / 200 / 204 / 207, 72 / 207, 76 / 144 / 197 / 200, 76 / 195 / 197 / 204 / 207 / 233, 76 / 197 / 207 / 233, 76 / 233, 81 / 139 / 144 / 195 / 200 / 204 / 207 / 233, 81 / 144 / 233, 81 / 197 / 200 / 207 / 233 / 338, 81 / 233 / 338, 81 / 338, 107, 107 / 2 59, 139 / 144 / 233, 144 / 233, 144 / 233 / 338, 156 / 407, 163 / 233 / 338, 200 / 204 / 207 / 233, 233 / 338, and 259, where these positions are numbered with reference to SEQ ID NO:20.In some additional embodiments, the engineered glycosyltransferase polypeptide sequence is 41E / 72P / 233Q / 338A, 41E / 72P / 338A, 41E / 144Q / 233T, 41E / 233Q, 41E / 233Q / 338V, 41E / 233T, 41E / 233T / 338V, 61D, 61D / 87K / 91L / 107L, 61D / 87K / 91L / 259T, 61D / 107V, 61D / 259T / 428I, 61D / 40 7T / 428I, 61E / 87K / 91L / 107V, 61E / 91L / 431M, 61E / 411T, 72P, 72P / 76S, 72P / 76S / 163A / 197K, 72P / 76S / 207V / 338V, 72P / 7 6T, 72P / 76T / 195Q / 233T, 72P / 76T / 197K / 204T, 72P / 76T / 207V / 233Q, 72P / 81T, 72P / 81T / 195Q / 233Q, 72P / 139N / 195Q / 204T , 72P / 144Q / 338V, 72P / 200R / 204T / 207V, 72P / 207V, 76S / 144Q / 197K / 200R, 76S / 195Q / 197K / 204T / 207V / 233T, 76S / 197K / 207V / 233Q, 76S / 233T, 81T / 139N / 144Q / 195Q / 200R / 204T / 207V / 233Q, 81T / 144Q / 233Q, 81T / 197K / 200R / 207V / 233Q / 338A, and at least one mutation or set of mutations selected from 81T / 233Q / 338V, 81T / 338V, 107V, 107V / 259T, 139N / 144Q / 233Q, 144Q / 233T, 144Q / 233T / 338V, 156S / 407T, 163A / 233T / 338A, 200R / 204T / 207V / 233T, 233Q / 338V, and 259T, wherein these positions are numbered with reference to SEQ ID NO:20.In some additional embodiments, the engineered glycosyltransferase polypeptide sequence is A41E / T72P / W233Q / C338A, A41E / T72P / C338A, A41E / M144Q / W233T, A41E / W233Q, A41E / W233Q / C338V, A41E / W233T, A41E / W233T / C338V, Q61D, Q61D / A87K / Q91L / A107L, Q61D / A87K / Q91L / E259T, Q61D / A107V, Q61D / E259T / K428I, Q61D / I407 T / K428I, Q61E / A87K / Q91L / A107V, Q61E / Q91L / D431M, Q61E / R411T, T72 P, T72P / R76S, T72P / R76S / L163A / Q197K, T72P / R76S / I207V / C338V, T72P / R76T, T72P / R76T / H195Q / W233T, T72P / R76T / Q197K / D204T, T72P / R76T / I207V / W233Q, T72P / H81T, T72P / H81T / H195Q / W233Q, T72P / K139N / H195Q / D204T, T72P / M144Q / C338V, T72P / K200R / D204T / I207V, T72P / I207V, R 76S / M144Q / Q197K / K200R, R76S / H195Q / Q197K / D204T / I207V / W233T, R76 S / Q197K / I207V / W233Q, R76S / W233T, H81T / K139N / M144Q / H195Q / K200R / D204T / I207V / W233Q, H81T / M144Q / W233Q, H81T / Q197K / K200R / I207V / W2 and at least one mutation or set of mutations selected from 33Q / C338A, H81T / W233Q / C338V, H81T / C338V, A107V, A107V / E259T, K139N / M144Q / W233Q, M144Q / W233T, M144Q / W233T / C338V, C156S / I407T, L163A / W233T / C338A, K200R / D204T / I207V / W233T, W233Q / C338V, and E259T, wherein these positions are numbered with reference to SEQ ID NO:20.

[0007] In some further embodiments, the polypeptide sequence of the engineered glycosyltransferase comprises at least one mutation or set of mutations at one or more positions selected from 71, 80, 81, 81 / 270, 83, 85, 97, 124, 263, 286, 334, 402, 420 and 456, which positions are numbered with reference to SEQ ID NO: 20. In some additional embodiments, the polypeptide sequence of the engineered glycosyltransferase comprises at least one mutation or set of mutations selected from 71G, 71T, 80D, 80P, 80Q, 81A, 81L, 81M, 81S, 81T, 81V, 81V / 270K, 83D, 85T, 85V, 97V, 124P, 263C, 286L, 334V, 402I, 420R and 456K, which positions are numbered with reference to SEQ ID NO: 20. In some additional embodiments, the polypeptide sequence of the engineered glycosyltransferase comprises at least one mutation or set of mutations selected from Q71G, Q71T, T80D, T80P, T80Q, H81A, H81L, H81M, H81S, H81T, H81V, H81V / Q270K, P83D, A85T, A85V, A97V, A124P, T263C, V286L, I334V, E402I, E420R and S456K, wherein these positions are numbered with reference to SEQ ID NO:20.

[0008] In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations at one or more positions selected from 41 / 72 / 233 / 338, 41 / 72 / 338, 61, 61 / 91 / 431, 61 / 259 / 428, 61 / 407 / 428, where these positions are numbered with reference to SEQ ID NO:20. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations selected from 41E / 72P / 233Q / 338A, 41E / 72P / 338A, 61D, 61D / 259T / 428I, 61D / 407T / 428I, 61E / 91L / 431M, 81T / 139N / 144Q / 195Q / 200R / 204T / 207V / 233Q, and 81T / 197K / 200R / 207V / 233Q / 338A, wherein these positions are numbered with reference to SEQ ID NO:20. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations selected from A41E / T72P / W233Q / C338A, A41E / T72P / C338A, Q61D, Q61D / E259T / K428I, Q61D / I407T / K428I, Q61E / Q91L / D431M, H81T / K139N / M144Q / H195Q / K200R / D204T / I207V / W233Q, and H81T / Q197K / K200R / I207V / W233Q / C338A, wherein these positions are numbered with reference to SEQ ID NO:20.

[0009] In yet some further embodiments, the engineered glycosyltransferase polypeptide sequence is selected from the group consisting of 73 / 81 / 144 / 207 / 259, 73 / 81 / 144 / 207 / 285 / 451, 73 / 81 / 259 / 285 / 426 / 451, 73 / 81 / 259 / 451, 73 / 111 / 285 / 451, 73 / 144 / 207 / 252 / 426 / 451, 73 / 144 / 207 / 259 / 285 / 426 / 451, 73 / 144 / 207 / 259 / 285 / 451, 73 / 144 / 207 / 259 / 285 / 451, 73 / 144 / 207 / 259 / 426 / 451, 73 / 144 / 259 / 285, 73 / 259 / 426 / 451, 73 / 259 / 451, 81 / 111 / 144 / 207 / 285 / 451, 81 / 111 / 259 / 451, 81 / 144 / 207 / 252 / 285 / 426, 81 / 144 / 207 / 259 / 451, 81 / 144 / 252 / 259 / 285 / 451, 81 / 144 / 259, 81 / 144 / 259 / 285, 81 / 144 / 259 / 451, 81 / 207 / 252 / 259 / 451, 81 / 207 / 285 / 426 / 451, 81 / 285, 81 / 285 / 451, 111 / 1 44 / 207 / 252 / 285 / 426 / 451, 111 / 144 / 252 / 259 / 285 / 451, 111 / 144 / 259 / 285 / 426 / 451, 111 / 207 / 285, 144 / 207 / 252 / 259, 144 / 207 / 259, 144 / 207 / 259 / 285 / 451, 144 / 207 / 259 / 426 / 451, 144 / 207 / 285 / 426, 144 / 207 / 451, 144 / 252 / 259 / 285, 144 / 252 / 259 / 285 / 451, 144 / 252 / 259 / 426, 144 / 252 / 2 and 285 / 451, wherein the positions are numbered with reference to SEQ ID NO:36.

[0010] In some additional embodiments, the engineered glycosyltransferase polypeptide sequence is selected from the group consisting of 73S / 81T / 144Q / 207V / 259T, 73S / 81T / 144Q / 207V / 285S / 451Q, 73S / 81T / 259T / 285S / 426I / 451Q, 73S / 81T / 259T / 451Q, 73S / 111T / 285S / 451Q, 73S / 144Q / 207V / 252P / 426I / 451Q, 73S / 144Q / 207V / 259T / 285S / 426I / 451Q, 73S / 144Q / 207V / 259T / 285S / 45 1Q, 73S / 144Q / 207V / 259T / 426I / 451Q, 73S / 144Q / 259T / 285S, 73S / 259T / 42 6I / 451Q, 73S / 259T / 451Q, 81T / 111T / 144Q / 207V / 285S / 451Q, 81T / 111T / 25 9T / 451Q, 81T / 144Q / 207V / 252P / 285S / 426I, 81T / 144Q / 207V / 259T / 451Q, 8 1T / 144Q / 252P / 259T / 285S / 451Q, 81T / 144Q / 259T, 81T / 144Q / 259T / 285S, 81 T / 144Q / 259T / 451Q, 81T / 207V / 252P / 259T / 451Q, 81T / 207V / 285S / 426I / 45 1Q, 81T / 285S, 81T / 285S / 451Q, 111T / 144Q / 207V / 252P / 285S / 426I / 451Q, 1 11T / 144Q / 252P / 259T / 285S / 451Q, 111T / 144Q / 259T / 285S / 426I / 451Q, 111 T / 207V / 285S, 144Q / 207V / 252P / 259T, 144Q / 207V / 259T, 144Q / 207V / 259T / 2 85S / 451Q, 144Q / 207V / 259T / 426I / 451Q, 144Q / 207V / 285S / 426I, 144Q / 207 V / 451Q, 144Q / 252P / 259T / 285S, 144Q / 252P / 259T / 285S / 451Q, 144Q / 252P / 259T / 426I, 144Q / 252P / 259T / 426I / 451Q, 144Q / 259T / 285S, 144Q / 285S, 14 4Q / 285S / 451Q, 144Q / 451Q, 207V, 207V / 252P / 259T / 451Q, 207V / 252P / 451Q,and at least one mutation or set of mutations selected from 207V / 259T, 207V / 259T / 426I / 451Q, 207V / 285S / 426I / 451Q, 207V / 451Q, 252P / 259T, 259T, 259T / 285S / 451Q, 285S, and 285S / 451Q, wherein these positions are numbered with reference to SEQ ID NO:36. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence is E73S / H81T / M144Q / I207V / E259T, E73S / H81T / M144Q / I207V / E285S / V451Q, E73S / H81T / E259T / E285S / A426I / V451Q, E73S / H81T / E259T / V451Q, E73S / S111T / E285S / V451Q, E73S / M144Q / I207V / S252P / A426I / V451Q, E73S / M144Q / I207V / E259T / E285S / A426I / V451Q, E73S / M144Q / I207V / E259T / E285S / V451Q, E73S / M144Q / I207V / E259T / A426I / V451Q, E73S / M144Q / E259T / E285S, E73S / E259T / A426I / V451Q, E73S / E259T / V451Q, H81T / S111T / M144Q / I207V / E285S / V451Q, H81T / S111T / E259T / V451Q, H81T / M144Q / I207V / S252P / E285S / A426I, H81T / M144Q / I207V / E259T / V451Q, H81T / M144Q / S252P / E259T / E285S / V451Q, H81T / M144Q / E259T, H81T / M144Q / E259T / E285S, H81T / M144Q / E259T / V451Q, H81T / I207V / S252P / E259T / V451Q, H81T / I207V / E285S / A426I / V451Q, H81T / E285S, H81T / E285S / V451Q, S111T / M144Q / I207V / S252P / E285 S / A426I / V451Q, S111T / M144Q / S252P / E259T / E285S / V451Q, S111T / M144Q / E259T / E285S / A426I / V451Q, S111T / I207V / E285S,M144Q / I207V / S252P / E259T, M144Q / I207V / E259T, M144Q / I207V / E259T / E285S / V4 51Q, M144Q / I207V / E259T / A426I / V451Q, M144Q / I207V / E285S / A426I, M144Q / I207V / V451Q, M144Q / S252P / E259T / E285S, M144Q / S252P / E259T / E285S / V451Q, M144Q / S 252P / E259T / A426I, M144Q / S252P / E259T / A426I / V451Q, M144Q / E259T / E285S, M144 and at least one mutation or set of mutations selected from Q / E285S, M144Q / E285S / V451Q, M144Q / V451Q, I207V, I207V / S252P / E259T / V451Q, I207V / S252P / V451Q, I207V / E259T, I207V / E259T / A426I / V451Q, I207V / E285S / A426I / V451Q, I207V / V451Q, S252P / E259T, E259T, E259T / E285S / V451Q, E285S, and E285S / V451Q, wherein these positions are numbered with reference to SEQ ID NO:36. In still some further embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations at one or more positions selected from 81 / 111 / 144 / 207 / 285 / 451, 144 / 207 / 252 / 259, 144 / 207 / 259 / 285 / 451, 144 / 252 / 259 / 426, 144 / 252 / 259 / 426 / 451, and 144 / 285 / 451, where these positions are numbered with reference to SEQ ID NO:36. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations selected from 81T / 111T / 144Q / 207V / 285S / 451Q, 144Q / 207V / 252P / 259T, 144Q / 207V / 259T / 285S / 451Q, 144Q / 252P / 259T / 426I, 144Q / 252P / 259T / 426I / 451Q, and 144Q / 285S / 451Q,In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations selected from H81T / S111T / M144Q / I207V / E285S / V451Q, M144Q / I207V / S252P / E259T, M144Q / I207V / E259T / E285S / V451Q, M144Q / S252P / E259T / A426I, M144Q / S252P / E259T / A426I / V451Q, and M144Q / E285S / V451Q, and these positions are numbered with reference to SEQ ID NO:36.

[0011] In still some further embodiments, the engineered glycosyltransferase polypeptide sequence is selected from the group consisting of 80 / 81, 80 / 81 / 85, 80 / 81 / 85 / 238, 80 / 81 / 85 / 251 / 259, 80 / 81 / 85 / 252 / 256 / 259, 80 / 81 / 85 / 259, 80 / 81 / 251 / 252 / 259, 80 / 85, 80 / 85 / 238, 80 / 85 / 238 / 251 / 259, 80 / 85 / 251, 80 / 85 / 251 / 252, 80 / 85 / 251 / 252 / 256 / 259, 80 / 85 / 251 / and at least one mutation or set of mutations at one or more positions selected from 252 / 259, 80 / 85 / 251 / 259, 80 / 85 / 259, 80 / 259, 81 / 85, 81 / 85 / 251 / 252, 81 / 85 / 259, 85, 85 / 238, 85 / 251, 85 / 251 / 252 / 256, 85 / 251 / 259, 85 / 256 / 259, 175 / 402, 251 / 252 / 259, 259, 270 / 402, and 420, wherein these positions are numbered with reference to SEQ ID NO:174.In some additional embodiments, the engineered glycosyltransferase polypeptide sequence is 80D / 81A, 80D / 81A / 85V, 80D / 81A / 85V / 238G, 80D / 81A / 85V / 251M / 259T, 80D / 81A / 85V / 252P / 256G / 259T, 80D / 85V, 80D / 85V / 23 8G, 80D / 85V / 238G / 251M / 259T, 80D / 85V / 251I / 252P / 256G / 259T, 80D / 85V / 251I / 259T, 8 0D / 85V / 251M, 80D / 85V / 251M / 252P, 80D / 85V / 251M / 252P / 256G / 259T, 80D / 85V / 259T, 80D / 259T, 80P / 81A / 85V / 259T, 80P / 81A / 251I / 252P / 259T, 80P / 85V, 80P / 85V / 251M / 252P / 2 59T, 81A / 85V, 81A / 85V / 251M / 252P, 81A / 85V / 259T, 85V, 85V / 238G, 85V / 251F, 85V / 251F / and at least one mutation or set of mutations selected from 259T, 85V / 251M / 252P / 256G, 85V / 256G / 259T, 175D / 402I, 251I / 252P / 259T, 259T, 270K / 402I, and 420R, wherein these positions are numbered with reference to SEQ ID NO:174.In some additional embodiments, the engineered glycosyltransferase polypeptide sequence is selected from the group consisting of T80D / T81A, T80D / T81A / A85V, T80D / T81A / A85V / A238G, T80D / T81A / A85V / A251M / E259T, T80D / T81A / A85V / S252P / L256G / E259T, T80D / A85V, T80D / A85V / A238 G, T80D / A85V / A238G / A251M / E259T, T80D / A85V / A251I / S252P / L256G / E259T, T80D / A85V / A251I / E259T, T 80D / A85V / A251M, T80D / A85V / A251M / S252P, T80D / A85V / A251M / S252P / L256G / E259T, T80D / A85V / E259T, T80D / E259T, T80P / T81A / A85V / E259T, T80P / T81A / A251I / S252P / E259T, T80P / A85V, T80P / A85V / A251M / S 252P / E259T, T81A / A85V, T81A / A85V / A251M / S252P, T81A / A85V / E259T, A85V, A85V / A238G, A85V / A251F, A and at least one mutation or set of mutations selected from 85V / A251F / E259T, A85V / A251M / S252P / L256G, A85V / L256G / E259T, E175D / E402I, A251I / S252P / E259T, E259T, Q270K / E402I, and E420R, wherein these positions are numbered with reference to SEQ ID NO:174. In some embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations at one or more positions selected from 3, 5, 7, 99, 153, 232 / 317, 252, 273, 299, 326, 393, 404, 409, 422, 443, 451 and 455, where these positions are numbered with reference to SEQ ID NO:174.In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations selected from 3S, 5A, 5H, 5L, 5T, 7A, 7Q, 99V, 153C, 232I / 317L, 252A, 273K, 299M, 326A, 326E, 326N, 326S, 393I, 404M, 404V, 409S, 422I, 443A, 451E, 451V, 455M and 455R, wherein these positions are numbered with reference to SEQ ID NO: 174. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations selected from N3S, S5A, S5H, S5L, S5T, T7A, T7Q, E99V, A153C, V232I / Y317L, S252A, S273K, V299M, G326A, G326E, G326N, G326S, V393I, G404M, G404V, A409S, M422I, S443A, Q451E, Q451V, S455M and S455R, wherein these positions are numbered with reference to SEQ ID NO: 174. In still some further embodiments, the polypeptide sequence of the engineered glycosyltransferase comprises at least one mutation or set of mutations at one or more positions selected from 80 / 81 / 85, 80 / 85, 80 / 85 / 251 / 259, 85, 85 / 251 / 252 / 256, 175 / 402, and 270 / 402, where these positions are numbered with reference to SEQ ID NO: 174. In some additional embodiments, the polypeptide sequence of the engineered glycosyltransferase comprises at least one mutation or set of mutations selected from 80D / 81A / 85V, 80D / 85V / 251I / 259T, 80P / 85V, 85V, 85V / 251M / 252P / 256G, 175D / 402I, and 270K / 402I, where these positions are numbered with reference to SEQ ID NO: 174.In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations selected from T80D / T81A / A85V, T80D / A85V / A251I / E259T, T80P / A85V, A85V, A85V / A251M / S252P / L256G, E175D / E402I, and Q270K / E402I, wherein these positions are numbered with reference to SEQ ID NO: 174.

[0012] In still some further embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations at one or more positions selected from 81 / 85, 81 / 85 / 175 / 259 / 402, 81 / 85 / 251, 81 / 85 / 251 / 259, 81 / 85 / 259, 81 / 85 / 259 / 402, 85, 85 / 175, 85 / 175 / 251, 85 / 175 / 251 / 259 / 402, 85 / 175 / 259, 85 / 175 / 259 / 402, 85 / 175 / 402, 85 / 259, 85 / 259 / 402, and 85 / 402, wherein these positions are numbered with reference to SEQ ID NO:406. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence is 81A / 85V, 81A / 85V / 175E / 259T / 402E, 81A / 85V / 251I, 81A / 85V / 251I / 259T, 81A / 85V / 259T, 81A / 85V / 259T / 402E, 85V, 85V / 175E, 85V / 175E / 251I, 85 and at least one mutation or set of mutations selected from V / 175E / 251I / 259T / 402E, 85V / 175E / 259T, 85V / 175E / 259T / 402E, 85V / 175E / 402E, 85V / 259T, 85V / 259T / 402E, and 85V / 402E, wherein these positions are numbered with reference to SEQ ID NO:406.In some additional embodiments, the engineered glycosyltransferase polypeptide sequence is selected from the group consisting of T81A / A85V, T81A / A85V / D175E / E259T / I402E, T81A / A85V / A251I, T81A / A85V / A251I / E259T, T81A / A85V / E259T, T81A / A85V / E259T / I402E, A85V, A85V / D175E, A85V / D175E / A251I , A85V / D175E / A251I / E259T / I402E, A85V / D175E / E259T, A85V / D175E / E259T / I402E, A85V / D175E / I402E, A85V / E259T, A85V / E259T / I402E, and A85V / I402E, wherein these positions are numbered with reference to SEQ ID NO:406.

[0013] In still some further embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations at one or more positions selected from 153, 153 / 326, 153 / 326 / 443, 153 / 326 / 443 / 455, 232, 232 / 273 / 299, 232 / 393 / 451, 299 / 451, 326, 404 and 451, where these positions are numbered with reference to SEQ ID NO:408. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations selected from 153C, 153C / 326S, 153C / 326S / 443A, 153C / 326S / 443A / 455M, 232I, 232I / 273K / 299M, 232I / 393I / 451E, 299M / 451E, 326E, 326S, 404V and 451E, wherein these positions are numbered with reference to SEQ ID NO:408. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations selected from A153C, A153C / G326S, A153C / G326S / S443A, A153C / G326S / S443A / S455M, V232I, V232I / S273K / V299M, V232I / V393I / Q451E, V299M / Q451E, G326E, G326S, G404V and Q451E, wherein these positions are numbered with reference to SEQ ID NO:408. In some further embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations at one or more positions selected from 25, 116, 146, 170, 173, 227, 296, 300, 315, 327, 330, 361, 408, 412, 438, 448 and 449, where these positions are numbered with reference to SEQ ID NO:408.In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations selected from 25A, 116D, 116R, 116S, 146G, 170C, 170G, 170P, 170S, 170T, 173S, 227T, 296V, 300S, 315T, 327Y, 330G, 330T, 361C, 408C, 408T, 412A, 412K, 412S, 438Q, 448H and 449S, wherein these positions are numbered with reference to SEQ ID NO:408. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations selected from L25A, E116D, E116R, E116S, S146G, D170C, D170G, D170P, D170S, D170T, R173S, S227T, R296V, A300S, K315T, F327Y, E330G, E330T, T361C, N408C, N408T, R412A, R412K, R412S, A438Q, E448H and R449S, wherein these positions are numbered with reference to SEQ ID NO:408. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations at one or more positions selected from 153, 232, 232 / 393 / 451, and 451, where these positions are numbered with reference to SEQ ID NO: 408. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations selected from 153C, 232I, 232I / 393I / 451E, and 451E, where these positions are numbered with reference to SEQ ID NO: 408. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations selected from A153C, V232I, V232I / V393I / Q451E, and Q451E, where these positions are numbered with reference to SEQ ID NO: 408.

[0014] In some embodiments, the polypeptide sequence of the engineered glycosyltransferase is selected from the group consisting of 146 / 170 / 196, 146 / 170 / 196 / 232, 146 / 170 / 196 / 232 / 423, 146 / 170 / 196 / 232 / 451 / 455, 146 / 170 / 196 / 408 / 451, 146 / 170 / 232, 146 / 170 / 232 / 423, 146 / 170 / 232 / 423 / 448 / 451 / 455, 146 / 170 / 259, 146 / 196, 146 / 196 / 2 32, 146 / 196 / 232 / 259 / 448 / 451, 146 / 196 / 455, 146 / 232 / 259 / 455, 146 / 232 / 315, 146 / 232 / 315 / 423 / 451 / 455, 146 / 232 / 326, 146 / 232 / 448, 146 / 232 / 451, and 146 / 413 / 451 / 455, wherein these positions are numbered with reference to SEQ ID NO:440. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence is 146G / 170S / 196I, 146G / 170S / 232I, 146G / 170S / 232I / 423K / 448H / 451E / 455M, 146G / 170T / 196I / 232I, 146G / 170T / 196I / 232I / 423K, 146G / 170T / 196I / 232I / 451E / 455R, 146G / 170T / 196I / 408T / 451E, 146G / 170T / 232I / 423K, 146G / 170T / 259E, 146G / 196I, and at least one mutation or set of mutations selected from 146G / 196I / 232I, 146G / 196I / 232I / 259E / 448H / 451E, 146G / 196I / 455R, 146G / 232I / 259E / 455M, 146G / 232I / 315S, 146G / 232I / 315S / 423K / 451E / 455M, 146G / 232I / 326E, 146G / 232I / 448H, 146G / 232I / 451E, and 146G / 413M / 451E / 455M, wherein these positions are numbered with reference to SEQ ID NO:440.In some additional embodiments, the engineered glycosyltransferase polypeptide sequence is S146G / D170S / W196I, S146G / D170S / V232I, S146G / D170S / V232I / R423K / E448H / Q451E / S455M, S146G / D170T / W196I / V232I, S 146G / D170T / W196I / V232I / R423K, S146G / D170T / W196I / V232I / Q451E / S455R, S146G / D1 70T / W196I / N408T / Q451E, S146G / D170T / V232I / R423K, S146G / D170T / T259E, S146G / W196 I, S146G / W196I / V232I, S146G / W196I / V232I / T259E / E448H / Q451E, S146G / W196I / S455R , S146G / V232I / T259E / S455M, S146G / V232I / K315S, S146G / V232I / K315S / R423K / Q451E / S 455M, S146G / V232I / G326E, S146G / V232I / E448H, S146G / V232I / Q451E, and S146G / V413M / Q451E / S455M, wherein these positions are numbered with reference to SEQ ID NO:440.

[0015] In some additional embodiments, the polypeptide sequence of the engineered glycosyltransferase comprises at least one mutation or set of mutations at one or more positions selected from 9, 12, 107, 131, 156, 161, 169, 199, 204, 209, 233, 262, 289, 337, and 417, where these positions are numbered with reference to SEQ ID NO: 440. In some additional embodiments, the polypeptide sequence of the engineered glycosyltransferase comprises at least one mutation or set of mutations selected from 9L, 12Q, 107C, 131A, 156S, 161E, 169T, 199S, 204M, 209Y, 233K, 262A, 289A, 337N, 337W, and 417V, where these positions are numbered with reference to SEQ ID NO: 440. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations selected from V9L, R12Q, A107C, P131A, C156S, L161E, E169T, A199S, D204M, E209Y, Q233K, S262A, K289A, S337N, S337W and P417V, wherein these positions are numbered with reference to SEQ ID NO:440. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations at one or more positions selected from 146 / 170 / 196, 146 / 170 / 196 / 232, 146 / 170 / 196 / 232 / 451 / 455, 146 / 170 / 232, 146 / 170 / 232 / 423 / 448 / 451 / 455, 146 / 196 / 232, 146 / 196 / 232 / 259 / 448 / 451, and 146 / 232 / 448, where these positions are numbered with reference to SEQ ID NO:440.In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations selected from 146G / 170S / 196I, 146G / 170S / 232I, 146G / 170S / 232I / 423K / 448H / 451E / 455M, 146G / 170T / 196I / 232I, 146G / 170T / 196I / 232I / 451E / 455R, 146G / 196I / 232I, 146G / 196I / 232I / 259E / 448H / 451E, and 146G / 232I / 448H, wherein these positions are numbered with reference to SEQ ID NO:440. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence is selected from the group consisting of S146G / D170S / W196I, S146G / D170S / V232I, S146G / D170S / V232I / R423K / E448H / Q451E / S455M, S146G / D170T / W196I / V232I, S146G / D170T / W and at least one mutation or set of mutations selected from 196I / V232I / Q451E / S455R, S146G / W196I / V232I, S146G / W196I / V232I / T259E / E448H / Q451E, and S146G / V232I / E448H, wherein these positions are numbered with reference to SEQ ID NO:440. In yet some further embodiments, the engineered glycosyltransferase polypeptide sequence is 9 / 12 / 107 / 161 / 199 / 209 / 337, 9 / 12 / 161 / 169 / 199 / 209 / 337, 9 / 12 / 199 / 204 / 209 / 337 / 455, 9 / 12 / 199 / 209 / 337 / 451 / 455, 9 / 107 / 131 / 20 4 / 259 / 417 / 451, 9 / 107 / 156 / 161 / 199 / 204 / 417 / 455, 9 / 107 / 161 / 209 / 259 / 289 / 451 / 455, 9 / 131 / 156 / 209 / 289 / 337, 9 / 131 / 204 / 337 / 451, 9 / 156 / 169 / 204 / 337, 9 / 169 / 204 / 289 / 337 / 451 / 455, 9 / 204, 9 / 204 / 259 / 289, 9 / 337, 12 / 14 / 107 / 204 / 289 / 455, 12 / 107 / 131 / 204 / 289 / 337 / 417 / 451 / 455, 12 / 107 / 156 / 209 / 289 / 455, 107 / 161 / 169 / 199 / 204 / 259 / 451, 107 / 199 / 204 / 289, 107 / 199 / 209 161 / 204 / 417, 204 / 451 / 455, 289, and 451 / 455, wherein these positions are numbered with reference to SEQ ID NO:520.In some additional embodiments, the engineered glycosyltransferase polypeptide sequence is 9L / 12Q / 107C / 161E / 199S / 209Y / 337N, 9L / 12Q / 161E / 169T / 199S / 209Y / 337W, 9L / 12Q / 199S / 204M / 209Y / 337N / 455R, 9L / 12Q / 199S / 209Y / 337W / 451E / 455R, 9L / 107C / 131A / 204M / 2 59E / 417V / 451E, 9L / 107C / 156S / 161E / 199S / 204M / 417V / 455R, 9L / 107C / 161E / 209Y / 259E / 289A / 451E / 455R, 9L / 13 1A / 156S / 209Y / 289A / 337N, 9L / 131A / 204M / 337N / 451E, 9L / 156S / 169T / 204M / 337N, 9L / 169T / 204M / 289A / 337W / 451 E / 455R, 9L / 204M, 9L / 204M / 259E / 289A, 9L / 337W, 12Q / 14I / 107C / 204M / 289A / 455R, 12Q / 107C / 131A / 204M / 289A / 33 7W / 417V / 451E / 455R, 12Q / 107C / 156S / 209Y / 289A / 455R, 107C / 161E / 169T / 199S / 204M / 259E / 451E, 107C / 199S / 204 M / 289A, 107C / 199S / 209Y / 259E / 451E / 455R, 107C / 204M, 156S / 169T / 199S / 204M / 209Y / 259E / 289A, 161E / 204M / 417V, 204M / 451E / 455R, 289A, and 451E / 455R, wherein these positions are numbered with reference to SEQ ID NO:520.In some additional embodiments, the engineered glycosyltransferase polypeptide sequence is selected from the group consisting of V9L / R12Q / A107C / L161E / A199S / E209Y / S337N, V9L / R12Q / L161E / E169T / A199S / E209Y / S337W, V9L / R12Q / A199S / D204M / E209Y / S337N / S455R, V9L / R12Q / A199S / E209Y / S337W / Q451E / S455R, V9L / A107C / P131A / D204M / T25 9E / P417V / Q451E, V9L / A107C / C156S / L161E / A199S / D204M / P417V / S455R, V9L / A107C / L161E / E209Y / T259E / K289A / Q451E / S455R, V9L / P 131A / C156S / E209Y / K289A / S337N, V9L / P131A / D204M / S337N / Q451E, V9L / C156S / E169T / D204M / S337N, V9L / E169T / D204M / K289A / S337W / Q451E / S455R, V9L / D204M, V9L / D204M / T259E / K289A, V9L / S337W, R12Q / V14I / A107C / D204M / K289A / S455R, R12Q / A107C / P131A / D204M / K 289A / S337W / P417V / Q451E / S455R, R12Q / A107C / C156S / E209Y / K289A / S455R, A107C / L161E / E169T / A199S / D204M / T259E / Q451E, A107C / A and at least one mutation or set of mutations selected from 199S / D204M / K289A, A107C / A199S / E209Y / T259E / Q451E / S455R, A107C / D204M, C156S / E169T / A199S / D204M / E209Y / T259E / K289A, L161E / D204M / P417V, D204M / Q451E / S455R, K289A, and Q451E / S455R, wherein these positions are numbered with reference to SEQ ID NO:520.In some further embodiments, the polypeptide sequence of the engineered glycosyltransferase comprises at least one mutation or set of mutations at one or more positions selected from 16, 53, 54, 78, 80, 95, 111, 221, 257, 336, 349, 391, 410, 413, 426, and 430, which positions are numbered with reference to SEQ ID NO: 520. In some additional embodiments, the polypeptide sequence of the engineered glycosyltransferase comprises at least one mutation or set of mutations selected from 16M, 53Q, 54P, 78V, 80A, 95W, 111C, 111G, 111S, 221A, 257A, 336R, 349V, 391R, 410V, 413L, 426S, and 430Q, which positions are numbered with reference to SEQ ID NO: 520. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations selected from L16M, K53Q, T54P, L78V, T80A, H95W, T111C, T111G, T111S, F221A, L257A, K336R, I349V, L391R, I410V, V413L, A426S and K430Q, wherein these positions are numbered with reference to SEQ ID NO:520. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations at one or more positions selected from 9 / 12 / 199 / 204 / 209 / 337 / 455, 9 / 131 / 156 / 209 / 289 / 337, 9 / 204, 12 / 14 / 107 / 204 / 289 / 455, 107 / 161 / 169 / 199 / 204 / 259 / 451, 107 / 199 / 204 / 289, and 204 / 451 / 455, where these positions are numbered with reference to SEQ ID NO:520.In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations selected from 9L / 12Q / 199S / 204M / 209Y / 337N / 455R, 9L / 131A / 156S / 209Y / 289A / 337N, 9L / 204M, 12Q / 14I / 107C / 204M / 289A / 455R, 107C / 161E / 169T / 199S / 204M / 259E / 451E, 107C / 199S / 204M / 289A, and 204M / 451E / 455R, wherein these positions are numbered with reference to SEQ ID NO:520. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations selected from V9L / R12Q / A199S / D204M / E209Y / S337N / S455R, V9L / P131A / C156S / E209Y / K289A / S337N, V9L / D204M, R12Q / V14I / A107C / D204M / K289A / S455R, A107C / L161E / E169T / A199S / D204M / T259E / Q451E, A107C / A199S / D204M / K289A, and D204M / Q451E / S455R, wherein these positions are numbered with reference to SEQ ID NO:520. In still some further embodiments, the engineered glycosyltransferase polypeptide sequence is 16, 16 / 59 / 80 / 413, 16 / 80, 16 / 80 / 111, 16 / 80 / 111 / 257, 16 / 80 / 221 / 257 / 336 / 410, 16 / 80 / 221 / 336 / 410, 16 / 80 / 257, 16 / 111 / 221, 16 / 111 / 221 / 257 / 391, 16 / 221, 16 / 221 / 257, 16 / and at least one mutation or set of mutations at one or more positions selected from 221 / 257 / 336 / 391, 16 / 221 / 410, 16 / 257, 16 / 257 / 336 / 413 / 420, 80 / 111 / 221 / 257 / 410, 111 / 221 / 257 / 336 / 391, 111 / 221 / 257 / 391, 221, and 221 / 257, where these positions are numbered with reference to SEQ ID NO:626. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence is 16M, 16M / 59Q / 80A / 413L, 16M / 80A, 16M / 80A / 111C, 16M / 80A / 111S / 257A, 16M / 80A / 221A / 257A / 336R / 410V, 16M / 80A / 221A / 336R / 410V, 16M / 80A / 257A, 16M / 111C / 221A / 257A / 391R, 16M / 111S / 221A, 16M / 221A, 16M / 221A / and at least one mutation or set of mutations selected from 257A, 16M / 221A / 257A / 336R / 391R, 16M / 221A / 410V, 16M / 257A, 16M / 257A / 336R / 413L / 420G, 80A / 111S / 221A / 257A / 410V, 111S / 221A / 257A / 336R / 391R, 111S / 221A / 257A / 391R, 221A, and 221A / 257A, wherein these positions are numbered with reference to SEQ ID NO:626.In some additional embodiments, the engineered glycosyltransferase polypeptide sequence is selected from the group consisting of L16M, L16M / H59Q / T80A / V413L, L16M / T80A, L16M / T80A / T111C, L16M / T80A / T111S / L257A, L16M / T80A / F221A / L257A / K336R / I410V, L16M / T80A / F221A / L257A / K336R / I410V, L16M / T80A / L257A, L16M / T111C / F221A / L257A / L391R, L16M / T111S / F221A, L16M ... and at least one mutation or set of mutations selected from: 1A / L257A, L16M / F221A / L257A / K336R / L391R, L16M / F221A / I410V, L16M / L257A, L16M / L257A / K336R / V413L / E420G, T80A / T111S / F221A / L257A / I410V, T111S / F221A / L257A / K336R / L391R, T111S / F221A / L257A / L391R, F221A, and F221A / L257A, wherein these positions are numbered with reference to SEQ ID NO:626. In some further embodiments, the polypeptide sequence of the engineered glycosyltransferase comprises at least one mutation or set of mutations at one or more positions selected from 5, 7, 14, 65, 91, 99, 102, 118, 138, 194, 254, 286, 416, 418, and 420, which positions are numbered with reference to SEQ ID NO: 626. In some additional embodiments, the polypeptide sequence of the engineered glycosyltransferase comprises at least one mutation or set of mutations selected from 5L, 5M, 5N, 5Q, 7L, 14L, 65V, 91V, 99L, 99S, 102R, 118S, 118V, 138I, 194P, 254G, 254T, 254V, 286L, 416E, 418D, and 420A, which positions are numbered with reference to SEQ ID NO: 626.In some additional embodiments, the polypeptide sequence of the engineered glycosyltransferase comprises at least one mutation or set of mutations selected from S5L, S5M, S5N, S5Q, T7L, V14L, I65V, Q91V, E99L, E99S, K102R, A118S, A118V, L138I, S194P, S254G, S254T, S254V, V286L, D416E, E418D and E420A, wherein these positions are numbered with reference to SEQ ID NO:626. In still some further embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations at one or more positions selected from 16 / 80 / 221 / 257 / 336 / 410, 16 / 111 / 221, 16 / 221 / 257, 16 / 221 / 410, 16 / 257, 80 / 111 / 221 / 257 / 410, and 221, where these positions are numbered with reference to SEQ ID NO:626. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations selected from 16M / 80A / 221A / 257A / 336R / 410V, 16M / 111S / 221A, 16M / 221A / 257A, 16M / 221A / 410V, 16M / 257A, 80A / 111S / 221A / 257A / 410V, and 221A, wherein these positions are numbered with reference to SEQ ID NO:626. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations selected from L16M / T80A / F221A / L257A / K336R / I410V, L16M / T111S / F221A, L16M / F221A / L257A, L16M / F221A / I410V, L16M / L257A, T80A / T111S / F221A / L257A / I410V, and F221A, wherein these positions are numbered with reference to SEQ ID NO:626.

[0016] In still some further embodiments, the engineered glycosyltransferase polypeptide sequence is selected from the group consisting of 5, 5 / 14 / 99, 5 / 14 / 416, 5 / 91, 5 / 91 / 102 / 118, 5 / 91 / 102 / 254 / 418, 5 / 91 / 106 / 254 / 286, 5 / 91 / 118 / 194 / 254 / 286, 5 / 91 / 118 / 194 / 254 / 41 8, 5 / 91 / 118 / 194 / 418, 5 / 91 / 118 / 254 / 286 / 418, 5 / 91 / 118 / 286, 5 / 91 / 194 / 254, 5 / 91 / 194 / 418, 5 / 91 / 254, 5 / 91 / 254 / 286, 5 / 102 / 418, 5 / 118 / 194 / 286 / 418, 5 / 118 / 254, 5 / 118 / 286, 5 / 194, 5 / 25 4 / 286, 5 / 418, 7 / 14, 7 / 14 / 65 / 99, 7 / 14 / 99 / 416, 14, 14 / 65 / 99 / 416, 14 / 99, 14 / 99 / 254, 14 / 99 / 254 / 416, 14 / 99 / 254 / 416 / 455, 14 / 99 / 416, 14 / 167, 14 / 254, 14 / 455, 91, 91 / 102 / 194, 91 / 118, 91 91 / 118 / 194, 91 / 118 / 194 / 286, 91 / 118 / 194 / 418, 91 / 118 / 286, 91 / 194 / 254 / 286, 91 / 286, and 118 / 286 / 418, wherein these positions are numbered with reference to SEQ ID NO:678.

[0017] In some additional embodiments, the engineered glycosyltransferase polypeptide sequence is 5L / 14L / 99L, 5L / 14L / 416E, 5N, 5N / 91V, 5N / 91V / 102R / 118V, 5N / 91V / 102R / 254T / 418D, 5N / 91V / 106I / 254T / 286L, 5N / 91V / 118S / 194P / 254T / 418D, 5N / 91V / 118S / 254T / 286L / 418D, 5N / 91 V / 118S / 286L, 5N / 91V / 118V / 194P / 254T / 286L, 5N / 91V / 118V / 194P / 418D, 5N / 91V / 194P / 254T, 5N / 91V / 194P / 418D, 5N / 91V / 254T, 5N / 91V / 254T / 286L, 5N / 102R / 418D, 5N / 118S / 194P / 286L / 418D, 5N / 118S / 286L, 5N / 118V / 254T, 5N / 194P, 5N / 254T / 286L, 5N / 418D, 7L / 14L, 7L / 14L / 65V / 99L, 7L / 14L / 99S / 416E, 14L, 14L / 65V / 99L / 416E, 14L / 99L, 14L / 99L / 254G , 14L / 99L / 254G / 416E, 14L / 99L / 254G / 416E / 455R, 14L / 99L / 416E, 14L / 99S / 416E, 14L / 167E, 14L / 254G, 14L / 455R, 91V , 91V / 102R / 194P, 91V / 118V, 91V / 118V / 194P, 91V / 118V / 194P / 286L, 91V / 118V / 194P / 418D, 91V / 118V / 286L, 91V / 194P / 254T / 286L, 91V / 286L, and 118V / 286L / 418D, wherein these positions are numbered with reference to SEQ ID NO:678.In some additional embodiments, the engineered glycosyltransferase polypeptide sequence is selected from the group consisting of S5L / V14L / E99L, S5L / V14L / D416E, S5N, S5N / Q91V, S5N / Q91V / K102R / A118V, S5N / Q91V / K102R / S254T / E418D, S5N / Q91V / S106I / S254T / V286L, S5N / Q91V / A118S / S194P / S254T / E418D, S5N / Q91V / A118S / S254T / V286L / E418D, S5N / Q91V / A118S / V286L, S5N / Q91V / A118V / S194P / S254T / V286L, S5N / Q91V / A118 V / S194P / E418D, S5N / Q91V / S194P / S254T, S5N / Q91V / S194P / E418D, S5N / Q91V / S254T, S5N / Q91V / S254T / V286L, S5N / K102R / E418D, S5N / A118S / S194P / V286L / E418D, S5N / A118S / V286L, S5N / A118V / S254T, S5N / S194P , S5N / S254T / V286L, S5N / E418D, T7L / V14L, T7L / V14L / I65V / E99L, T7L / V14L / E99S / D416E, V14L, V14L / I65V / E99L / D416E, V14L / E99L, V14L / E 99L / S254G, V14L / E99L / S254G / D416E, V14L / E99L / S254G / D416E / S455R , V14L / E99L / D416E, V14L / E99S / D416E, V14L / D167E, V14L / S254G, V14L / S455R, Q91V, Q91V / K102R / S194P, Q91V / A118V, Q91V / A118V / S194P, Q91V / A118V / S194P / V286L, Q91V / A118V / S194P / E418D, Q91V / A118V / V286L, Q91V / S194P / S254T / V286L, Q91V / V286L, and A118V / V286L / E418D, wherein these positions are numbered with reference to SEQ ID NO:678.In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations at one or more positions selected from 5 / 91 / 106 / 254 / 286, 5 / 91 / 118 / 286, 5 / 91 / 254 / 286, 14, 14 / 65 / 99 / 416, 14 / 99, and 14 / 99 / 254, where these positions are numbered with reference to SEQ ID NO:678. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations selected from 5N / 91V / 106I / 254T / 286L, 5N / 91V / 118S / 286L, 5N / 91V / 254T / 286L, 14L, 14L / 65V / 99L / 416E, 14L / 99L, and 14L / 99L / 254G, wherein these positions are numbered with reference to SEQ ID NO:678. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations selected from S5N / Q91V / S106I / S254T / V286L, S5N / Q91V / A118S / V286L, S5N / Q91V / S254T / V286L, V14L, V14L / I65V / E99L / D416E, V14L / E99L, and V14L / E99L / S254G, wherein these positions are numbered with reference to SEQ ID NO:678.

[0018] The present invention also provides engineered glycosyltransferases comprising a polypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:12. In some embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations at one or more positions selected from 16, 16 / 127 / 169, 16 / 134, 16 / 143 / 423, 16 / 169, 16 / 169 / 398 / 399, 16 / 169 / 423, 16 / 398, 16 / 398 / 399, 16 / 398 / 427, 16 / 423, 16 / 423 / 427, 134, 143, 399 / 423, and 423 / 427, where these positions are numbered with reference to SEQ ID NO:24. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations selected from 16T, 16T / 127V / 169E, 16T / 134S, 16T / 143G / 423L, 16T / 169E, 16T / 169E / 398M / 399K, 16T / 169E / 423L, 16T / 398M, 16T / 398M / 399K, 16T / 398M / 427S, 16T / 423L, 16T / 423L / 427S, 134S, 143G, 399K / 423L, and 423L / 427S, wherein these positions are numbered with reference to SEQ ID NO:24. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence is L16T, L16T / Q127V / P169E, L16T / A134S, L16T / P143G / R423L, L16T / P169E, L16T / P169E / A398M / Q399K, L16T / P169E / R423L, L16T / A398M, L16 and at least one mutation or set of mutations selected from T / A398M / Q399K, L16T / A398M / R427S, L16T / R423L, L16T / R423L / R427S, A134S, P143G, Q399K / R423L, and R423L / R427S, where these positions are numbered with reference to SEQ ID NO:24.In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations at one or more positions selected from 3, 8, 11, 41, 44, 56, 60, 122, 135, 137, 138, 139, 158, 164, 176, 221, 232, 233, 235, 248, 249, 281, 284, 285, 301, 322, 372, 392, 400, 421, 426, 427, 433, 440, 443, and 446, where these positions are numbered with reference to SEQ ID NO:24. In some additional embodiments, the polypeptide sequence of the engineered glycosyltransferase is 3I, 3V, 8R, 11L, 11P, 41M, 44E, 56A, 60T, 122C, 135V, 137T, 138K, 138R, 139T, 158H, 158V, 164A, 164E, 164N, 164Q, 164S, 176S, 221D, 232A, 233S, 233T, 235L, 235 M, 248M, 249R, 281S, 284V, 285A, 301G, 322R, 372S, 392V, 400C, 421A, 421G, 426S, 427A, 427W, 433G, 433T, 440L, 440M, 443G, 443H, and 446L, wherein these positions are numbered with reference to SEQ ID NO:24. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence is selected from the group consisting of H3I, H3V, V8R, Q11L, Q11P, F41M, Y44E, K56A, P60T, L122C, L135V, Q137T, G138K, G138R, I139T, F158H, F158V, H164A, H164E, H164N, H164Q, H164S, R176S, E221D, P232A, P233S, P233T, Q235L, Q236R, Q237R, Q238R, Q238R, Q239T, H240A, H240B, H240C, H240D, H240E, H240N, H240Q, H240S, R176S, E221D, P232A, P233S, P233T, Q235L, Q236R, Q236S ... 235M, I248M, D249R, G281S, L284V, S285A, Q301G, L322R, A372S, I392V, V400C, S421A, S421G, A426S, R427A, R427W, N433G, N433T, I440L, I440M, C443G, C443H, and R446L, wherein these positions are numbered with reference to SEQ ID NO:24.In some further embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations at one or more positions selected from 16, 16 / 127 / 169, and 16 / 169, where these positions are numbered with reference to SEQ ID NO: 24. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations selected from 16T, 16T / 127V / 169E, and 16T / 169E, where these positions are numbered with reference to SEQ ID NO: 24. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations selected from L16T, L16T / Q127V / P169E, and L16T / P169E, where these positions are numbered with reference to SEQ ID NO: 24.

[0019] In some further embodiments, the engineered glycosyltransferase polypeptide sequence is selected from the group consisting of 3 / 22 / 198 / 421, 3 / 22 / 421, 3 / 201 / 248 / 322, 3 / 201 / 248 / 322 / 392, 13 / 233 / 331 / 440, 13 / 440 / 443, 22 / 32 / 56 / 60 / 198 / 248 / 322 / 392 / 421, 22 / 56, 22 / 56 / 137 / 198 / 201 / 248 / 322 / 392, 22 / 56 / 137 / 198 / 248 / 392 / 421, 22 / 56 / 137 / 322, 22 / 56 / 137 / 322 / 392 / 423, 22 / 56 / 198 / 202 / 248, 22 / 56 / 198 / 248 / 322 / 392 / 421, 22 / 56 / 198 / 248 / 421 / 423, 22 / 56 / 421 / 423, 22 / 60 / 392 / 421, 22 / 137, 22 / 137 / 198 / 202 / 248, 22 / 198 / 202 / 248 / 392, 22 / 248 / 392, 22 / 392, 22 / 421, 22 / 421 / 423, 22 / 423, 44 / 164 / 233, 44 / 164 / 233 / 329 / 331, 44 / 331, 125, 125 / 233 / 443 / 446, 164 / 221 / 233 / 331 / 440 / 446, 164 / 233 / and at least one mutation or set of mutations at one or more positions selected from 331 / 440, 164 / 233 / 446, 164 / 331, 164 / 440, 164 / 443, 198 / 202 / 392, 221 / 329 / 331, 233, 233 / 446, 329, 421 / 423, and 443 / 446, which positions are numbered with reference to SEQ ID NO:858. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence is 3I / 22G / 198Q / 421G, 3I / 22G / 421G, 3I / 201A / 248M / 322R, 3I / 201A / 248M / 322R / 392V, 13Q / 233S / 331V / 440M, 13Q / 440M / 4 43G, 22G / 32L / 56A / 60T / 198Q / 248M / 322R / 392V / 421G, 22G / 56A, 22G / 56A / 137T / 198Q / 201A / 248M / 322R / 392V, 22G / 56A / 137T / 198Q / 248M / 392V / 421G, 22G / 56A / 137T / 322R,22G / 56A / 137T / 322R / 392V / 423L, 22G / 56A / 198Q / 202Y / 248M, 22G / 56A / 198Q / 248M / 32 2R / 392V / 421G, 22G / 56A / 198Q / 248M / 421G / 423L, 22G / 56A / 421G / 423L, 22G / 60T / 392V / 421G, 22G / 137T, 22G / 137T / 198Q / 202Y / 248M, 22G / 198Q / 202Y / 248M / 392V, 22G / 248M / 3 92V, 22G / 392V, 22G / 421G, 22G / 421G / 423L, 22G / 423L, 44E / 164A / 233S, 44E / 164E / 233S , 44E / 164E / 233S / 329L / 331V, 44E / 331V, 125L, 125L / 233S / 443G / 446L, 164A / 233S / 446 L, 164A / 331V, 164A / 440M, 164A / 443G, 164E / 221D / 233S / 331V / 440M / 446L, 164E / 233S / The amino acid sequence includes at least one mutation or set of mutations selected from 331V / 440M, 198Q / 202Y / 392V, 221D / 329L / 331V, 233S, 233S / 446L, 329L, 421G / 423L, and 443G / 446L, wherein these positions are numbered with reference to SEQ ID NO:858. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence is selected from the group consisting of H3I / A22G / G198Q / S421G, H3I / A22G / S421G, H3I / G201A / I248M / L322R, H3I / G201A / I248M / L322R / I392V, L13Q / P233S / I331V / I440M, L13Q / I440M / C443G, A22G / I32L / K56A / P60T / G198Q / I248 M / L322R / I392V / S421G, A22G / K56A, A22G / K56A / Q137T / G198Q / G201A / I248M / L322R / I392V, A22G / K56A / Q137T / G198Q / I2 48M / I392V / S421G, A22G / K56A / Q137T / L322R, A22G / K56A / Q137T / L322R / I392V / R423L, A22G / K56A / G198Q / I202Y / I248M,A22G / K56A / G198Q / I248M / L322R / I392V / S421G, A22G / K56A / G198Q / I248M / S421G / R423L, A22 G / K56A / S421G / R423L, A22G / P60T / I392V / S421G, A22G / Q137T, A22G / Q137T / G198Q / I202Y / I24 8M, A22G / G198Q / I202Y / I248M / I392V, A22G / I248M / I392V, A22G / I392V, A22G / S421G, A22G / S 421G / R423L, A22G / R423L, Y44E / H164A / P233S, Y44E / H164E / P233S, Y44E / H164E / P233S / A329L / I331V, Y44E / I331V, F125L, F125L / P233S / C443G / R446L, H164A / P233S / R446L, H164A / I331V , H164A / I440M, H164A / C443G, H164E / E221D / P233S / I331V / I440M / R446L, H164E / P233S / I331V / I440M, G198Q / I202Y / I392V, E221D / A329L / I331V, P233S, P233S / R446L, A329L, S421G / R423L, and C443G / R446L, and these positions are numbered with reference to SEQ ID NO:858. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations at one or more positions selected from 3 / 22 / 198 / 421, 13 / 440 / 443, 22 / 56 / 198 / 202 / 248, 22 / 137, 22 / 198 / 202 / 248 / 392, 164 / 221 / 233 / 331 / 440 / 446, 164 / 233 / 331 / 440, and 233 / 446, where these positions are numbered with reference to SEQ ID NO:858. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence is 3I / 22G / 198Q / 421G, 13Q / 440M / 443G, 22G / 56A / 198Q / 202Y / 248M, 22G / 137T, 22G / 198Q / 202Y / 248M / 392V,and at least one mutation or set of mutations selected from 164E / 221D / 233S / 331V / 440M / 446L, 164E / 233S / 331V / 440M, and 233S / 446L, wherein these positions are numbered with reference to SEQ ID NO:858. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations selected from H3I / A22G / G198Q / S421G, L13Q / I440M / C443G, A22G / K56A / G198Q / I202Y / I248M, A22G / Q137T, A22G / G198Q / I202Y / I248M / I392V, H164E / E221D / P233S / I331V / I440M / R446L, H164E / P233S / I331V / I440M, and P233S / R446L, wherein these positions are numbered with reference to SEQ ID NO:858.

[0020] In some further embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations at one or more positions selected from 83 / 202 / 233, 164, 164 / 202, 164 / 202 / 233 / 331, 164 / 202 / 248 / 272, 164 / 202 / 331, 164 / 202 / 331 / 423, 164 / 423, 202 / 233, 202 / 233 / 248, 202 / 233 / 248 / 423, 202 / 248, 202 / 331, 202 / 421 / 423, 202 / 423, 202 / 446, 233, 248, and 423, wherein these positions are numbered with reference to SEQ ID NO:994. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence is 83Y / 202I / 233S, 164E, 164E / 202I, 164E / 202I / 233S / 331V, 164E / 202I / 248I / 272C, 164E / 202I / 331V, 164E / 202I / 331V / 423L, 164E / 423L, 202I / 233S ... and at least one mutation or set of mutations selected from 02I / 233S / 248I, 202I / 233S / 248I / 423L, 202I / 248I, 202I / 331V, 202I / 421G / 423L, 202I / 423L, 202I / 446L, 233S, 248I, and 423L, wherein these positions are numbered with reference to SEQ ID NO:994.In some additional embodiments, the engineered glycosyltransferase polypeptide sequence is selected from the group consisting of H83Y / Y202I / P233S, H164E, H164E / Y202I, H164E / Y202I / P233S / I331V, H164E / Y202I / M248I / R272C, H164E / Y202I / I331V, H164E / Y202I / I331V / R423L, H164E / R423L, Y202I / P233S , Y202I / P233S / M248I, Y202I / P233S / M248I / R423L, Y202I / M248I, Y202I / I331V, Y202I / S421G / R423L, Y202I / R423L, Y202I / R446L, P233S, M248I, and R423L, wherein these positions are numbered with reference to SEQ ID NO:994. In some embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations at one or more positions selected from 7, 9, 10, 54, 73, 84, 106, 115, 116, 132, 165, 286, 309, 389, 406, 422 and 438, where these positions are numbered with reference to SEQ ID NO:994. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations selected from 7T, 9L, 10P, 54L, 54M, 73R, 73S, 84L, 106S, 115A, 116I, 132R, 165P, 286G, 309E, 389E, 406H, 406M, 406Q, 422A, 422T, 422V, 438A, and 438T, wherein these positions are numbered with reference to SEQ ID NO:994.In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations selected from G7T, S9L, D10P, S54L, S54M, A73R, A73S, Y84L, A106S, N115A, L116I, K132R, E165P, N286G, K309E, D389E, S406H, S406M, S406Q, K422A, K422T, K422V, E438A, and E438T, wherein these positions are numbered with reference to SEQ ID NO:994. In some further embodiments, the polypeptide sequence of the engineered glycosyltransferase comprises at least one mutation or set of mutations at one or more positions selected from 83 / 202 / 233, 164, 164 / 202, 164 / 423, 202 / 233 / 248, and 233, where these positions are numbered with reference to SEQ ID NO: 994. In some additional embodiments, the polypeptide sequence of the engineered glycosyltransferase comprises at least one mutation or set of mutations selected from 83Y / 202I / 233S, 164E, 164E / 202I, 164E / 423L, 202I / 233S / 248I, and 233S, where these positions are numbered with reference to SEQ ID NO: 994. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations selected from H83Y / Y202I / P233S, H164E, H164E / Y202I, H164E / R423L, Y202I / P233S / M248I, and P233S, wherein these positions are numbered with reference to SEQ ID NO:994.

[0021] In yet some further embodiments, the engineered glycosyltransferase polypeptide sequence is selected from the group consisting of 7 / 9 / 73 / 165 / 286, 7 / 9 / 165 / 286, 7 / 9 / 422, 7 / 116 / 165 / 286, 7 / 165, 9 / 73 / 116 / 165 / 286 / 422, 9 / 286 / 389, 54, 54 / 84, 54 / 406, 73, 73 / 116 / 165 / 286 / 389, 73 / 116 / 286, 73 / 116 / 286 / 422, 73 / 165 / 28 and 422, wherein the positions are numbered with reference to SEQ ID NO: 1080. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence is selected from the group consisting of 7T / 9L / 73R / 165P / 286G, 7T / 9L / 165P / 286G, 7T / 9L / 422A, 7T / 116I / 165P / 286G, 7T / 165P, 9L / 73R / 116I / 165P / 286G / 422A, 9L / 286G / 389E, 54L / 84L, 54M, 54M / 406M, 73R, 73R / 116I / 165P / 286G / 389E, 73R / 116I / 286G, 73R / 116I / 286G / 422A, 73R / 165P 165P / 286G, 165P / 389E, 165P / 389E / 422T, 286G, 286G / 422A, 389E / 422A, and 422T, wherein these positions are numbered with reference to SEQ ID NO: 1080.In some additional embodiments, the engineered glycosyltransferase polypeptide sequence is G7T / S9L / A73R / E165P / N286G, G7T / S9L / E165P / N286G, G7T / S9L / K422A, G7T / L116I / E165P / N286G, G7T / E165P, S9L / A73R / L 116I / E165P / N286G / K422A, S9L / N286G / D389E, S54L / Y84L, S54M, S54M / S406M, A73R, A7 3R / L116I / E165P / N286G / D389E, A73R / L116I / N286G, A73R / L116I / N286G / K422A, A73R / E165P / N286G, A73R / N286G / D389E, A73R / N286G / K422A, A73R / N286G / K422T, A73R / K422 T, Y84L, N115A, L116I, L116I / E165P, L116I / E165P / N286G / K422A, L116I / D389E, E165P , E165P / N286G, E165P / D389E, E165P / D389E / K422T, N286G, N286G / K422A, D389E / K422A, and K422T, where these positions are numbered with reference to SEQ ID NO: 1080. In some further embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations at one or more positions selected from 185 / 190, 219, 220, 255, 257, 302, 385, 395, 395 / 437, 399, 401, 409, 412, 416, 434, 441, 445, 447, and 449, where these positions are numbered with reference to SEQ ID NO: 1080.In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations selected from 185L / 190P, 219L, 220A, 255R, 257H, 302A, 302H, 302L, 302R, 302T, 385M, 385P, 385S, 395A, 395D, 395H, 395N, 395S / 437L, 399R, 401V, 409L, 412K, 416N, 434I, 441A, 441K, 441L, 441R, 441S, 445K, 445R, 447A, 447R, 447S, 447V, 449A, 449H, and 449T, wherein these positions are numbered with reference to SEQ ID NO: 1080. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence is selected from the group consisting of F185L / T190P, C219L, T220A, P255R, N257H, N302A, N302H, N302L, N302R, N302T, V385M, V385P, V385S, G395A, G395D, G395H, G395N, G395S / E437L, Q399R, L401V ... , T409L, I412K, K416N, A434I, Q441A, Q441K, Q441L, Q441R, Q441S, N445K, N445R, N447A, N447R, N447S, N447V, Y449A, Y449H, and Y449T, and these positions are numbered with reference to SEQ ID NO: 1080. In some further embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations at one or more positions selected from 9 / 73 / 116 / 165 / 286 / 422, 54, 54 / 84, 54 / 406, 73 / 116 / 286 / 422, 116 / 165 / 286 / 422, and 286 / 422, where these positions are numbered with reference to SEQ ID NO: 1080.In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations selected from 9L / 73R / 116I / 165P / 286G / 422A, 54L / 84L, 54M, 54M / 406M, 73R / 116I / 286G / 422A, 116I / 165P / 286G / 422A, and 286G / 422A, wherein these positions are numbered with reference to SEQ ID NO: 1080. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations selected from S9L / A73R / L116I / E165P / N286G / K422A, S54L / Y84L, S54M, S54M / S406M, A73R / L116I / N286G / K422A, L116I / E165P / N286G / K422A, and N286G / K422A, wherein these positions are numbered with reference to SEQ ID NO: 1080.

[0022] In yet some further embodiments, the polypeptide sequence of the engineered glycosyltransferase is selected from the group consisting of 54, 54 / 185 / 190, 54 / 185 / 190 / 219 / 257 / 302 / 385 / 389 / 395 / 445 / 447 / 449, 54 / 185 / 190 / 219 / 385, 54 / 185 / 190 / 219 / 385 / 389 / 395 / 399 / 441 / 445 / 447 / 449, 54 / 185 / 190 / 219 / 385 / 395 / 399 / 441 / 445 / 447 / 449, 54 / 185 / 190 / 219 / 385 / 445 / 447, 54 / 185 / 190 / 219 / 385 / 445 / 447, 5 / 190 / 219 / 389 / 395 / 399 / 441 / 445 / 447 / 449, 54 / 185 / 190 / 302, 54 / 185 / 190 / 302 / 395 / 399, 54 / 185 / 190 / 385 / 447 / 449, 54 / 185 / 190 / 389 / 395, 54 / 185 / 190 / 389 / 441 / 445 / 447 / 449, 54 / 185 / 190 / 389 / 445, 54 / 185 / 190 / 395 / 399 / 441 / 445 / 447 / 449, 54 / 185 / 219 / 389, 54 / 185 / 257 / 389 / 441 / 445 / 447 / 449, 5 4 / 185 / 302 / 385 / 399 / 445 / 447, 54 / 185 / 385 / 389 / 395 / 441 / 445 / 449, 54 / 185 / 385 / 395 / 399 / 445 / 447 / 449, 54 / 185 / 389 / 395 / 445 / 447, 54 / 190 / 219 / 257 / 302, 54 / 190 / 219 / 257 / 395 / 445 / 447 / 449, 54 / 190 / 257, 54 / 190 / 257 / 302 / 399, 54 / 190 / 257 / 309 / 385 / 389 / 395 / 399 / 445, 54 / 190 / 257 / 385 / 389, 54 / 19 0 / 257 / 395 / 445 / 449, 54 / 190 / 302 / 389, 54 / 190 / 302 / 389 / 395 / 399 / 445 / 447, 54 / 190 / 385 / 389 / 395 / 441 / 445, 54 / 190 / 385 / 395, 54 / 190 / 385 / 445 / 447 / 449, 54 / 190 / 395, 54 / 190 / 395 / 445 / 447, 54 / 219 / 302 / 395 / 441 / 445 / 447, 54 / 219 / 385 / 389 / 395 / 441 / 445 / 447, 54 / 257, 54 / 257 / 385 / 449, 54 / 257 / 389,54 / 257 / 399、54 / 257 / 441 / 447、54 / 257 / 441 / 449、54 / 302 / 385 / 399 / 441 / 445 / 447、54 / 302 / 385 / 399 / 441 / 445 / 449、54 / 385、54 / 385 / 389 / 441 / 445 / 447 / 449、54 / 389 / 395 / 399 / 447 / 449、185 / 190 / 219 / 257 / 389 / 395 / 445 / 447、185 / 190 / 257 / 302 / 395 / 441 / 445 / 447、185 / 190 / 257 / 385 / 389 / 399、185 / 190 / 257 / 385 / 399 / 445 / 447、185 / 190 / 257 / 389 / 395、185 / 190 / 257 / 389 / 395 / 399 / 441 / 447 / 449、185 / 190 / 385 / 389 / 441 / 445 / 447、185 / 190 / 389 / 447 / 449、185 / 190 / 395 / 399、185 / 219 / 257 / 399 / 445 / 447、185 / 257 / 385 / 395 / 399 / 445 / 447、185 / 302 / 395 / 399 / 441 / 445 / 447 / 449、185 / 395 / 399 / 441 / 445 / 447、190 / 219 / 257 / 385 / 389 / 441 / 445 / 447 / 449、190 / 219 / 302 / 385 / 399 / 445、190 / 257 / 302 / 385 / 399、190 / 257 / 385 / 389 / 399、190 / 257 / 385 / 389 / 445 / 447、190 / 257 / 385 / 395、190 / 257 / 385 / 441 / 445 / 447 / 449、190 / 257 / 389 / 395 / 441 / 445 / 447、190 / 257 / 399 / 447 / 449、190 / 302 / 385 / 389 / 395 / 399 / 441 / 445 / 447、190 / 389、219 / 257 / 385 / 389 / 395 / 441 / 447 / 449、219 / 257 / 395、219 / 385 / 389 / 399 / 445 / 449、219 / 395、257 / 385 / 389 / 399、257 / 389 / 395 / 399 / 445 / 447、257 / 389 / 395 / 399 / 445 / 449、257 / 389 / 399、302 / 389 / 395 / 445、385 / 389、385 / 395、389 / 395 / 445 / 447、395 / 399、and 399, wherein these positions are numbered with reference to SEQ ID NO: 1216.

[0023] In some additional embodiments, the engineered glycosyltransferase polypeptide sequence is 54M, 54M / 185L / 190P, 54M / 185L / 190P / 219L / 257H / 302R / 385P / 389E / 395H / 445K / 447A / 449H, 54M / 185L / 190P / 219L / 385P / 445R / 447A, 54M / 185L / 190P / 219L / 385S, 54M / 185L / 190P / 219L / 385S / 389E / 395D / 399R / 441R / 445R / 447A / 449H, 54M / 185L / 190P / 219L / 385S / 389E / 395D / 399R / 441R / 445R / 447A / 449H, 90P / 219L / 385S / 395A / 399R / 441R / 445K / 447A / 449H, 54M / 185L / 190P / 219 L / 389E / 395H / 399R / 441S / 445K / 447A / 449H, 54M / 185L / 190P / 302H, 54M / 18 5L / 190P / 302R / 395H / 399R, 54M / 185L / 190P / 385P / 447A / 449H, 54M / 185L / 1 90P / 389E / 395H, 54M / 185L / 190P / 389E / 441R / 445R / 447A / 449H, 54M / 185L / 190P / 389E / 445K, 54M / 185L / 190P / 395D / 399R / 441R / 445K / 447A / 449H, 54 M / 185L / 219L / 389E, 54M / 185L / 257H / 389E / 441S / 445R / 447A / 449H, 54M / 18 5L / 302H / 385S / 399R / 445R / 447A, 54M / 185L / 385S / 389E / 395D / 441R / 445K / 449H, 54M / 185L / 385S / 395D / 399R / 445R / 447A / 449H, 54M / 185L / 389E / 395D / 445K / 447A, 54M / 190P / 219L / 257H / 302H, 54M / 190P / 219L / 257H / 395H / 445 R / 447A / 449H, 54M / 190P / 257H, 54M / 190P / 257H / 302H / 399R, 54M / 190P / 257 H / 309N / 385S / 389E / 395D / 399R / 445K, 54M / 190P / 257H / 385S / 389E, 54M / 19 0P / 257H / 395D / 445K / 449H, 54M / 190P / 302H / 389E / 395D / 399R / 445K / 447S,<h2 style=";text-align:left;direction:ltr">54M / 190P / 302R / 389E、54M / 190P / 385P / 395D、54M / 190P / 385P / 445R / 447A / 449H、54M / 190P / 385S / 389E / 395H / 441S / 445R、54M / 190P / 395A、54M / 190P / 395H / 445K / 447A、54M / 219L / 302R / 395H / 441S / 445R / 447A、54M / 219L / 385P / 389E / 395D / 441R / 445K / 447A、54M / 257H、54M / 257H / 385S / 449H、54M / 257H / 389E, 54M / 257H / 399R, 54M / 257H / 441R / 447A, 54M / 257H / 441R / 449H, 54M / 302H / 385P / 399R / 441S / 445K / 447S, 54M / 302H / 385S / 399R / 441S / 445R / 449H 54M / 385P, 54M / 385P / 389E / 441R / 445K / 447A / 449H, 54M / 389E / 395D / 399R / 447A / 449H, 185L / 190P / 219L / 257H / 389E / 395A / 445K / 447A, 185L / 190P / 25 7H / 302R / 395D / 441S / 445R / 447S、185L / 190P / 257H / 385P / 389E / 399R、185L / 190P / 257H / 385P / 399R / 445R / 447A、185L / 190P / 257H / 389E / 395A、185L / 1 90P / 257H / 389E / 395H / 399R / 441S / 447S / 449H、185L / 190P / 385S / 389E / 441 R / 445K / 447S、185L / 190P / 389E / 447S / 449H、185L / 190P / 395D / 399R、185L / 1 90P / 395H / 399R、185L / 219L / 257H / 399R / 445R / 447A、185L / 257H / 385S / 395A / 399R / 445R / 447A、185L / 302R / 395H / 399R / 441R / 445K / 447S / 449H、185L / 395D / 399R / 441R / 445R / 447A、190P / 219L / 257H / 385S / 389E / 441S / 445R / 447A / 449H、190P / 219L / 302H / 385P / 399R / 445R、190P / 257H / 302H / 385P / 399R、190P / 257H / 385P / 389E / 399R, 190P / 257H / 385S / 389E / 445K / 447A, 190P / 257H / 385S / 395D, 190P / 257H / 385S / 441S / 445R / 447S / 449H, 190P / 257H / 389E / 395H / 441R / 445 R / 447A, 190P / 257H / 399R / 447A / 449H, 190P / 302H / 385P / 389E / 395D / 399R / 441S / 44 5K / 447S, 190P / 389E, 219L / 257H / 385S / 389E / 395D / 441R / 447A / 449H, 219L / 257H / 39 5H, 219L / 385S / 389E / 399R / 445R / 449H, 219L / 395H, 257H / 385P / 389E / 399R, 257H / 389E / 395D / 399R / 445K / 447A, 257H / 389E / 395D / 399R / 445K / 449H, 257H / 389E / 399R, 302H / 389E / 395H / 445R, 385S / 389E, 385S / 395D, 389E / 395D / 445R / 447S, 395D / 399R, and 399R, wherein these positions are numbered with reference to SEQ ID NO: 1216.

[0024] In some additional embodiments, the engineered glycosyltransferase polypeptide sequence is S54M, S54M / F185L / T190P, S54M / F185L / T190P / C219L / N257H / N302R / V385P / D389E / G395H / N445K / N447A / Y449H, S54M / F185L / T190P / C219L / V385P / N445R / N447A, S54M / F185L / T190P / C219L / V385S, S54M / F185L / T190P / C219L / V385S / D389E / G395D / Q399R / Q441R / N445R / N447A / Y449H, S54M / F185L / T190P / C219L / V385S / G39 5A / Q399R / Q441R / N445K / N447A / Y449H, S54M / F185L / T190P / C219L / D389E / G395H / Q399R / Q441S / N445K / N447A / Y449H, S54M / F185L / T190P / N302H, S54 M / F185L / T190P / N302R / G395H / Q399R, S54M / F185L / T190P / V385P / N447A / Y 449H, S54M / F185L / T190P / D389E / G395H, S54M / F185L / T190P / D389E / Q441R / N445R / N447A / Y449H, S54M / F185L / T190P / D389E / N445K, S54M / F185L / T19 0P / G395D / Q399R / Q441R / N445K / N447A / Y449H, S54M / F185L / C219L / D389E, S54M / F185L / N257H / D389E / Q441S / N445R / N447A / Y449H, S54M / F185L / N302 H / V385S / Q399R / N445R / N447A, S54M / F185L / V385S / D389E / G395D / Q441R / N 445K / Y449H, S54M / F185L / V385S / G395D / Q399R / N445R / N447A / Y449H, S54M / F185L / D389E / G395D / N445K / N447A, S54M / T190P / C219L / N257H / N302H, S5 4M / T190P / C219L / N257H / G395H / N445R / N447A / Y449H, S54M / T190P / N257H,<h2 style=";text-align:left;direction:ltr">S54M / T190P / N257H / N302H / Q399R、S54M / T190P / N257H / K309N / V385S / D38 9E / G395D / Q399R / N445K、S54M / T190P / N257H / V385S / D389E、S54M / T190P / N 257H / G395D / N445K / Y449H、S54M / T190P / N302H / D389E / G395D / Q399R / N445K / N447S、S54M / T190P / N302R / D389E、S54M / T190P / V385P / G395D、S54M / T19 0P / V385P / N445R / N447A / Y449H、S54M / T190P / V385S / D389E / G395H / Q441S / N445R、S54M / T190P / G395A、S54M / T190P / G395H / N445K / N447A、S54M / C219L / N302R / G395H / Q441S / N445R / N447A、S54M / C219L / V385P / D389E / G395D / Q441R / N445K / N447A、S54M / N257H、S54M / N257H / V385S / Y449H、S54M / N257H / D 389E, S54M / N257H / Q399R, S54M / N257H / Q441R / N447A, S54M / N257H / Q441R / Y449H, S54M / N302H / V385P / Q399R / Q441S / N445K / N447S, S54M / N302H / V38 5S / Q399R / Q441S / N445R / Y449H、S54M / V385P、S54M / V385P / D389E / Q441R / N 445K / N447A / Y449H、S54M / D389E / G395D / Q399R / N447A / Y449H、F185L / T190 P / C219L / N257H / D389E / G395A / N445K / N447A、F185L / T190P / N257H / N302R / G395D / Q441S / N445R / N447S、F185L / T190P / N257H / V385P / D389E / Q399R、F1 85L / T190P / N257H / V385P / Q399R / N445R / N447A、F185L / T190P / N257H / D389E / G395A、F185L / T190P / N257H / D389E / G395H / Q399R / Q441S / N447S / Y449H、<h2 style=";text-align:left;direction:ltr">F185L / T190P / V385S / D389E / Q441R / N445K / N447S、F185L / T190P / D389E / N 447S / Y449H、F185L / T190P / G395D / Q399R、F185L / T190P / G395H / Q399R、F18 5L / C219L / N257H / Q399R / N445R / N447A、F185L / N257H / V385S / G395A / Q399R / N445R / N447A、F185L / N302R / G395H / Q399R / Q441R / N445K / N447S / Y449H、 F185L / G395D / Q399R / Q441R / N445R / N447A、T190P / C219L / N257H / V385S / D 389E / Q441S / N445R / N447A / Y449H、T190P / C219L / N302H / V385P / Q399R / N44 5R、T190P / N257H / N302H / V385P / Q399R、T190P / N257H / V385P / D389E / Q399R、T190P / N257H / V385S / D389E / N445K / N447A、T190P / N257H / V385S / G395D、 T190P / N257H / V385S / Q441S / N445R / N447S / Y449H、T190P / N257H / D389E / G 395H / Q441R / N445R / N447A、T190P / N257H / Q399R / N447A / Y449H、T190P / N30 2H / V385P / D389E / G395D / Q399R / Q441S / N445K / N447S、T190P / D389E、C219L / N257H / V385S / D389E / G395D / Q441R / N447A / Y449H、C219L / N257H / G395H、 C219L / V385S / D389E / Q399R / N445R / Y449H, C219L / G395H, N257H / V385P / D389E / Q399R, N257H / D389E / G395D / Q399R / N445K / N447A, N257H / D389E / G39 5D / Q399R / N445K / Y449H、N257H / D389E / Q399R、N302H / D389E / G395H / N445R、V385S / D389E、V385S / G395D、D389E / G395D / N445R / N447S、G395D / Q399R、and Q399R, where these positions are numbered with reference to SEQ ID NO: 1216. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations at one or more positions selected from 5, 14, 96, 108, 157, 181, 188, 278, 293 and 341, where these positions are numbered with reference to SEQ ID NO: 1216. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations selected from 5S, 14T, 96K, 96P, 108E, 157Q, 181L, 188L, 278L, 293V and 341L.

[0025] In some additional embodiments, the polypeptide sequence of the engineered glycosyltransferase comprises at least one mutation or set of mutations selected from H5S, R14T, G96K, G96P, N108E, N157Q, M181L, E188L, V278L, A293V and I341L, wherein these positions are numbered with reference to SEQ ID NO: 1216. In some further embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations at one or more positions selected from 54 / 185 / 190 / 219 / 257 / 302 / 385 / 389 / 395 / 445 / 447 / 449, 54 / 185 / 190 / 385 / 447 / 449, 54 / 190 / 257 / 302 / 385 / 395 / 399, 54 / 190 / 302 / 389 / 395 / 399 / 445 / 447, 54 / 257 / 385 / 449, 54 / 257 / 389, and 54 / 389 / 395 / 399 / 447 / 449, where these positions are numbered with reference to SEQ ID NO: 1216. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence is 54M / 185L / 190P / 219L / 257H / 302R / 385P / 389E / 395H / 445K / 447A / 449H, 54M / 185L / 190P / 385P / 447A / 449H, 54M / 190P / 257H / 302R / 385P / 395A / 399R, and at least one mutation or set of mutations selected from 54M / 190P / 302H / 389E / 395D / 399R / 445K / 447S, 54M / 257H / 385S / 449H, 54M / 257H / 389E, and 54M / 389E / 395D / 399R / 447A / 449H, wherein these positions are numbered with reference to SEQ ID NO:1216.In some additional embodiments, the engineered glycosyltransferase polypeptide sequence is S54M / F185L / T190P / C219L / N257H / N302R / V385P / D389E / G395H / N445K / N447A / Y449H, S54M / F185L / T190P / V385P / N447A / Y449H, S54M / T190P / N257H / N302R / V385P / G395A / Q399 R, S54M / T190P / N302H / D389E / G395D / Q399R / N445K / N447S, S54M / N257H / V385S / Y449H, S54M / N257H / D389E, and S54M / D389E / G395D / Q399R / N447A / Y449H, wherein these positions are numbered with reference to SEQ ID NO: 1216.

[0026] In some further embodiments, the engineered glycosyltransferase polypeptide sequence is selected from the group consisting of 14 / 42 / 51 / 108 / 181, 14 / 42 / 51 / 157 / 341, 14 / 42 / 341, 14 / 51 / 96 / 157, 14 / 51 / 96 / 157 / 341, 14 / 51 / 96 / 341, 14 / 51 / 108, 14 / 51 / 341, 14 / 96, 14 / 96 / 108 / 133, 14 / 96 / 108 / 181 / 341, 14 / 96 / 108 / 188, 14 / 96 / 34 and 293, wherein the positions are numbered with reference to SEQ ID NO: 1488. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence is 14T / 42T / 51V / 108E / 181L, 14T / 42T / 51V / 157Q / 341L, 14T / 42T / 341L, 14T / 51V / 96K / 157Q, 14T / 51V / 96P / 157Q / 341L, 14T / 51V / 96P / 341L, 14T / 51V / 108E, 14T / 51V / 341L, 14T / 96K, 14T / 96K / 108E / 133I, 14T / 96K / 108E / 188V, 14T / 96K / 341L, 14T / 96P / 1 and at least one mutation or set of mutations selected from 08E / 181L / 341L, 14T / 108E / 157Q, 14T / 108E / 157Q / 188L, 14T / 157Q / 181L / 278L, 14T / 278L, 42T / 96P / 157Q / 341L, 42T / 188L / 341L, 96K / 188L, 96P / 108E / 181L / 293V, 96P / 108E / 188L, 96P / 108E / 188L / 341L, 181L, 188L, and 293V, wherein these positions are numbered with reference to SEQ ID NO: 1488.In some additional embodiments, the engineered glycosyltransferase polypeptide sequence is R14T / V42T / N51V / N108E / M181L, R14T / V42T / N51V / N157Q / I341L, R14T / V42T / I341L, R14T / N51V / G96K / N157Q, R14T / N51V / G 96P / N157Q / I341L, R14T / N51V / G96P / I341L, R14T / N51V / N108E, R14T / N51V / I341L, R14T / G96K, R14T / G96K / N108E / V133I, R14T / G96K / N108E / E188V, R14T / G96K / I341L, R14T / G9 6P / N108E / M181L / I341L, R14T / N108E / N157Q, R14T / N108E / N157Q / E188L, R14T / N157Q / M181L / V278L, R14T / V278L, V42T / G96P / N157Q / I341L, V42T / E188L / I341L, G96K / E188L, G96P / N108E / M181L / A293V, G96P / N108E / E188L, G96P / N108E / E188L / I341L, M181L, E188L, and A293V, where these positions are numbered with reference to SEQ ID NO: 1488. In some further embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations at one or more positions selected from 122, 144, 147, 187, 196, 197, 198, 199, 201, 268, and 324, where these positions are numbered with reference to SEQ ID NO: 1488. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations selected from 122V, 144V, 147F, 187K, 196M, 196P, 196V, 197V, 198A, 198F, 199G, 199Q, 199S, 201P, 268A, 324K and 324R, wherein these positions are numbered with reference to SEQ ID NO: 1488.In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations selected from L122V, L144V, S147F, T187K, A196M, A196P, A196V, P197V, Q198A, Q198F, N199G, N199Q, N199S, G201P, Y268A, H324K and H324R, where these positions are numbered with reference to SEQ ID NO: 1488. In some further embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations at one or more positions selected from 14 / 42 / 341, 14 / 96, 14 / 96 / 108 / 133, 14 / 96 / 108 / 181 / 341, and 188, where these positions are numbered with reference to SEQ ID NO: 1488. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations selected from 14T / 42T / 341L, 14T / 96K, 14T / 96K / 108E / 133I, 14T / 96P / 108E / 181L / 341L, and 188L, where these positions are numbered with reference to SEQ ID NO: 1488. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations selected from R14T / V42T / I341L, R14T / G96K, R14T / G96K / N108E / V133I, R14T / G96P / N108E / M181L / I341L, and E188L, where these positions are numbered with reference to SEQ ID NO: 1488.

[0027] In yet some additional embodiments, the engineered glycosyltransferase polypeptide sequence is 147 / 188 / 196 / 201, 152 / 187 / 188 / 324, 152 / 188, 152 / 188 / 196 / 198 / 199 / 324, 152 / 188 / 196 / 199, 152 / 188 / 196 / 201 / 324, 152 / 188 / 324, 188, 188 / 196 / 198 / 199 / 201, 188 / 196 / 198 / 199 / 201 / 324, 188 / 196 / 1 and at least one mutation or set of mutations at one or more positions selected from 98 / 201, 188 / 196 / 198 / 324, 188 / 196 / 201, 188 / 198 / 199 / 201 / 324, 188 / 198 / 199 / 268, 188 / 198 / 201 / 324, 188 / 198 / 324, 188 / 199 / 201, 188 / 201, 188 / 201 / 324, and 188 / 324, wherein these positions are numbered with reference to SEQ ID NO:1516. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence is 147F / 188E / 196V / 201P, 152Y / 187K / 188E / 324K, 152Y / 188E, 152Y / 188E / 196V / 198A / 199Q / 324K, 152Y / 188E / 196V / 199Q, 152Y / 188E / 196V / 201P / 324K, 152Y / 188E / 324K, 188E, 188E / 196V / 198A / 199Q / 201P, 188E / 196V / 198A / 324K, 188E / 196V / 198A / 324K, 188E / 196V / 198A / 324K, 188E / 196V / 198 and 188E / 324K, 188E / 196V / 198F / 201P, 188E / 196V / 201P, 188E / 198A / 199Q / 268A, 188E / 198A / 201P / 324K, 188E / 198A / 324K, 188E / 198F / 199Q / 201P / 324K, 188E / 199Q / 201P, 188E / 201P, 188E / 201P / 324K, and 188E / 324K, wherein these positions are numbered with reference to SEQ ID NO: 1516.In some additional embodiments, the engineered glycosyltransferase polypeptide sequence is S147F / L188E / A196V / G201P, F152Y / T187K / L188E / H324K, F152Y / L188E, F152Y / L188E / A196V / Q198A / N199Q / H324K, F152Y / L188E, F152Y / L188E / A196V / Q198A / N199Q / H324K, F152Y / L188E, 2Y / L188E / A196V / N199Q, F152Y / L188E / A196V / G201P / H324K, F152Y / L188E / H324K, L1 88E, L188E / A196V / Q198A / N199Q / G201P, L188E / A196V / Q198A / H324K, L188E / A196V / Q1 98F / N199Q / G201P / H324K, L188E / A196V / Q198F / G201P, L188E / A196V / G201P, L188E / Q 198A / N199Q / Y268A, L188E / Q198A / G201P / H324K, L188E / Q198A / H324K, L188E / Q198F / and at least one mutation or set of mutations selected from N199Q / G201P / H324K, L188E / N199Q / G201P, L188E / G201P, L188E / G201P / H324K, and L188E / H324K, wherein these positions are numbered with reference to SEQ ID NO: 1516. In some further embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations at one or more positions selected from 152 / 188 / 196 / 198 / 199 / 324, 188 / 196 / 198 / 199 / 201, 188 / 196 / 198 / 201, 188 / 196 / 201, 188 / 198 / 199 / 268, 188 / 201, and 188 / 324, where these positions are numbered with reference to SEQ ID NO:1516.In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations selected from 152Y / 188E / 196V / 198A / 199Q / 324K, 188E / 196V / 198A / 199Q / 201P, 188E / 196V / 198F / 201P, 188E / 196V / 201P, 188E / 198A / 199Q / 268A, 188E / 201P, and 188E / 324K, wherein these positions are numbered with reference to SEQ ID NO: 1516. In some additional embodiments, the engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations selected from F152Y / L188E / A196V / Q198A / N199Q / H324K, L188E / A196V / Q198A / N199Q / G201P, L188E / A196V / Q198F / G201P, L188E / A196V / G201P, L188E / Q198A / N199Q / Y268A, L188E / G201P, and L188E / H324K, wherein these positions are numbered with reference to SEQ ID NO: 1516.

[0028] The present invention also provides engineered glycosyltransferases provided herein, the engineered glycosyltransferases being selected from beta-1,2-glycosyltransferases and beta-1,3-glycosyltransferases. In some embodiments, the engineered glycosyltransferases are NDP-glycosyltransferases selected from ADP-glucose-dependent glycosyltransferases (AGT), CDP-glucose-dependent glycosyltransferases (CGT), GDP-glucose-dependent glycosyltransferases (GGT), TDP-glucose-dependent glycosyltransferases (TGT) and IDP-glucose-dependent glycosyltransferases (IGT). In some additional embodiments, the engineered glycosyltransferases are ADP-glucose-dependent glycosyltransferases. In some further embodiments, the engineered glycosyltransferases preferentially use sugar donors other than uracil diphosphate.

[0029] The present invention also provides engineered polynucleotides encoding at least one engineered glycosyltransferase polypeptide provided herein. The present invention also provides vectors comprising at least one engineered polynucleotide encoding at least one glycosyltransferase provided herein. In some embodiments, the vector further comprises at least one regulatory sequence. The present invention also provides host cells comprising at least one engineered polynucleotide encoding at least one glycosyltransferase provided herein. The present invention further provides host cells comprising at least one vector provided herein. In some embodiments, the host cell is selected from eukaryotes and prokaryotes.

[0030] The present invention also provides methods for producing at least one engineered glycosyltransferase provided herein, comprising culturing a host cell provided herein under conditions such that the engineered glycosyltransferase is produced by the host cell. In some embodiments, the method further comprises recovering the engineered glycosyltransferase. The present invention also provides compositions comprising at least one engineered glycosyltransferase provided herein.

[0031] The present invention also provides an engineered sucrose synthase comprising a polypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 6. In some embodiments, an engineered sucrose synthase comprising a polypeptide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:22. In some embodiments, the engineered sucrose synthase polypeptide sequence comprises at least one mutation or set of mutations at one or more positions selected from 3 / 548, 4, 7, 12, 41, 42, 44, 47, 52, 57, 59, 71, 81, 85, 93, 97, 122, 129, 134, 136, 139, 154, 175, 215, 266, 270, 343, 358, 381, 388, 434, 442, 519, 524, 532, 536, 570, 589, 603, 606, 615, 635, 641, 652, 724, 727, and 738, which positions are numbered with reference to SEQ ID NO:22.In some additional embodiments, the engineered sucrose synthase polypeptide sequence is 3G / 548R, 4Q, 4T, 7H, 7L, 12K, 41Q, 41S, 42D, 42E, 42I, 44A, 44I, 44L, 44T, 47A, 47G, 47R, 47T, 52G, 52Q, 52S, 52T, 52V, 57H, 57Y, 59M, 59T, 71Q, 71T, 81H, 81P, 81Q, 85T, 93I, 97S, 97T, 122P, 129Q, 134P, 134Q, 134R, 136G, 139G, 154A, 175S, 215L, 266R, 270L, 343L, and at least one mutation or set of mutations selected from 358P, 358Q, 381K, 381R, 381T, 381Y, 388A, 388R, 388S, 434G, 434L, 442Y, 519C, 519V, 524R, 524S, 532E, 532T, 532Y, 536T, 570K, 589Q, 603M, 603T, 606A, 606P, 615T, 635G, 635R, 641I, 641L, 652R, 652Y, 724A, 724S, 727H, 738A, and 738K, wherein these positions are numbered with reference to SEQ ID NO:22.In some additional embodiments, the engineered sucrose synthase polypeptide sequence is selected from the group consisting of E3G / P548R, E4Q, E4T, Q7H, Q7L, S12K, K41Q, K41S, T42D, T42E, T42I, R44A, R44I, R44L, R44T, P47A, P47G, P47R, P47T, P52G, P52Q, P52 S, P52T, P52V, W57H, W57Y, A59M, A59T, R71Q, R71T, L81H, L81P, L81Q, V85T, V93I, A97S, A9 7T, A122P, E129Q, V134P, V134Q, V134R, Q136G, K139G, H154A, G175S, F215L, N266R, V270L, H343L, E358P, E358Q, S381K, S381R, S381T, S381Y, K388A, K388R, K388S, Y434G, Y434L, H4 42Y, T519C, T519V, A524R, A524S, S532E, S532T, S532Y, E536T, R570K, S589Q, Q603M, Q603 T, M606A, M606P, R615T, S635G, S635R, V641I, V641L, G652R, G652Y, K724A, K724S, E727H, S738A, and S738K, where these positions are numbered with reference to SEQ ID NO: 22. In some embodiments, the engineered sucrose synthase polypeptide sequence comprises at least one mutation or set of mutations at one or more positions selected from 41, 52, 134, 381, 442, 519, 524, and 724, where these positions are numbered with reference to SEQ ID NO: 22. In some additional embodiments, the engineered sucrose synthase polypeptide sequence comprises at least one mutation or set of mutations selected from 41S, 52V, 134R, 381R, 442Y, 519V, 524R and 724S, wherein these positions are numbered with reference to SEQ ID NO:22.In some additional embodiments, the engineered sucrose synthase polypeptide sequence comprises at least one mutation or set of mutations selected from K41S, P52V, V134R, S381R, H442Y, T519V, A524R and K724S, wherein the positions are numbered with reference to SEQ ID NO:22.

[0032] In some embodiments, the polypeptide sequence of the engineered sucrose synthase is selected from the group consisting of 25 / 44 / 52 / 134 / 434 / 724, 41 / 44 / 52 / 97 / 442 / 719 / 724, 41 / 44 / 52 / 134 / 442, 41 / 44 / 52 / 434 / 442 / 532 / 724, 41 / 44 / 52 / 434 / 724, 41 / 44 / 136 / 329, 41 / 44 / 136 / 442, 41 / 44 / 532, 41 / 52 / 97 / 434 / 442 / 532 / 724, 41 / 52 / 134 / 136 / 434, 41 / 52 / 134 / 442 / 724, 41 / 52 / 13 6, 41 / 52 / 434, 41 / 52 / 434 / 442, 41 / 52 / 434 / 442 / 724, 41 / 52 / 442, 41 / 97 / 134 / 434 / 442 / 532 / 553, 41 / 134 / 442 / 532, 41 / 329 / 442, 41 / 434 / 442 / 532, 41 / 434 / 442 / 532 / 724, 41 / 434 / 532, 41 / 532, 44 / 52 / 97 / 434 / 442 / 724, 44 / 52 / 97 / 442 / 724, 44 / 52 / 134 / 136 / 329 / 434 / 442 / 532, 44 / 52 / 134 / 434 / 532, 44 / 136 / 329 / 434 / 532, 44 / 136 / 532, 44 / 434 / 442 / 553, 52, 52 / 97, 52 / 97 / 434 / 442, 52 / 97 / 442, 52 / 97 / 532, 52 / 134, 52 / 134 / 136 / 434, 52 / 134 / 136 / 442, 5 2 / 134 / 329 / 434, 52 / 134 / 329 / 532, 52 / 134 / 434 / 442 / 532 / 553, 52 / 134 / 442 / 724, 52 / 136, 52 / 136 / 434, 52 / 136 / 434 / 442, 52 / 136 / 434 / 442 / 532, 52 / 13 6 / 434 / 724, 52 / 136 / 442, 52 / 434, 52 / 434 / 442 / 532, 52 / 434 / 442 / 724, 52 / 434 / 532, 52 / 442, 52 / 442 / 532 / 724, 52 / 442 / 553 / 724, 52 / 442 / 724, 52 / 532 , 52 / 553, 57 / 97 / 434 / 442 / 724, 97 / 134 / 136 / 442 / 532, 97 / 134 / 136 / 532, 97 / 134 / 442, 97 / 136 / 434 / 442, 97 / 329 / 724, 97 / 442, 134, 134 / 136 / 434 / 442,and at least one mutation or set of mutations at one or more positions selected from 134 / 136 / 434 / 442 / 532 / 553 / 724, 134 / 136 / 434 / 442 / 553 / 724, 134 / 136 / 434 / 532 / 724, 134 / 136 / 442 / 532, 134 / 434 / 442 / 724, 136, 136 / 442, 136 / 442 / 724, 136 / 532 / 724, 434, 434 / 442, 442, 442 / 724, and 532 / 724, wherein these positions are numbered with reference to SEQ ID NO: 1652. In some additional embodiments, the engineered sucrose synthase polypeptide sequence is 25T / 44I / 52G / 134R / 434G / 724S, 41S / 44I / 52G / 97T / 442Y / 719T / 724S, 41S / 44I / 52G / 434G / 442Y / 532Y / 724S, 41S / 44I / 52G / 434G / 724S, 41S / 44I / 52V / 134R / 442Y, 41S / 44I / 136G / 329Q, 41S / 44I / 136G / 442Y, 41S / 44I / 532Y, 41S / 52G / 134R / 442Y / 724S, 41S / 52G / 136G, 41S / 52V / 97T / 434G / 442Y / 532Y / 724S, 41S / 52V / 134R / 136G / 434G, 41S / 52V / 434G, 41S / 52V / 434G / 442Y, 41S / 52V / 434G / 442Y / 724S, 41S / 5 2V / 442Y, 41S / 97T / 134R / 434G / 442Y / 532Y / 553A, 41S / 134R / 442Y / 532Y, 41S / 329Q / 442Y, 41S / 434G / 442Y / 532Y, 41S / 434G / 442Y / 532Y / 724S, 41S / 434G / 532Y, 41S / 532Y, 44I / 52G / 134R / 136G / 329Q / 434G / 442Y / 532Y, 44I / 52V / 97T / 434G / 442Y / 724S, 44I / 52V / 97T / 442Y / 724S, 44I / 52V / 134R / 434G / 532Y, 44I / 136G / 329Q / 434G / 532Y, 44I / 1 36G / 532Y, 44I / 434G / 442Y / 553A, 52G, 52G / 97T, 52G / 97T / 434G / 442Y, 52G / 97T / 442Y, 52G / 97T / 532Y, 52G / 134R,52G / 134R / 136G / 434G, 52G / 134R / 136G / 442Y, 52G / 134R / 329Q / 434G, 52G / 134R / 434G / 442Y / 532Y / 553A, 52G / 136G, 52G / 434G / 4 42Y / 532Y, 52G / 442Y, 52G / 442Y / 553A / 724S, 52G / 442Y / 724S, 52G / 532Y, 52G / 553A, 52V / 134R / 329Q / 532Y, 52V / 134R / 442Y / 724 S, 52V / 136G / 434G, 52V / 136G / 434G / 442Y, 52V / 136G / 434G / 442Y / 532Y, 52V / 136G / 434G / 724S, 52V / 136G / 442Y, 52V / 434G, 52V / 434G / 442Y / 724S, 52V / 434G / 532Y, 52V / 442Y, 52V / 442Y / 532Y / 724S, 52V / 442Y / 553A / 724S, 52V / 442Y / 724S, 52V / 532Y, 57C / 97 T / 434G / 442Y / 724S, 97T / 134R / 136G / 442Y / 532Y, 97T / 134R / 136G / 532Y, 97T / 134R / 442Y, 97T / 136G / 434G / 442Y, 97T / 329Q / 724 S, 97T / 442Y, 134R, 134R / 136G / 434G / 442Y, 134R / 136G / 434G / 442Y / 532Y / 553A / 724S, 134R / 136G / 434G / 442Y / 553A / 724S, 134R / 136G / 434G / 532Y / 724S, 134R / 136G / 442Y / 532Y, 134R / 434G / 442Y / 724S, 136G, 136G / 442Y, 136G / 442Y / 724S, 136G / 532Y / 724S, 434G, 434G / 442Y, 442Y, 442Y / 724S, and 532Y / 724S, wherein these positions are numbered with reference to SEQ ID NO: 1652. In some additional embodiments, the engineered sucrose synthase polypeptide sequence is A25T / R44I / P52G / V134R / Y434G / K724S, K41S / R44I / P52G / A97T / H442Y / A719T / K724S, K41S / R44I / P52G / Y434G / H442Y / S532Y / K724S,K41S / R44I / P52G / Y434G / K724S、K41S / R44I / P52V / V134R / H442Y、K41S / R44I / Q136G / E329Q、K41S / R44I / Q136G / H442Y、K41S / R44I / S532Y、K41S / P52G / V134R / H442Y / K724S、K41S / P52G / Q136G、K41S / P52V / A97T / Y434G / H442Y / S532Y / K724S、K41S / P52V / V134R / Q136G / Y434G、K41S / P52V / Y434G、K41S / P52V / Y434G / H442Y、K41S / P52V / Y434G / H442Y / K724S、K41S / P52V / H442Y、K41S / A97T / V134R / Y434G / H442Y / S532Y / V553A、K41S / V134R / H442Y / S532Y、K41S / E329Q / H442Y、K41S / Y434G / H442Y / S532Y、K41S / Y434G / H442Y / S532Y / K724S、K41S / Y434G / S532Y、K41S / S532Y、R44I / P52G / V134R / Q136G / E329Q / Y434G / H442Y / S532Y、R44I / P52V / A97T / Y434G / H442Y / K724S、R44I / P52V / A97T / H442Y / K724S、R44I / P52V / V134R / Y434G / S532Y、R44I / Q136G / E329Q / Y434G / S532Y、R44I / Q136G / S532Y、R44I / Y434G / H442Y / V553A、P52G、P52G / A97T、P52G / A97T / Y434G / H442Y、P52G / A97T / H442Y、P52G / A97T / S532Y、P52G / V134R、P52G / V134R / Q136G / Y434G、P52G / V134R / Q136G / H442Y、P52G / V134R / E329Q / Y434G、P52G / V134R / Y434G / H442Y / S532Y / V553A、P52G / Q136G、P52G / Y434G / H442Y / S532Y、P52G / H442Y、P52G / H442Y / V553A / K724S、P52G / H442Y / K724S、P52G / S532Y、P52G / V553A、P52V / V134R / E329Q / S532Y、P52V / V134R / H442Y / K724S, P52V / Q136G / Y434G, P52V / Q136G / Y434G / H442Y, P52V / Q136G / Y434G / H442Y / S532Y, P52V / Q136G / Y434G / K724S, P52V / Q136G / H442Y, P52V / Y434G, P52V / Y434G / H442Y / K724S, P52V / Y434G / S532Y, P52V / H442 Y, P52V / H442Y / S532Y / K724S, P52V / H442Y / V553A / K724S, P52V / H442Y / K724S, P52V / S532Y, W57C / A97T / Y434G / H442 Y / K724S, A97T / V134R / Q136G / H442Y / S532Y, A97T / V134R / Q136G / S532Y, A97T / V134R / H442Y, A97T / Q136G / Y434G / H4 42Y, A97T / E329Q / K724S, A97T / H442Y, V134R, V134R / Q136G / Y434G / H442Y, V134R / Q136G / Y434G / H442Y / S532Y / V553 A / K724S, V134R / Q136G / Y434G / H442Y / V553A / K724S, V134R / Q136G / Y434G / S532Y / K724S, V134R / Q136G / H442Y / S532 and at least one mutation or set of mutations selected from Y, V134R / Y434G / H442Y / K724S, Q136G, Q136G / H442Y, Q136G / H442Y / K724S, Q136G / S532Y / K724S, Y434G, Y434G / H442Y, H442Y, H442Y / K724S, and S532Y / K724S, wherein these positions are numbered with reference to SEQ ID NO: 1652. In some additional embodiments, the engineered sucrose synthase polypeptide sequence comprises at least one mutation or set of mutations at one or more positions selected from 3, 4, 22, 32, 34, 38, 47 / 488, 51, 51 / 433, 62, 75 / 169, 101, 169, 195 / 213, 708 and 718, which positions are numbered with reference to SEQ ID NO: 1652. In some additional embodiments, the engineered sucrose synthase polypeptide sequence comprises at least one mutation or set of mutations at one or more positions selected from 3A, 4V, 22L ...at least one mutation or set of mutations selected from 22T, 32S, 34E, 34S, 38L, 38N, 38V, 38W, 47S / 488N, 51A, 51H, 51H / 433P, 51T, 62I, 75I / 169A, 101V, 169E, 195K / 213T, 708V and 718H, wherein these positions are numbered with reference to SEQ ID NO: 1652; In some additional embodiments, the engineered sucrose synthase polypeptide sequence comprises at least one mutation or set of mutations selected from E3A, E4V, H22L, H22T, L32S, T34E, T34S, R38L, R38N, R38V, R38W, P47S / D488N, Y51A, Y51H, Y51H / L433P, Y51T, V62I, M75I / Q169A, L101V, Q169E, S195K / A213T, T708V, and A718H, wherein these positions are numbered with reference to SEQ ID NO: 1652. In some further embodiments, the engineered sucrose synthase polypeptide sequence comprises at least one mutation or set of mutations at one or more positions selected from 44 / 52 / 97 / 442 / 724, 52 / 97 / 442, 97 / 442, and 434 / 442, which positions are numbered with reference to SEQ ID NO: 1652. In some additional embodiments, the engineered sucrose synthase polypeptide sequence comprises at least one mutation or set of mutations selected from 44I / 52V / 97T / 442Y / 724S, 52G / 97T / 442Y, 97T / 442Y, and 434G / 442Y, which positions are numbered with reference to SEQ ID NO: 1652. In some additional embodiments, the engineered sucrose synthase polypeptide sequence comprises at least one mutation or set of mutations selected from R44I / P52V / A97T / H442Y / K724S, P52G / A97T / H442Y, A97T / H442Y, and Y434G / H442Y, wherein the positions are numbered with reference to SEQ ID NO: 1652.

[0033] In yet some further embodiments, the engineered sucrose synthase polypeptide sequence is 4 / 22, 4 / 22 / 34, 4 / 22 / 34 / 38, 4 / 22 / 34 / 38 / 169 / 708, 4 / 22 / 34 / 47 / 51 / 169 / 213, 4 / 22 / 51 / 708, 4 / 22 / 101, 4 / 34 / 38 / 101, 4 / 34 / 708, 4 / 62 / 433 / 708, 4 / 169, 4 / 169 / 433, 4 / 169 / 708, 4 / 433, 4 / 708, 22 / 34, 22 22 / 34 / 101 / 169, 22 / 34 / 101 / 169 / 195, 22 / 34 / 169 / 708, 22 / 47 / 169 / 433, 22 / 75, 34 / 38 / 62, 34 / 62 / 169 / 433, 34 / 101, 62 / 708, 75 / 169, 169 / 708, and 195 / 708, wherein these positions are numbered with reference to SEQ ID NO: 1822. In some additional embodiments, the engineered sucrose synthase polypeptide sequence is 4V / 22L / 34E, 4V / 22L / 51A / 708V, 4V / 22L / 101V, 4V / 22T, 4V / 22T / 34E / 38N, 4V / 22T / 34E / 38V / 169A / 708V, 4V / 22T / 34E / 47S / 51H / 169A / 213T, 4V / 34E / 38N / 101V, 4V / 34E / 708V, 4V / 62I / 433P / 708V, 4V / 169A, 4V / 169A / 433P, 4V / 169A / 708V, 4V / 433P, 4V / 708V , 22L / 34E, 22L / 34S / 169A / 708V, 22L / 75I, 22T / 34E / 38V, 22T / 34E / 101V / 169E, 22T / 34E / 101V / 169E / 195K, 22T / 47S / 169A / 433P, 34E / 62I / 169E / 433P, 34E / 101V, 34S / 38L / 62I, 62I / 708V, 75I / 169A, 169A / 708V, and 195K / 708V, wherein these positions are numbered with reference to SEQ ID NO: 1822.In some additional embodiments, the engineered sucrose synthase polypeptide sequence is E4V / H22L / T34E, E4V / H22L / Y51A / T708V, E4V / H22L / L101V, E4V / H22T, E4V / H22T / T34E / R38N, E4V / H22T / T34E / R38V / Q169A / T708V, E4V / H22T / T34E / P47S / Y51H / Q169A / A213T, E4V / T34E / R38N / L101V, E4V / T34E / T708V, E4 V / V62I / L433P / T708V, E4V / Q169A, E4V / Q169A / L433P, E4V / Q169A / T708V, E4V / L433P, E4V / T708V, H22L / T34E, H22L / T34S / Q169A / T708V, H22L / M75I, H22T / T34E / R38V, H22T / T34E / L101V / Q169E, H22T / T34E / L101V / Q169E / S195K, H22T / P47S / Q169A / L433P, T34E / V62I / Q and at least one mutation or set of mutations selected from 169E / L433P, T34E / L101V, T34S / R38L / V62I, V62I / T708V, M75I / Q169A, Q169A / T708V, and S195K / T708V, wherein these positions are numbered with reference to SEQ ID NO:1822. In some additional embodiments, the engineered sucrose synthase polypeptide sequence comprises at least one mutation or set of mutations at one or more positions selected from 4, 22, 34, 121, 169, 341, 411, 433, 441, 518, 526, 527, 528, 544, 557, 558, 565, 585, 602, 604, 623, 708, 731 and 770, which positions are numbered with reference to SEQ ID NO: 1822.In some additional embodiments, the engineered sucrose synthase polypeptide sequence comprises at least one mutation or set of mutations selected from 4V, 22L, 22T, 34E, 121D, 169A, 341N, 411I, 433P, 441A, 441L, 518S, 526H, 526V, 527A, 527Q, 528Q, 544H, 557P, 558G, 565V, 585A, 602P, 604A, 623A, 623K, 623Q, 623R, 708V, 731A, 731M, 731Q, 731T, and 770T, wherein these positions are numbered with reference to SEQ ID NO: 1822. In some additional embodiments, the engineered sucrose synthase polypeptide sequence comprises at least one mutation or set of mutations selected from E4V, H22L, H22T, T34E, E121D, Q169A, P341N, L411I, L433P, D441A, D441L, R518S, T526H, T526V, E527A, E527Q, E528Q, R544H, R557P, Q558G, M565V, S585A, V602P, N604A, H623A, H623K, H623Q, H623R, T708V, D731A, D731M, D731Q, D731T, and V770T, wherein these positions are numbered with reference to SEQ ID NO: 1822. In some additional embodiments, the engineered sucrose synthase polypeptide sequence comprises at least one mutation or set of mutations at one or more positions selected from 4, 4 / 22 / 34 / 38, 4 / 169, 4 / 169 / 433, 4 / 433, 62 / 708, and 169 / 708, where these positions are numbered with reference to SEQ ID NO: 1822. In some additional embodiments, the engineered sucrose synthase polypeptide sequence comprises at least one mutation or set of mutations selected from 4V, 4V / 22T / 34E / 38N, 4V / 169A, 4V / 169A / 433P, 4V / 433P, 62I / 708V, and 169A / 708V, where these positions are numbered with reference to SEQ ID NO: 1822.In some additional embodiments, the engineered sucrose synthase polypeptide sequence comprises at least one mutation or set of mutations selected from E4V, E4V / H22T / T34E / R38N, E4V / Q169A, E4V / Q169A / L433P, E4V / L433P, V62I / T708V, and Q169A / T708V, wherein the positions are numbered with reference to SEQ ID NO: 1822.

[0034] In yet some additional embodiments, the engineered sucrose synthase polypeptide sequence is 34 / 62 / 411 / 526 / 557, 34 / 62 / 411 / 557 / 602 / 604, 34 / 62 / 441 / 557 / 623, 34 / 62 / 518 / 623, 34 / 62 / 623 / 731, 34 / 121 / 441 / 544 / 604 / 623, 34 / 121 / 526 / 604 / 731, 34 / 411 / 441, 34 / 411 / 441 / 518 / 526 / 557 / 565 / 731, 34 / 411 / 441 / 518 / 544 / 557 / 565 / 708 / 731, 34 / 411 / 441 / 518 / 585 / 604 / 770, 34 / 441 / 518 / 526 / 585 / 604 / 731, 34 / 441 / 526 / 565 / 708 / 770, 34 / 441 / 544 / 557 / 585 / 602 / 623, 34 / 441 / 585 / 623 / 708 / 7 31, 34 / 526 / 585 / 623, 62 / 121 / 329 / 518 / 557 / 565 / 623 / 708, 62 / 121 / 411 / 441 / 518 / 544 / 557 / 585 / 604 / 623, 62 / 121 / 441 / 518 / 526 / 623 / 770, 62 / 121 / 44 1 / 565 / 585, 62 / 121 / 518 / 557 / 585 / 604 / 708 / 770, 62 / 411 / 526 / 565 / 604 / 623, 62 / 411 / 585 / 731, 62 / 441 / 518 / 557 / 604 / 623 / 708 / 731, 62 / 441 / 623 / 708 / 770, 62 / 441 / 770, 121 / 441 / 518 / 526 / 602 / 604, 121 / 441 / 518 / 526 / 623 / 708, 121 / 441 / 526 / 557 / 565 / 708, 121 / 604 / 708 / 731 / 770, 411 / 441 / 518 / 557 / 623, 411 / 441 / 518 / 708, 411 / 441 / 604 / 623 / 708 / 731, 411 / 518 / 526 / 604 / 623 / 731, 411 / 565 / 604 / 623, 411 / 585 / 623, 441 / 518 / 526 / 557 / 565 / 602 / 604 / 623 / 708, 441 / 518 / 526 / 557 / 585 / 604 / 623 / 708, 441 / 518 / 526 / 604 / 623, 441 / 518 / 557 / 565 / 585, 441 / 518 / 565 / 623 / 731 / 770, 441 / 518 / 585, 441 / 585,At least one mutation or set of mutations at one or more positions selected from 441 / 585 / 602 / 604 / 623 / 708 / 731, 441 / 708 / 731, 557 / 602 / 604, 557 / 604, 585 / 623 / 708, and 623, where these positions are numbered with reference to SEQ ID NO: 2092.

[0035] In some additional embodiments, the engineered sucrose synthase polypeptide sequence is 34E / 62I / 411I / 526H / 557P, 34E / 62I / 411I / 557P / 602P / 604A, 34E / 62I / 441L / 557P / 623K, 34E / 62I / 518S / 623K, 34E / 62I / 623K / 731Q, 34E / 121D / 441L / 544H / 604A / 623K, 34E / 121D / 526H / 604A / 731Q, 34E / 411I / 441L, 34E / 411I / 441L / 518S / 526H / 557P / 565V / 731Q, 34E / 411I / 441L / 518S / 544H / 557P / 565V / 708V / 731Q, 34E / 41 1I / 441L / 518S / 585A / 604A / 770T, 34E / 441L / 518S / 526H / 585A / 604A / 731Q , 34E / 441L / 526H / 565V / 708V / 770T, 34E / 441L / 544H / 557P / 585A / 602P / 62 3K, 34E / 441L / 585A / 623K / 708V / 731Q, 34E / 526H / 585A / 623K, 62I / 121D / 32 9Q / 518S / 557P / 565V / 623R / 708V, 62I / 121D / 411I / 441L / 518S / 544H / 557P / 585A / 604A / 623K, 62I / 121D / 441L / 518S / 526H / 623R / 770T, 62I / 121D / 44 1L / 565V / 585A, 62I / 121D / 518S / 557P / 585A / 604A / 708V / 770T, 62I / 411I / 526H / 565V / 604A / 623K, 62I / 411I / 585A / 731Q, 62I / 441L / 518S / 557P / 604 A / 623K / 708V / 731Q, 62I / 441L / 623R / 708V / 770T, 62I / 441L / 770T, 121D / 4 41L / 518S / 526H / 602P / 604A, 121D / 441L / 518S / 526H / 623K / 708V, 121D / 44 1L / 526H / 557P / 565V / 708V, 121D / 604A / 708V / 731Q / 770T, 411I / 441L / 518 S / 557P / 623K, 411I / 441L / 518S / 708V, 411I / 441L / 604A / 623K / 708V / 731Q,411I / 518S / 526H / 604A / 623K / 731Q, 411I / 565V / 604A / 623R, 411I / 585A / 623K, 441L / 518S / 526H / 557P / 565V / 602P / 604A / 623 K / 708V, 441L / 518S / 526H / 557P / 585A / 604A / 623R / 708V, 441L / 518S / 526H / 604A / 623R, 441L / 518S / 557P / 565V / 585A, 441L / 51 and at least one mutation or set of mutations selected from 8S / 565V / 623R / 731Q / 770T, 441L / 518S / 585A, 441L / 585A, 441L / 585A / 602P / 604A / 623R / 708V / 731Q, 441L / 708V / 731Q, 557P / 602P / 604A, 557P / 604A, 585A / 623R / 708V, and 623R, wherein these positions are numbered with reference to SEQ ID NO:2092. In some additional embodiments, the engineered sucrose synthase polypeptide sequence is selected from the group consisting of T34E / V62I / L411I / T526H / R557P, T34E / V62I / L411I / R557P / V602P / N604A, T34E / V62I / D441L / R557P / H623K, T34E / V62I / R51 8S / H623K, T34E / V62I / H623K / D731Q, T34E / E121D / D441L / R544H / N604A / H623K, T34E / E121D / T526H / N604A / D731Q, T34E / L411I / D441L, T34E / L411I / D441L / R518S / T526H / R5 57P / M565V / D731Q, T34E / L411I / D441L / R518S / R544H / R557P / M565V / T708V / D731Q, T3 4E / L411I / D441L / R518S / S585A / N604A / V770T, T34E / D441L / R518S / T526H / S585A / N60 4A / D731Q, T34E / D441L / T526H / M565V / T708V / V770T, T34E / D441L / R544H / R557P / S585 A / V602P / H623K, T34E / D441L / S585A / H623K / T708V / D731Q, T34E / T526H / S585A / H623K,V62I / E121D / E329Q / R518S / R557P / M565V / H623R / T708V、V62I / E121D / L411I / D441L / R518S / R544H / R557P / S585A / N604A / H623K、V62I / E121D / D441L / R518S / T526H / H623R / V770T、V62I / E121D / D441L / M565V / S585A、V62I / E121D / R518S / R557P / S585A / N604A / T708V / V770T、V62I / L411I / T526H / M565V / N604A / H623K、V62I / L411I / S585A / D731Q、V62I / D441L / R518S / R557P / N604A / H623K / T708V / D731Q、V62I / D441L / H623R / T708V / V770T、V62I / D441L / V770T、E121D / D441L / R518S / T526H / V602P / N604A、E121D / D441L / R518S / T526H / H623K / T708V、E121D / D441L / T526H / R557P / M565V / T708V、E121D / N604A / T708V / D731Q / V770T、L411I / D441L / R518S / R557P / H623K、L411I / D441L / R518S / T708V、L411I / D441L / N604A / H623K / T708V / D731Q、L411I / R518S / T526H / N604A / H623K / D731Q、L411I / M565V / N604A / H623R、L411I / S585A / H623K、D441L / R518S / T526H / R557P / M565V / V602P / N604A / H623K / T708V、D441L / R518S / T526H / R557P / S585A / N604A / H623R / T708V、D441L / R518S / T526H / N604A / H623R、D441L / R518S / R557P / M565V / S585A、D441L / R518S / M565V / H623R / D731Q / V770T、D441L / R518S / S585A、D441L / S585A、D441L / S585A / V602P / N604A / H623R / T708V / D731Q、D441L / T708V / D731Q、R557P / V602P / N604A、R557P / N604A、S585A / H623R / T708V, and H623R, where these positions are numbered with reference to SEQ ID NO: 2092. In still some further embodiments, the engineered sucrose synthase polypeptide sequence comprises at least one mutation or set of mutations at one or more positions selected from 63, 65, 269, 323, 406, 416, 469, 511 and 640, where these positions are numbered with reference to SEQ ID NO: 2092. In some additional embodiments, the engineered sucrose synthase polypeptide sequence comprises at least one mutation or set of mutations selected from 63G, 63S, 63T, 65C, 65L, 269V, 323G, 323S, 323T, 406G, 416F, 469S, 511L, 640A, 640E, 640H, 640N, 640R, 640V and 640W, wherein these positions are numbered with reference to SEQ ID NO:2092. In some additional embodiments, the engineered sucrose synthase polypeptide sequence comprises at least one mutation or set of mutations selected from A63G, A63S, A63T, T65C, T65L, I269V, A323G, A323S, A323T, N406G, L416F, T469S, V511L, T640A, T640E, T640H, T640N, T640R, T640V and T640W, wherein the positions are numbered with reference to SEQ ID NO:2092. In some further embodiments, the engineered sucrose synthase polypeptide sequence comprises at least one mutation or set of mutations at one or more positions selected from 34 / 62 / 441 / 557 / 623, 34 / 441 / 585 / 623 / 708 / 731, 62 / 441 / 623 / 708 / 770, 411 / 441 / 518 / 557 / 623, 411 / 441 / 604 / 623 / 708 / 731, 441 / 518 / 526 / 557 / 585 / 604 / 623 / 708, 441 / 518 / 557 / 565 / 585, and 441 / 585 / 602 / 604 / 623 / 708 / 731, wherein these positions are numbered with reference to SEQ ID NO:2092. In some additional embodiments, the polypeptide sequence of the engineered sucrose synthase is34E / 62I / 441L / 557P / 623K, 34E / 441L / 585A / 623K / 708V / 731Q, 62I / 441L / 623R / 708V / 770 T, 411I / 441L / 518S / 557P / 623K, 411I / 441L / 604A / 623K / 708V / 731Q, 441L / 518S / 526H / 55 and at least one mutation or set of mutations selected from 7P / 585A / 604A / 623R / 708V, 441L / 518S / 557P / 565V / 585A, and 441L / 585A / 602P / 604A / 623R / 708V / 731Q, wherein these positions are numbered with reference to SEQ ID NO:2092. In some additional embodiments, the engineered sucrose synthase polypeptide sequence is selected from the group consisting of T34E / V62I / D441L / R557P / H623K, T34E / D441L / S585A / H623K / T708V / D731Q, V62I / D441L / H623R / T708V / V770T, L411I / D441L / R518S / R557P / H623K, L411I / D441L / N604A / H623K / T708V / D731Q, D441L / R518S / T526H / R557P / S585A / N604A / H623R / T708V, D441L / R518S / R557P / M565V / S585A, and D441L / S585A / V602P / N604A / H623R / T708V / D731Q, wherein these positions are numbered with reference to SEQ ID NO: 2092.

[0036] In some further embodiments, the engineered sucrose synthase polypeptide sequence is 34, 34 / 62, 34 / 62 / 65 / 640, 34 / 62 / 323, 34 / 63 / 65 / 323 / 528 / 640, 34 / 63 / 65 / 406 / 528 / 640 / 713, 34 / 63 / 323 / 526 / 528 / 640, 34 / 63 / 406 / 528 / 640 / 731, 34 / 65, 34 / 65 / 528 / 640, 34 / 323 / 406 / 640, 34 / 323 / 640, 34 / 640, 38 / 640, 62 / 63, 62 / 63 / 65 / 528 / 640, 62 / 63 / 69 / 323 / 406 / 640, 62 / 63 / 526 / 640 / 731, 62 / 323 / 640, 63 / 6 5 / 323 / 406 / 526 / 528 / 640 / 731, 63 / 65 / 406 / 731, 63 / 65 / 528 / 640 / 731, 63 / 65 / 640, 63 / 323 / 406 / 640, 63 / 406, 63 / 406 / 640, 63 / 526 / 528 / 640 / 731, 63 / 731, 323 / 406 / 640, 323 / 406 / 64 and at least one mutation or set of mutations at one or more positions selected from: 0 / 731, 323 / 526 / 528 / 640, 323 / 526 / 640, 323 / 640 / 731, 406 / 640, 526 / 528 / 640 / 731, and 731, where these positions are numbered with reference to SEQ ID NO:2182.In some additional embodiments, the engineered sucrose synthase polypeptide sequence is 34E, 34E / 62I, 34E / 62I / 65C / 640R, 34E / 62I / 323G, 34E / 63S / 65C / 406G / 528D / 640R / 713V, 34E / 63S / 323G / 526T / 528D / 640R, 34E / 63S / 406G / 528D / 640N / 731Q, 34E / 63T / 65C / 3 23G / 528D / 640R, 34E / 65C, 34E / 65C / 528D / 640R, 34E / 323G / 406G / 640A, 34E / 323G / 640A, 34E / 640A, 34E / 640R, 38H / 640R, 62I / 63S / 65C / 528D / 640A, 62I / 63S / 526T / 640R / 731Q, 62I / 63T, 62I / 63T / 69F / 323G / 406G / 640R, 62 I / 323G / 640R, 63S / 65C / 640R, 63S / 323G / 406G / 640A, 63S / 406G, 63S / 526T / 528D / 640R / 731Q, 63S / 731Q, 63T / 6 5C / 323G / 406G / 526T / 528D / 640N / 731Q, 63T / 65C / 406G / 731Q, 63T / 65C / 528D / 640A / 731Q, 63T / 406G / 640A, 323 and at least one mutation or set of mutations selected from: G / 406G / 640N, 323G / 406G / 640R / 731Q, 323G / 526T / 528D / 640R, 323G / 526T / 640R, 323G / 640A / 731Q, 406G / 640R, 526T / 528D / 640A / 731Q, and 731Q, wherein these positions are numbered with reference to SEQ ID NO:2182.In some additional embodiments, the engineered sucrose synthase polypeptide sequence is selected from the group consisting of T34E, T34E / V62I, T34E / V62I / T65C / T640R, T34E / V62I / A323G, T34E / A63S / T65C / N406G / E528D / T640R / A713V, T34E / A63S / A323G / H526T / E528D / T640R, T34E / A63S / N406G / E528D / T640N / D731Q, T34E / A63T / T65C / A323G ...A323G / H526T / E528D / T640R, T34E / A63S / A323G / H526T / E528D / T640R, T34E / A63S / A323G / H526T / E528D 23G / E528D / T640R, T34E / T65C, T34E / T65C / E528D / T640R, T34E / A323G / N406G / T640A, T34E / A323G / T640A, T34E / T640A, T34E / T640 R, R38H / T640R, V62I / A63S / T65C / E528D / T640A, V62I / A63S / H526T / T640R / D731Q, V62I / A63T, V62I / A63T / I69F / A323G / N406G / T64 0R, V62I / A323G / T640R, A63S / T65C / T640R, A63S / A323G / N406G / T640A, A63S / N406G, A63S / H526T / E528D / T640R / D731Q, A63S / D731 Q, A63T / T65C / A323G / N406G / H526T / E528D / T640N / D731Q, A63T / T65C / N406G / D731Q, A63T / T65C / E528D / T640A / D731Q, A63T / N406G / T640A, A323G / N406G / T640N, A323G / N406G / T640R / D731Q, A323G / H526T / E528D / T640R, A323G / H526T / T640R, A323G / T640A / D731Q, N406G / T640R, H526T / E528D / T640A / D731Q, and D731Q, wherein these positions are numbered with reference to SEQ ID NO:2182.In some further embodiments, the engineered sucrose synthase polypeptide sequence comprises at least one mutation or set of mutations at one or more positions selected from 28, 30, 30 / 158, 37, 59, 102, 108, 158, 164, 183, 191, 206, 235, 307, 311, 409, 419, 533, 543, 546, 559, 683, 710, 752 and 793, which positions are numbered with reference to SEQ ID NO:2182. In some additional embodiments, the polypeptide sequence of the engineered sucrose synthase is 28H, 30A, 30H / 158H, 30M, 30Q, 37L, 37M, 59T, 102N, 102T, 108R, 158A, 158K, 164G, 164Q, 164S, 164T, 183P, 191L, 206I, 235A, 307E, 307H, 311L, 31 1S, 409S, 419L, 419V, 533K, 533R, 543A, 546N, 546Q, 546T, 559R, 683L, 683V, 710A, 710G, 710S, 710V, 752R, and 793G, wherein these positions are numbered with reference to SEQ ID NO:2182. In some additional embodiments, the engineered sucrose synthase polypeptide sequence is R28H, S30A, S30H / R158H, S30M, S30Q, Q37L, Q37M, A59T, S102N, S102T, Q108R, R158A, R158K, A164G, A164Q, A164S, A164T, N183P, T191L, L206I, V235A, N307E, N307H, Q311L, , Q311S, A409S, T419L, T419V, L533K, L533R, G543A, P546N, P546Q, P546T, K559R, R683L, R683V, E710A, E710G, E710S, E710V, T752R, and E793G, wherein these positions are numbered with reference to SEQ ID NO:2182.In some further embodiments, the engineered sucrose synthase polypeptide sequence comprises at least one mutation or set of mutations at one or more positions selected from 34 / 62, 34 / 65, 34 / 323 / 640, 34 / 640, 38 / 640, and 63 / 65 / 406 / 731, where these positions are numbered with reference to SEQ ID NO: 2182. In some additional embodiments, the engineered sucrose synthase polypeptide sequence comprises at least one mutation or set of mutations selected from 34E / 62I, 34E / 65C, 34E / 323G / 640A, 34E / 640A, 38H / 640R, and 63T / 65C / 406G / 731Q, where these positions are numbered with reference to SEQ ID NO: 2182. In some additional embodiments, the engineered sucrose synthase polypeptide sequence comprises at least one mutation or set of mutations selected from T34E / V62I, T34E / T65C, T34E / A323G / T640A, T34E / T640A, R38H / T640R, and A63T / T65C / N406G / D731Q, wherein these positions are numbered with reference to SEQ ID NO:2182.

[0037] In yet some further embodiments, the engineered sucrose synthase polypeptide sequence is 34 / 65 / 323 / 406 / 411 / 565, 34 / 323 / 406 / 411 / 565, 34 / 323 / 406 / 565 / 731, 62 / 65 / 323 / 406 / 565, 62 / 65 / 323 / 406 / 565 / 731, 62 / 65 / 323 / 411 / 565, 62 / 65 / 406 / 411 / 565 / 731, 62 / 323 / 406 / 411 , 62 / 323 / 406 / 411 / 565, 62 / 323 / 411 / 565, 63 / 65 / 323 / 406 / 565 / 731, 63 / 65 / 406 / 411 / 565, 65 / 323 / 406 / 565, 323 / 406 / 411 / 565, 323 / 411 / 565, and 636, wherein these positions are numbered with reference to SEQ ID NO:2322. In some additional embodiments, the engineered sucrose synthase polypeptide sequence is 34E / 65C / 323G / 406G / 411I / 565V, 34E / 323G / 406G / 411I / 565V, 34E / 323G / 406G / 565V / 731Q, 62I / 65C / 323G / 406G / 565V, 62I / 65C / 323G / 406G / 565V / 731Q, 62I / 65C / 323G / 411I / 565V, 62I / 65C / 406G / 411I / 565V / 731Q, 62I / 323G / 406G / 4 and at least one mutation or set of mutations selected from 11I, 62I / 323G / 406G / 411I / 565V, 62I / 323G / 411I / 565V, 63T / 65C / 323G / 406G / 565V / 731Q, 63T / 65C / 406G / 411I / 565V, 65C / 323G / 406G / 565V, 323G / 406G / 411I / 565V, 323G / 411I / 565V, 636A, 636H, and 636N, wherein these positions are numbered with reference to SEQ ID NO:2322.In some additional embodiments, the engineered sucrose synthase polypeptide sequence is selected from the group consisting of T34E / T65C / A323G / N406G / L411I / M565V, T34E / A323G / N406G / L411I / M565V, T34E / A323G / N406G / M565V / D731Q, V62I / T65C / A323G / N406G / M565V, V62I / T65C / A323G / N406G / M565V / D731Q, V62I / T65C / A323G / L411I / M565V, V62I / T65C / N406G / L411I / M565V / D731Q, V62I / A323G / N406 and at least one mutation or set of mutations selected from: G / L411I, V62I / A323G / N406G / L411I / M565V, V62I / A323G / L411I / M565V, A63T / T65C / A323G / N406G / M565V / D731Q, A63T / T65C / N406G / L411I / M565V, T65C / A323G / N406G / M565V, A323G / N406G / L411I / M565V, A323G / L411I / M565V, Q636A, Q636H, and Q636N, wherein these positions are numbered with reference to SEQ ID NO:2322. In some further embodiments, the engineered sucrose synthase polypeptide sequence comprises at least one mutation or set of mutations at one or more positions selected from 34 / 65 / 323 / 406 / 411 / 565, 34 / 323 / 406 / 411 / 565, 62 / 65 / 323 / 406 / 565, 62 / 65 / 406 / 411 / 565 / 636 / 731, 62 / 65 / 406 / 411 / 565 / 731, 62 / 323 / 406 / 411, 62 / 323 / 406 / 411 / 565, and 62 / 323 / 411 / 565, wherein these positions are numbered with reference to SEQ ID NO:2322.In some additional embodiments, the engineered sucrose synthase polypeptide sequence comprises at least one mutation or set of mutations selected from 34E / 65C / 323G / 406G / 411I / 565V, 34E / 323G / 406G / 411I / 565V, 62I / 65C / 323G / 406G / 565V, 62I / 65C / 406G / 411I / 565V / 636H / 731Q, 62I / 65C / 406G / 411I / 565V / 731Q, 62I / 323G / 406G / 411I, 62I / 323G / 406G / 411I / 565V, and 62I / 323G / 411I / 565V, wherein these positions are numbered with reference to SEQ ID NO:2322. In some additional embodiments, the engineered sucrose synthase polypeptide sequence is selected from the group consisting of T34E / T65C / A323G / N406G / L411I / M565V, T34E / A323G / N406G / L411I / M565V, V62I / T65C / A323G / N406G / M565V, V62I / T65C / N406G / L411I / M565V / Q636H / D731Q, V ... and at least one mutation or set of mutations selected from 2I / T65C / N406G / L411I / M565V / D731Q, V62I / A323G / N406G / L411I, V62I / A323G / N406G / L411I / M565V, and V62I / A323G / L411I / M565V, wherein these positions are numbered with reference to SEQ ID NO: 2322.

[0038] The present invention also provides engineered polynucleotides encoding at least one engineered sucrose synthase polypeptide provided herein. The present invention further provides vectors comprising at least one engineered polynucleotide encoding at least one engineered sucrose synthase provided herein. In some embodiments, the vector further comprises at least one regulatory sequence. The present invention also provides host cells comprising at least one engineered polynucleotide encoding at least one sucrose synthase. In some embodiments, the host cell comprises at least one vector comprising at least one polynucleotide encoding at least one sucrose synthase. In some embodiments, the host cell is selected from eukaryotes and prokaryotes. The present invention also provides methods for producing at least one engineered sucrose synthase variant provided herein, comprising culturing a host cell provided herein under conditions such that the engineered sucrose synthase variant is produced by the host cell. In some embodiments, the method further comprises recovering the engineered sucrose synthase variant. The present invention also provides compositions comprising at least one engineered sucrose synthase variant provided herein.

[0039] The present invention also provides a method for glycosylation of a substrate, the method comprising providing at least one substrate, at least one engineered glycosyltransferase provided herein, and contacting the substrate with the glycosyltransferase under conditions such that the substrate is glycosylated to produce at least one glycosylation product. In some embodiments, the substrate comprises at least one steviol glycoside. In some additional embodiments, the glycosylation product comprises at least one monoglycosylated and / or polyglycosylated product.

[0040] The present invention also provides a method for producing rebaudioside M, the method including providing a rebaudioside D and / or rebaudioside I substrate, NDP-glucose, and at least one engineered glycosyltransferase provided herein, combining the rebaudioside D and rebaudioside I substrates, NDP-glucose, and glycosyltransferase under conditions such that rebaudioside M is produced.

[0041] The present invention also provides a method for producing rebaudioside A and / or rebaudioside I, the method including providing a stevioside substrate, NDP-glucose, and at least one engineered glycosyltransferase provided herein, combining the stevioside substrate, NDP-glucose, and glycosyltransferase under conditions such that rebaudioside A and / or rebaudioside I is produced.

[0042] The present invention also provides a method for producing rebaudioside D, the method including providing a stevioside substrate, NDP-glucose, and at least one engineered glycosyltransferase provided herein, combining the stevioside substrate, NDP-glucose, glycosyltransferase under conditions such that rebaudioside D is produced.

[0043] In some embodiments of the methods provided herein, the NDP-glucose is selected from ADP-glucose, CDP-glucose, TDP-glucose, GDP-glucose, and / or IDT-glucose. In some further embodiments, the NDP-glucose is not UDP-glucose.

[0044] The present invention also provides a method for producing rebaudioside M, the method including providing a rebaudioside D and / or rebaudioside I substrate, ADP-glucose, and at least one engineered glycosyltransferase provided herein, combining the rebaudioside D and / or rebaudioside I substrate, ADP-glucose, and glycosyltransferase under conditions such that rebaudioside M is produced.

[0045] The present invention also provides methods for producing rebaudioside A and / or rebaudioside I, the method including providing a stevioside substrate, ADP-glucose, and at least one engineered ADP-glycosyltransferase provided herein, combining the stevioside substrate, ADP-glucose, and glycosyltransferase under conditions such that rebaudioside A and / or rebaudioside I is produced.

[0046] The present invention also provides a method for producing rebaudioside D, the method including providing a stevioside substrate, ADP-glucose, and at least one engineered glycosyltransferase provided herein, combining the stevioside substrate, ADP-glucose, and glycosyltransferase under conditions such that rebaudioside D is produced.

[0047] The present invention also provides a method for producing rebaudioside M, the method including providing a rebaudioside D and / or rebaudioside I substrate, an NDP, sucrose, sucrose synthase, and at least one engineered glycosyltransferase provided herein, combining the rebaudioside D substrate, NDP, sucrose, sucrose synthase, and glycosyltransferase under conditions such that rebaudioside M is produced.

[0048] The present invention also provides a method for producing rebaudioside A and / or rebaudioside I, the method including providing a stevioside substrate, an NDP, sucrose, sucrose synthase, and at least one engineered glycosyltransferase provided herein, combining the stevioside substrate, NDP, sucrose, sucrose synthase, and glycosyltransferase under conditions such that rebaudioside A and / or rebaudioside I is produced.

[0049] The present invention also provides a method for producing rebaudioside D, the method including providing a stevioside substrate, an NDP, sucrose, a sucrose synthase, and at least one engineered glycosyltransferase provided herein, combining the stevioside substrate, the NDP, sucrose, sucrose synthase, and the glycosyltransferase under conditions such that rebaudioside D is produced.

[0050] The present invention also provides a method for producing rebaudioside M, the method comprising providing a stevioside substrate comprising at least one stevioside and / or a mixture of stevioside and rebA, an NDP, sucrose, a sucrose synthase, and at least one engineered glycosyltransferase provided herein, combining the stevioside substrate, NDP, sucrose, sucrose synthase, and glycosyltransferase under conditions such that rebaudioside M is produced.

[0051] The present invention also provides a method for producing rebaudioside M, the method including providing a stevioside substrate, an NDP, sucrose, at least one sucrose synthase, and at least one engineered glycosyltransferase provided herein, combining the stevioside substrate, the NDP, and the glycosyltransferase under conditions such that rebaudioside A is produced first, followed by rebaudioside D and / or rebaudioside I, and finally by rebaudioside M.

[0052] In some embodiments of the methods provided herein, the sucrose synthase is an engineered sucrose synthase provided herein. In some further embodiments of the methods provided herein, the method is performed as a one-pot reaction. In some alternative embodiments, the method is performed sequentially. In yet some additional embodiments, the method further comprises repeating the steps of the method. In some further embodiments, sucrose is recycled throughout the repeated steps. In some additional embodiments, the engineered glycosyltransferase and / or other reaction components are recycled. In yet some further embodiments, the stevioside substrate is extracted from Stevia rebaudiana. In some additional embodiments, the stevioside substrate is produced synthetically. In yet some further embodiments, the glycosyltransferase and / or sucrose synthase are immobilized. In some further embodiments, the method produces a reaction product comprising fructose. In yet some further embodiments, fructose is removed from the reaction product. In some embodiments, the method further comprises a washing step. In some additional embodiments, the method further comprises at least one column chromatography step. In yet some additional embodiments of the method, the at least one engineered glycosyltransferase is a beta-1,2-glycosyltransferase selected from the glycosyltransferases provided herein. In some additional embodiments of the method, the at least one engineered glycosyltransferase is a beta-1,3-glycosyltransferase selected from the glycosyltransferases provided herein. In some embodiments of the method, the at least one engineered glycosyltransferase is a beta-1,2-glycosyltransferase selected from the glycosyltransferases provided herein, further comprising a beta-1,3-glycosyltransferase selected from the glycosyltransferases provided herein. In some embodiments, the method further comprises at least one engineered sucrose synthase provided herein.

[0053] The present invention also provides at least one rebaudioside produced according to the methods provided herein. The present invention also provides compositions comprising a rebaudioside produced according to the methods provided herein. In some embodiments, rebaudioside M is produced according to the methods provided herein. The present invention also provides compositions comprising rebaudioside M produced according to the methods provided herein.

[0054] In some embodiments, rebaudioside A is produced according to the methods provided herein. The present invention also provides compositions comprising rebaudioside A produced according to the methods provided herein. In some embodiments, rebaudioside I is produced according to the methods provided herein. The present invention also provides compositions comprising rebaudioside I produced according to the methods provided herein. In some embodiments, rebaudioside D is produced according to the methods provided herein. The present invention also provides compositions comprising rebaudioside D produced according to the methods provided herein. In some embodiments, a mixture of rebaudiosides is produced according to the methods provided herein. The present invention also provides compositions comprising a mixture of rebaudiosides produced according to the methods provided herein. In some embodiments, the mixture comprises at least two compositions comprising at least one rebaudioside produced according to the methods provided herein. The present invention provides compositions comprising a mixture of at least two rebaudiosides provided herein. [Brief description of the drawings]

[0055] [Figure 1]1 provides an enzymatic reaction scheme in which a glycosyltransferase catalyzes the transfer of a glucosyl group from a nucleoside diphosphoglucose (NDP-glucose), e.g., ADP-glucose, to an acceptor, e.g., R-OH, where R is any glycosyl, alkoxy, carboxy, aminocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, aminoalkyl, haloalkyl, alkylthioalkyl, heterocycloalkyl, heteroaryl, or heteroarylalkyl group. In a further embodiment, R-OH is stevioside or rebaudioside D and the product is rebaudioside A, rebaudioside I, or rebaudioside M. A nucleoside diphosphate-dependent synthase catalyzes the transfer of a glucosyl group from a glucose donor (e.g., sucrose) to a nucleoside diphosphate, regenerating NDP-glucose and releasing a by-product (e.g., fructose).

[0056] [Diagram 2] FIG. 2 provides the structure of rebaudioside M with carbon numbering.

[0057] [Diagram 3] FIG. 3 provides the structure of rebaudioside I with the carbons numbered. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0058] The present invention provides engineered glycosyltransferase (GT) enzymes, polypeptides having GT activity, and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. The present invention provides engineered sucrose synthase (SuS) enzymes, polypeptides having SuS activity, and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. The present invention further provides compositions comprising GT enzymes, and methods of using engineered GT enzymes to generate products with β-glucose linkages. The present invention further provides compositions and methods for the production of rebaudioside (e.g., rebaudioside M, rebaudioside A, rebaudioside I, and rebaudioside D). The present invention also provides compositions comprising SuS enzymes, and methods of using them. Methods of producing GT and SuS enzymes are also provided.

[0059] Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by those skilled in the art to which this invention belongs. In general, the nomenclature used herein and the experimental procedures of cell culture, molecular genetics, microbiology, organic chemistry, analytical chemistry and nucleic acid chemistry described below are well known and commonly used in the art. Such techniques are well known and described in numerous textbooks and references well known to those skilled in the art. Standard techniques or their modified forms are used for chemical synthesis and chemical analysis. All patents, patent applications, papers and publications mentioned herein both above and below are hereby expressly incorporated by reference into this specification.

[0060] Any suitable method and material similar or equivalent to those described herein can be used in the practice of the present invention, and some methods and materials are described herein.It should be understood that the present invention is not limited to the specific methodology, protocols and reagents described, since these methodologies, protocols and reagents can vary depending on the context in which they are used by those skilled in the art.Therefore, the terms defined immediately below are more fully explained by referring to the present invention in general.

[0061] It should be understood that both the summary above and the detailed description below are exemplary and explanatory only and are not restrictive of the present invention. The section headings used herein are for organizational purposes only and should not be considered as limiting the subject matter described. Numerical ranges are inclusive of the numbers that define the range. Thus, every numerical range disclosed herein is intended to include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein. Every maximum (or minimum) numerical limit disclosed herein is also intended to include every lower (upper) numerical limit, as if such lower (upper) numerical limit were expressly written herein.

[0062] Abbreviations and Definitions Abbreviations used for genetically encoded amino acids are conventional and are as follows: alanine (Ala or A), arginine (Are 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). When three-letter abbreviations are used, the amino acids may be in the L-configuration or the D-configuration about the alpha carbon (Cα), unless specifically preceded by "L" or "D" or otherwise clear from the context in which the abbreviation is used. For example, "Ala" indicates alanine with no configuration specified about the alpha carbon, while "D-Ala" and "L-Ala" indicate D-alanine and L-alanine, respectively. When one-letter abbreviations are used, capital letters indicate amino acids in the L-configuration about the alpha carbon, and lower case letters indicate amino acids in the D-configuration about the alpha carbon. For example, "A" indicates L-alanine and "a" indicates D-alanine. When a polypeptide sequence is presented as a series of one-letter or three-letter abbreviations (or mixtures thereof), the sequence is presented in the amino (N) to carboxy (C) direction according to common convention.

[0063] The abbreviations used for genetically encoded nucleosides are conventional and are as follows: adenosine (A), guanosine (G), cytidine (C), thymidine (T), and uridine (U). Unless otherwise specified, the abbreviated nucleosides may be ribonucleosides or 2'-deoxyribonucleosides. Nucleosides may be designated individually or collectively as either ribonucleosides or 2'-deoxyribonucleosides. When a nucleic acid sequence is presented as a series of one-letter abbreviations, the sequence is presented in the 5' to 3' direction according to common convention, with no phosphate indicated.

[0064] With respect to the present invention, technical and scientific terms used in the description herein shall have the meanings commonly understood by those skilled in the art unless otherwise defined. Accordingly, the following terms are intended to have the following meanings:

[0065] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, "a polypeptide" includes more than one polypeptide.

[0066] Similarly, "comprise," "comprises," "comprising," "include," "includes," and "including" are interchangeable and are not intended to be limiting. Thus, as used herein, the term "comprising" and its cognates are used in their inclusive sense (i.e., equivalent to the term "including" and its corresponding cognates).

[0067] Those skilled in the art will understand that where the description of various embodiments uses the term "comprising," in some specific instances the embodiments may instead be described using the words "consisting essentially of" or "consisting of."

[0068] As used herein, the term "about" refers to an acceptable error for a particular value. In some cases, "about" refers to within 0.05%, 0.5%, 1.0%, or 2.0% of a given value range. In some cases, "about" refers to within 1 standard deviation, 2 standard deviations, 3 standard deviations, or 4 standard deviations of a given value.

[0069] As used herein, "EC" numbers refer to the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) enzyme nomenclature. The IUBMB biochemical classification is a numerical classification system for enzymes based on the chemical reactions they catalyze.

[0070] As used herein, "ATCC" refers to the American Type Culture Collection, whose biorepository collection includes genes and strains.

[0071] As used herein, "NCBI" refers to the National Center for Biological Information and the sequence databases provided therein.

[0072] "Protein," "polypeptide," and "peptide" are used interchangeably herein to refer to a polymer of at least two amino acids covalently linked by amide bonds, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). This definition includes D-amino acids, L-amino acids, mixtures of D- and L-amino acids, polymers comprising D-amino acids, polymers comprising L-amino acids, and polymers comprising mixtures of D- and L-amino acids.

[0073] "Amino acids" are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted one-letter codes.

[0074] As used herein, "polynucleotide" and "nucleic acid" refer to two or more nucleotides that are covalently linked together. A polynucleotide may be composed entirely of ribonucleotides (i.e., RNA), entirely of 2' deoxyribonucleotides (i.e., DNA), or a mixture of ribonucleotides and 2' deoxyribonucleotides. Nucleosides are typically linked together by standard phosphodiester linkages, although a polynucleotide may contain one or more non-standard linkages. A polynucleotide may be single-stranded or double-stranded, or may contain both single-stranded and double-stranded regions. Additionally, a polynucleotide is typically composed of naturally occurring coding nucleobases (i.e., adenine, guanine, uracil, thymine, and cytosine), but may also contain one or more modified and / or synthetic nucleobases, such as, for example, inosine, xanthine, hypoxanthine, and the like. In some embodiments, such modified or synthetic nucleobases are nucleobases that code for an amino acid sequence.

[0075] As used herein, "nucleoside" refers to a glycosylamine that includes a nucleobase (i.e., a nitrogenous base) and a five-carbon sugar (e.g., ribose or deoxyribose). Non-limiting examples of nucleosides include cytidine, uridine, adenosine, guanosine, thymidine, and inosine. In contrast, the term "nucleotide" refers to a glycosylamine that includes a nucleobase, a five-carbon sugar, and one or more phosphate groups. In some embodiments, a nucleoside can be phosphorylated by a kinase to produce a nucleotide.

[0076] As used herein, "nucleoside diphosphate" refers to a glycosylamine that includes a nucleobase (i.e., a nitrogenous base), a five-carbon sugar (e.g., ribose or deoxyribose), and a diphosphate (i.e., pyrophosphate) moiety. In some embodiments herein, "nucleoside diphosphate" is abbreviated as "NDP." Non-limiting examples of nucleoside diphosphate include cytidine diphosphate (CDP), uridine diphosphate (UDP), adenosine diphosphate (ADP), guanosine diphosphate (GDP), thymidine diphosphate (TDP), and inosine diphosphate. The terms "nucleoside" and "nucleotide" may be used interchangeably in some contexts.

[0077] As used herein, "coding sequence" refers to that portion of a nucleic acid (eg, a gene) that codes for the amino acid sequence of a protein.

[0078] As used herein, the terms "biocatalysis," "biocatalysis," "biotransformation," and "biosynthesis" refer to the use of enzymes to carry out chemical reactions on organic compounds.

[0079] As used herein, "glycosyltransferase" (GT) refers to a polypeptide that is enzymatically capable of transferring glycosyl residues from an activated sugar donor to monomeric and multimeric acceptor molecules. In some embodiments, glycosyltransferases are referred to as "glycosyltransferase variants" or "glycosyltransferase combination variants." In some embodiments, "glycosyltransferase" refers to UDP-glucuronosyltransferase enzymes of classification EC 2.4.1.17 that catalyze the transfer of glucose (also known as UDP-glucuronoside) from UDP-α-D-glucuronic acid to an acceptor, releasing UDP to form an acceptor β-D-glucuronoside. The Carbohydrate-Associated Enzymes Database (CAZy) provides a continuously updated list of glycosyltransferase families. In some embodiments, glycosyltransferases include, but are not limited to, enzymes classified in the GT1 family. In some preferred embodiments, the glycosyltransferase variants of the present invention preferentially utilize ADP-glucose. In some additional embodiments, the glycosyltransferase enzyme of the present invention does not utilize UDP-glucose. In some further embodiments, the glycosyltransferase variant of the present invention utilizes ADP-glucose, CDP-glucose, TDP-glucose, GDP-glucose and / or IDT-glucose, but does not utilize UDP-glucose. Thus, in some preferred embodiments, the present invention provides ADP-glucose-dependent glycosyltransferase (ADP-glycosyltransferase; AGT), CDP-glucose-dependent glycosyltransferase (CDP-glycosyltransferase; CGT), GDP-glucose-dependent glycosyltransferase (GDP-glycosyltransferase; GGT), TDP-glucose-dependent glycosyltransferase (TDP-glycosyltransferase; TGT), and IDP-glucose-dependent glycosyltransferase (IDP-glycosyltransferase; IGT).

[0080] As used herein, "NDP-glycosyltransferase" (NDP-GT) refers to a polypeptide that has the enzymatic ability to transfer glycosyl residues from an activated sugar donor, which is an NDP, to monomeric and polymeric acceptor molecules. In some embodiments, NDP-glycosyltransferases are generally referred to as "glycosyltransferases". Indeed, the term "glycosyltransferase" as used herein encompasses NDP-glycosyltransferases, including but not limited to ADP-glucose-dependent glycosyltransferase (ADP-glycosyltransferase; AGT), CDP-glucose-dependent glycosyltransferase (CDP-glycosyltransferase; CGT), GDP-glucose-dependent glycosyltransferase (GDP-glycosyltransferase; GGT), TDP-glucose-dependent glycosyltransferase (TDP-glycosyltransferase; TGT), and IDP-glucose-dependent glycosyltransferase (IDP-glycosyltransferase; IGT). In some embodiments, the glycosyltransferase enzymes of the invention utilize ADP-glucose, CDP-glucose, TDP-glucose, GDP-glucose and / or IDT-glucose, but not UDP-glucose, hi some additional embodiments, these enzymes are referred to as "variants" or "combination variants" (e.g., ADP-glycosyltransferase variants).

[0081] As used herein, "transglycosylation" refers to a reaction in which a glycosyl residue is transferred from a di-, tri-, or oligosaccharide donor to a non-glycosylated or glycosylated donor molecule.

[0082] As used herein, "transglycosylation" refers to a transglycosylation reaction in which the transferred glycosyl residue is glucose and the di-, tri- or oligosaccharide donor contains glucose.

[0083] As used herein, "glycosylation" refers to the formation of a glycosidic bond between a glycosyl residue and an acceptor molecule.

[0084] As used herein, "glycosylation" refers to the formation of a glycosidic bond between a glucose residue and an acceptor molecule.

[0085] As used herein, "glycosyl" refers to an organic group that is a monovalent free radical or substituent structure obtained by removal of the hemiacetal hydroxyl group from a cyclic form of a monosaccharide, lower oligosaccharide, or oligosaccharide derivative. Glycosyl groups react with inorganic acids (e.g., phosphoric acid) to form esters (e.g., glucose 1-phosphate).

[0086] As used herein, "glycoside" refers to a molecule in which a carbohydrate (e.g., a sugar) is linked to another functional group by a glycosidic bond. Glycosides can be hydrolyzed to produce sugar and non-sugar (i.e., aglycone) components.

[0087] As used herein, the term "steviol glycoside" refers to naturally occurring steviol glycosides (e.g., stevioside, steviol monoside, steviol bioside, rubusoside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside G, rebaudioside C, rebaudioside F, rebaudioside A, rebaudioside I, rebaudioside E, rebaudioside F, rebaudioside I ... Rebaudioside H, Rebaudioside L, Rebaudioside K, Rebaudioside J, Rebaudioside M (also called Rebaudioside X), Rebaudioside D, Rebaudioside N, Rebaudioside O), and synthetic steviol glycosides (e.g., enzymatically glucosylated steviol glycosides), and combinations thereof. The chemical structures of steviol and its glycosides are provided below (see WO 2013 / 176738).

[0088] As used herein, "stevioside substrate" refers to any suitable substance that contains at least one steviol glycoside. [Table A-1] [Table A-2]

[0089] As used herein, "sucrose synthase" refers to a glycosyltransferase enzyme (EC 2.4.1.1.13) that reversibly catalyzes the chemical reaction of NDP-glucose + D-fructose to NDP and sucrose. In some embodiments, the present invention provides variants of Acidithiobacillus caldus sucrose synthase ("AcSuS"). In some embodiments, these enzymes are referred to as "sucrose synthase variants", "SuS", "SUS", "SuS variants", "SUS variants", "sucrose synthase combination variants", or "SuS combination variants", or "SUS combination variants". In some embodiments, these variants preferentially utilize NDPs other than uridine (i.e., ADP-glucose, CDP-glucose, TDP-glucose, GDP-glucose and / or IDP-glucose are utilized rather than UDP-glucose). In some embodiments, these variants do not utilize UDP-glucose.

[0090] As used herein, the term "one-pot reaction" refers to the production of a rebaudioside of interest in one reaction vessel. In some embodiments, the term is used in reference to the production of rebM from starting materials, including but not limited to rebA and / or stevioside, with the intermediate production of other rebaudiosides (e.g., rebD and / or rebI). In some embodiments, the conversion of stevioside to RebA, the conversion of RebA to RebD and / or RebI, and the conversion of RebD and / or RebI to RebM are carried out as a multiple enzyme cascade in one reaction vessel.

[0091] As used herein, "wild-type" and "naturally occurring" refer to forms found in nature. For example, a wild-type polypeptide or polynucleotide sequence is one that is present in an organism, that can be isolated from a source in nature, and that has not been intentionally modified by human manipulation.

[0092] As used herein, "recombinant," "engineered," and "non-naturally occurring" when used with respect to a cell, nucleic acid, or polypeptide refer to a material that has been modified in a manner that would not otherwise occur in nature, or a material that corresponds to the natural or native form of the material. In some embodiments, the cell, nucleic acid, or polypeptide is identical to a naturally occurring cell, nucleic acid, or polypeptide, but is produced or derived from synthetic material and / or by manipulation using recombinant techniques. Non-limiting examples include recombinant cells that express genes not found in the native (non-recombinant) form of the cell, or that express native genes that would otherwise be expressed at different levels, among others.

[0093] The term "percent (%) sequence identity" is used herein to refer to a comparison between polynucleotides or polypeptides, and is determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide or polypeptide sequence within the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence due to the optimal alignment of these two sequences. The percentage can be calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the sequence identity percentage. Alternatively, the percentage can be calculated by determining the number of positions where either the same nucleic acid base or amino acid residue occurs in both sequences, or the number of positions where the nucleic acid base or amino acid residue is aligned with a gap to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the sequence identity percentage. Those skilled in the art will appreciate that there are many established algorithms available for aligning two sequences. Optimal alignment of sequences for comparison can be performed by any suitable method, as known in the art, including, but not limited to, the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 2:482

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

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

[1988] ), by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA and TFASTA in the GCG Wisconsin Software Package), or by visual inspection.Examples of algorithms suitable for determining percent sequence identity and percent 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] , respectively). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in a query sequence that match or meet some positive threshold score T when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold (see Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. The cumulative score is calculated using the parameters M (reward score for a matching residue pair, always >0) and N (penalty score for mismatching residues, always <0) for nucleotide sequences. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. The extension of the word hits in each direction is stopped when the cumulative alignment score falls by an amount X from its maximum achieved value; when the cumulative score becomes zero or less due to the accumulation of one or more negatively scoring residue alignments; or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. The BLASTP program for amino acid sequences uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915

[1989] ). Exemplary determinations of sequence alignment and percent sequence identity can utilize the BESTFIT or GAP programs of the GCG Wisconsin Software package (Accelrys, Madison WI) using the default parameters provided.

[0094] As used herein, "reference sequence" refers to a defined sequence used as a standard for sequence and / or activity comparison. A reference sequence may be a subset of a larger sequence, such as a segment of a full-length gene or polypeptide sequence. In general, 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. Since two polynucleotides or polypeptides each may (1) contain a sequence that is similar between the two sequences (i.e., a portion of the complete sequence), and (2) further contain a sequence that differs between the two sequences, sequence comparison between two (or more) polynucleotides or polypeptides is typically performed by comparing the sequences of the two polynucleotides or polypeptides over a "comparison window" to identify and compare local regions of sequence similarity. In some embodiments, a "reference sequence" may be based on a primary amino acid sequence, in which case the reference sequence is a sequence that may have one or more changes in the primary sequence.

[0095] As used herein, a "comparison window" refers to a conceptual segment of at least about 20 contiguous nucleotide positions or amino acid residues within which a sequence can be compared to a reference sequence of at least 20 contiguous nucleotides or amino acids, and the portion of the sequence within the comparison window may contain 20 percent or less additions or deletions (i.e., gaps) compared to the reference sequence (which contains no additions or deletions) for optimal alignment of the two sequences. The comparison window may be longer than 20 contiguous residues, including windows of 30, 40, 50, 100 or more, as appropriate.

[0096] As used herein, "corresponding to," "to," and "compared to" when used in the context of numbering a given amino acid or polynucleotide sequence refer to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue numbers or residue positions of a given polymer are indicated relative to the reference sequence, not according to the actual number positions of the residues in the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, e.g., an engineered glycosyltransferase, can be aligned with a reference sequence by introducing gaps to optimize residue matches between the two sequences. In these cases, although gaps exist, the numbering of the residues in the given amino acid or polynucleotide sequence is done relative to the reference sequence to which it is aligned.

[0097] As used herein, "substantial identity" refers to a polynucleotide or polypeptide sequence having at least 80 percent sequence identity, at least 85 percent identity, between at least 89-95 percent sequence identity, or more usually at least 99 percent sequence identity, relative to a reference sequence, over a comparison window of at least 20 residue positions, often over a window of at least 30-50 residues, where the sequence identity percentage is calculated by comparing the reference sequence over the comparison window to a sequence that contains deletions or additions totaling 20 percent or less of the reference sequence. In some specific embodiments as applied to polypeptides, the term "substantial identity" means that two polypeptide sequences share at least 80 percent sequence identity, preferably at least 89 percent sequence identity, at least 95 percent sequence identity or higher sequence identity (e.g., 99 percent sequence identity), when optimally aligned, e.g., optimally aligned by the programs GAP or BESTFIT using default gap weights. In some embodiments, residue positions that are not identical in the compared sequences differ by conservative amino acid substitutions.

[0098] As used herein, "amino acid difference" and "residue difference" refer to the difference in an amino acid residue at a position of a polypeptide sequence compared to the amino acid residue at the corresponding position in a reference sequence. In some cases, the reference sequence has a histidine tag, but the numbering is maintained compared to an equivalent reference sequence without the histidine tag. The position of the amino acid difference is generally referred to herein as "Xn", where n refers to the corresponding position in the reference sequence to which the residue difference is based. For example, "residue difference at position X93 compared to SEQ ID NO:4" refers to the difference in the amino acid residue at the polypeptide position corresponding to position 93 of SEQ ID NO:4. Thus, if the reference polypeptide of SEQ ID NO:4 has a serine at position 93, then "residue difference at position X93 compared to SEQ ID NO:4" refers to the amino acid substitution of any residue other than serine at the polypeptide position corresponding to position 93 of SEQ ID NO:4. In most cases herein, a particular amino acid residue difference at a position is indicated as "XnY", where "Xn" designates the corresponding position as described above, and "Y" is a one-letter identifier of the amino acid found in the engineered polypeptide (i.e., the residue that differs from that in the reference polypeptide). In some cases (e.g., in the tables presented in the examples), the present invention also provides specific amino acid differences, which are indicated by the conventional designation "AnB", where A is the one-letter identifier of the residue in the reference sequence, "n" is the number of the residue position in the reference sequence, and B is the one-letter identifier of the residue substitution in the sequence of the engineered polypeptide. In some cases, the polypeptides of the present invention contain one or more amino acid residue differences compared to the reference sequence, which are indicated by a list of the specific positions at which the residue difference exists compared to the reference sequence. In some embodiments, when more than one amino acid can be used at a particular residue position of the 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 multiple substitutions within a given variant (i.e., more than one substitution exists in a given sequence, e.g., in a combination variant).In some embodiments, the invention includes engineered polypeptide sequences that include one or more amino acid differences, including conservative or non-conservative amino acid substitutions. In some additional embodiments, the invention provides engineered polypeptide sequences that include both conservative and non-conservative amino acid substitutions.

[0099] As used herein, "conservative amino acid substitution" refers to the substitution of a residue with a different residue that has a similar side chain, and thus typically includes the substitution of an amino acid in a polypeptide with an amino acid within the same or similar defined class of amino acid.By way of example, and without limitation, in some embodiments, an amino acid with an aliphatic side chain is replaced with another aliphatic amino acid (e.g., alanine, valine, leucine and isoleucine); an amino acid with a hydroxyl side chain is replaced with another amino acid with a hydroxyl side chain (e.g., serine and threonine); an amino acid with an aromatic side chain is replaced with another amino acid with an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan and histidine); an amino acid with a basic side chain is replaced with another amino acid with a basic side chain (e.g., lysine and arginine); an amino acid with an acidic side chain is replaced with another amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid); and / or a hydrophobic or hydrophilic amino acid is replaced with another hydrophobic or hydrophilic amino acid, respectively.

[0100] As used herein, "non-conservative substitution" refers to the substitution of an amino acid in a polypeptide with an amino acid that has significantly different side chain properties. Non-conservative substitutions may use amino acids between defined groups rather than within defined groups, and affect (a) the structure of the peptide backbone in the area of ​​the substitution (e.g., substitution of glycine with proline), (b) the charge or hydrophobicity, or (c) the bulk of the side chain. By way of example, and not limitation, exemplary non-conservative substitutions may be an acidic amino acid substituted with a basic or aliphatic amino acid; an aromatic amino acid substituted with a small amino acid; and a hydrophilic amino acid substituted with a hydrophobic amino acid.

[0101] As used herein, "deletion" refers to the modification of a polypeptide by the removal of one or more amino acids from a reference polypeptide. Deletion can 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% of the total number of amino acids, or up to 20% of the total number of amino acids, constituting the reference enzyme, while retaining the enzymatic activity of the engineered glycosyltransferase enzyme and / or retaining improved properties. Deletion can concern internal and / or terminal parts of the polypeptide. In various embodiments, deletion can include a contiguous segment or can be discontinuous. Deletion is typically indicated by "-" in the amino acid sequence.

[0102] As used herein, "insertion" refers to the modification of a polypeptide by the addition of one or more amino acids from a reference polypeptide. The insertion may be in the internal part of the polypeptide or at the carboxy or amino terminus. As used herein, insertion includes fusion proteins as known in the art. The insertion may be a contiguous segment of amino acids in a naturally occurring polypeptide or may be separated by one or more amino acids.

[0103] "Functional fragment" and "biologically active fragment" are used interchangeably herein to refer to a polypeptide that has an amino-terminal and / or carboxy-terminal deletion and / or an internal deletion, but in which the remaining amino acid sequence is identical to the corresponding positions in a sequence to which it is compared (e.g., a full-length engineered glycosyltransferase of the invention), and which retains substantially all of the activity of the full-length polypeptide.

[0104] As used herein, an "isolated polypeptide" refers to a polypeptide that is substantially separated from other contaminants that naturally accompany it (e.g., proteins, lipids, and polynucleotides). The term encompasses polypeptides that have been removed or purified from their naturally occurring environment or expression system (e.g., within a host cell or by in vitro synthesis). Recombinant glycosyltransferase polypeptides may be present within a cell, in cell culture medium, or prepared in various forms, such as a lysate or isolated preparation. Thus, in some embodiments, a recombinant glycosyltransferase polypeptide may be an isolated polypeptide.

[0105] As used herein, a "substantially pure polypeptide" or "purified protein" refers to a composition in which the polypeptide species is the predominant species present (i.e., it is more abundant than any other individual macromolecular species in the composition, on a molar or weight basis), and generally is a substantially purified composition when the species of interest constitutes at least about 50 percent, by molar or weight percent, of the macromolecular species present. However, in some embodiments, a composition comprising a glycosyltransferase comprises a glycosyltransferase that is less than 50% pure (e.g., about 10%, about 20%, about 30%, about 40%, or about 50%). Generally, a substantially pure glycosyltransferase composition comprises 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 percent, of the total macromolecular species present in the composition. In some embodiments, the target species is purified to essential homogeneity such that the composition consists essentially of a single macromolecular species (i.e., contaminant species cannot be detected in the composition by conventional detection methods). Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered macromolecular species. In some embodiments, an isolated recombinant glycosyltransferase polypeptide is a substantially pure polypeptide composition.

[0106] As used herein, "improved enzymatic properties" refers to at least one improved property of an enzyme. In some embodiments, the invention provides engineered glycosyltransferase polypeptides that exhibit an improvement in any enzymatic property compared to a reference glycosyltransferase polypeptide and / or a wild-type glycosyltransferase polypeptide and / or another engineered glycosyltransferase polypeptide. Thus, the level of "improvement" can be determined and compared among various glycosyltransferase polypeptides, including wild-type as well as engineered glycosyltransferases. Improved properties include, but are not limited to, properties such as increased protein expression, increased thermal activity, increased thermostability, increased pH activity, increased stability, increased enzymatic activity, increased substrate specificity or affinity, increased specific activity, increased resistance to substrate or end-product inhibition, increased chemical stability, improved chemical selectivity, improved solvent stability, increased resistance to acidic pH, increased resistance to proteolytic activity (i.e., reduced susceptibility to proteolysis), reduced aggregation, increased solubility, and altered temperature profile. In additional embodiments, the term is used in reference to at least one improved property of a sucrose synthase enzyme. In some embodiments, the present invention provides engineered sucrose synthase polypeptides that show an improvement in any enzymatic property compared to a reference sucrose synthase polypeptide and / or a wild-type sucrose synthase polypeptide and / or another engineered sucrose synthase polypeptide. Thus, the level of "improvement" can be determined and compared between various sucrose synthase polypeptides, including wild-type as well as engineered sucrose synthases.

[0107] As used herein, "increased enzyme activity" and "enhanced catalytic activity" refer to improved properties of an engineered polypeptide, which may be represented by an increase in specific activity (e.g., product produced / time / weight of protein) or an increase in the percent conversion of substrate to product (e.g., the percent conversion of a starting amount of substrate to product within a specified period of time using a specified amount of enzyme) compared to a reference enzyme. In some embodiments, these terms refer to improved properties of an engineered glycosyltransferase polypeptide provided herein, which may be represented by an increase in specific activity (e.g., product produced / time / weight of protein) or an increase in the percent conversion of substrate to product (e.g., the percent conversion of a starting amount of substrate to product within a specified period of time using a specified amount of glycosyltransferase) compared to a reference glycosyltransferase enzyme. In some embodiments, these terms are used in reference to the improved sucrose synthase enzymes provided herein. Exemplary methods for determining the enzymatic activity of engineered glycosyltransferases and sucrose synthases of the invention are provided in the Examples. K m , V max or k cat Any property related to enzyme activity can be affected, including classical enzyme properties (these changes can lead to increased enzyme activity). For example, improvements in enzyme activity can be from about 1.1-fold the enzyme activity of the corresponding wild-type enzyme to as much as 2-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold or more higher enzyme activity than the naturally occurring glycosyltransferase or another engineered glycosyltransferase from which the glycosyltransferase polypeptide is derived.

[0108] As used herein, "conversion" refers to the enzymatic conversion (or biotransformation) of a substrate to a corresponding product. "Percent conversion" refers to the percent of a substrate that is converted to a product under specified conditions within a period of time. Thus, the "enzyme activity" or "activity" of a glycosyltransferase polypeptide can be expressed as the "percent conversion" of a substrate to a product within a particular period of time.

[0109] An enzyme with "generalist properties" (or "generalist enzyme") refers to an enzyme that exhibits improved activity against a broad range of substrates compared to the parent sequence. A generalist enzyme does not necessarily demonstrate improved activity against every possible substrate. In some embodiments, the present invention provides glycosyltransferase variants that have generalist properties because they exhibit similar or improved activity against a broad range of sterically and electronically diverse substrates compared to the parent gene. In addition, the generalist enzymes provided herein have been engineered to be improved across a broad range of diverse molecules to increase production of metabolites / products.

[0110] The term "stringent hybridization conditions" is used herein to refer to conditions under which nucleic acid hybrids are stable. As known to those skilled in the art, hybrid stability is determined by the melting temperature (T m In general, hybrid stability is a function of ionic strength, temperature, G / C content, and the presence of chaotropic agents. m Values ​​can be calculated using known methods for predicting melting temperatures (e.g., Baldino et al., Meth. Enzymol., 168:761-777

[1989] ; Bolton et al., 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] (misprint, Nucl. Acids Res., 19:698

[1991] ); Sambrook et al., supra; Suggs et al., 1981, in Developmental Biology Using Purified Genes, Brown et (see, e.g., J. Am. Soc. 1999, 11:111-115, 1992; Wetmur, Crit. Rev. Biochem. Mol. Biol. 26:227-259, 1991). In some embodiments, the polynucleotides encode the polypeptides disclosed herein and hybridize under defined conditions, such as moderately stringent or highly stringent conditions, to the complement of a sequence encoding an engineered glycosyltransferase enzyme of the invention.

[0111] As used herein, "hybridization stringency" refers to hybridization conditions, such as washing conditions, during nucleic acid hybridization. Generally, hybridization reactions are carried out under conditions of lower stringency, followed by washing of various but higher stringency. The term "moderately stringent hybridization" refers to conditions that allow target DNA to bind to complementary nucleic acids that have about 60% identity to target DNA, preferably about 75% identity, about 85% identity, and more than about 90% identity to target polynucleotide. Exemplary moderately stringent conditions are those that correspond to hybridization in 50% formamide, 5x Denhardt's solution, 5x SSPE, 0.2% SDS at 42°C, followed by washing in 0.2x SSPE, 0.2% SDS at 42°C. "High stringency hybridization" refers to a high stringency hybridization that is achieved by detecting a thermal melting temperature, T, determined under solution conditions for a defined polynucleotide sequence. m In some embodiments, high stringency conditions generally refer to conditions that are about 10° C. to about 0.018 M. It refers to conditions that allow hybridization of only those nucleic acid sequences that form stable hybrids at 65°C in NaCl (i.e., if a hybrid is not stable at 65°C in 0.018M NaCl, it will not be stable under high stringency conditions as contemplated herein). High stringency conditions can be provided, for example, by hybridization in conditions equivalent to 50% formamide, 5x Denhardt's solution, 5x SSPE, 0.2% SDS at 42°C, followed by washing in 0.1x SSPE and 0.1% SDS at 65°C. Another high stringency condition is hybridization in 5x SSC containing 0.1% (w / v) SDS at 65°C and washing in 0.1x SSC containing 0.1% SDS at 65°C. Other high stringency hybridization conditions and moderately stringent conditions are described in the above references.

[0112] As used herein, "codon-optimized" refers to the change of codons in a polynucleotide encoding a protein to those preferentially used in that particular organism such that the encoded protein is efficiently expressed in that organism. Although the genetic code is degenerate in that most amino acids are represented by a few codons, called "synonymous" or "synonymous" codons, it is well known that the codon usage by a particular organism is not random, but is biased toward certain codon triplets. This bias in codon usage may be more severe for a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and aggregated protein-coding regions of the genome of an organism. In some embodiments, a polynucleotide encoding a glycosyltransferase enzyme may be codon-optimized for optimal production in the host organism selected for expression.

[0113] As used herein, "preferred" codons, "optimal" codons and "high codon usage bias" codons, when used alone or in combination, refer synonymously to codons that are used in protein coding regions at a higher frequency than other codons that code for the same amino acid. Preferred codons may be determined with respect to codon usage frequency in a single gene, a set of genes of common function or origin, highly expressed genes, with respect to codon frequency in aggregate protein coding regions across organisms, with respect to codon frequency in aggregate protein coding regions of related organisms, or with respect to combinations thereof. Codons whose frequency increases with gene expression levels are typically optimal codons for expression. A variety of methods are known for determining codon frequencies (e.g., codon usage, relative synonymous codon usage) and codon preferences in a particular organism, including multivariate analyses, for example using cluster analysis or correspondence analysis, as well as for determining the effective number of codons used in a gene (see, e.g., GCG CodonPreference, Genetics Computer Group Wisconsin Package; CodonW, Peden, University of Nottingham; McInerney, Bioinform., 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., supra; Henaut and Danchin, in Escherichia coli and Salmonella, Neidhardt, et al. (eds.), ASM Press, Washington DC, p. 2047-2066

[1996] ). The data source for obtaining codon usage can be based on any available nucleotide sequence capable of encoding a protein. These datasets include nucleic acid sequences that are actually known to encode expressed proteins (e.g., complete protein coding sequences-CDS), to encode expressed sequence tags (ESTS), or to encode predicted coding regions of genomic sequences (see, e.g., Mount, Bioinformatics: Sequence and Genome Analysis, Chapter 8, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

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

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

[1997] ).

[0114] As used herein, "control sequences" include all components necessary or advantageous for the expression of a polynucleotide and / or polypeptide of the present invention. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, a polyadenylation sequence, a propeptide sequence, a promoter sequence, a signal peptide sequence, an initiation sequence, and a transcription terminator. At a minimum, control sequences include a promoter, and transcriptional and translational stop signals. Control sequences may also be provided with linkers to introduce specific restriction sites facilitating ligation of the control sequence with the coding region of the nucleic acid sequence encoding the polypeptide.

[0115] "Operably linked" is defined herein as a configuration in which a control sequence is positioned relative to a polynucleotide of interest such that the control sequence is suitable (i.e., in a functional relationship) to direct or regulate expression of the polynucleotide and / or polypeptide of interest.

[0116] "Promoter sequence" refers to a nucleic acid sequence, e.g., a coding sequence, that is recognized by a host cell for expression of a polynucleotide of interest. The promoter sequence contains a transcriptional control sequence that mediates the expression of the polynucleotide of interest. The promoter can be any nucleic acid sequence that exhibits transcriptional activity in a selected host cell, including mutant, truncated and hybrid promoters, and the promoter can be derived from a gene encoding an extracellular or intracellular polypeptide, either homologous or heterologous to the host cell.

[0117] The phrase "suitable reaction conditions" refers to conditions in an enzymatic conversion reaction solution (e.g., ranges of enzyme loading, substrate loading, temperature, pH, buffers, co-solvents, etc.) under which a glycosyltransferase polypeptide of the invention can convert a substrate into a desired product compound. Some exemplary "suitable reaction conditions" are provided herein.

[0118] As used herein, "loading," such as in "compound loading" or "enzyme loading," refers to the concentration or amount of a component in the reaction mixture at the start of the reaction.

[0119] As used herein, a "substrate" in the context of an enzymatic conversion reaction process refers to a compound or molecule that is acted upon by an engineered enzyme (e.g., an engineered glycosyltransferase polypeptide) provided herein.

[0120] As used herein, the terms "biomass," "biomass substrate," "cellulosic biomass," "cellulosic feedstock," and "cellulosic substrate" refer to any material that contains cellulose. Biomass can be derived from plants, animals, or microorganisms and can include, but is not limited to, agricultural, industrial, and forestry residues, industrial and municipal waste, and terrestrial and aquatic crops grown for energy purposes. Examples of cellulosic substrates include, but are not limited to, wood, wood pulp, paper pulp, corn fiber, corn kernels, corn cobs, crop residues such as corn husks, corn stover, grasses, wheat, wheat straw, barley, barley straw, hay, rice, rice straw, switchgrass, waste paper, paper processing and pulping waste, woody or herbaceous plants, fruit or vegetable pulp, corn cobs, brewer's grains, grasses, rice husks, cotton, hemp, flax, sisal, sugar cane pomace, sorghum, soybeans, switchgrass; ingredients obtained from grinding grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs, bushes, vegetables, fruits and flowers; and any suitable mixtures thereof. In some embodiments, cellulosic biomass includes, but is not limited to, cultivated crops (e.g., grasses, including C4 grasses, such as switchgrass, cordgrass, ryegrass, miscanthus, reed canary grass, or any combination thereof), sugar mill residues, such as, but not limited to, marc (e.g., sugar cane marc, beet pulp [e.g., sugar beet], or combinations thereof), agricultural residues (e.g., soybean stover, corn stover, corn fiber, rice straw, sugar cane straw, rice, rice husk, barley straw, corn cob, wheat straw, canola straw, oat straw, oat husk, corn fiber, hemp, flax, sisal, cotton, or any combinations thereof), fruit pulp, vegetable pulp, brewer's grains, forestry biomass (e.g., wood, wood pulp, paper pulp, recycled wood pulp fiber, sawdust, hardwoods, such as poplar wood, softwoods, or combinations thereof). Additionally, in some embodiments, cellulosic biomass includes cellulosic and / or forestry waste, including, but not limited to, paper processing and pulp processing waste, newsprint, cardboard, and the like.In some embodiments, the cellulosic biomass comprises one type of fiber, while in some alternative embodiments, the cellulosic biomass comprises a mixture of fibers derived from different cellulosic biomass. In some embodiments, the biomass may also comprise transgenic plants expressing ligninase and / or cellulase enzymes (see, e.g., U.S. Patent Application Publication No. 2008 / 0104724, which is incorporated herein by reference).

[0121] As used herein, the term "slurry" refers to an aqueous solution in which one or more solid components, such as a cellulosic substrate, are dispersed.

[0122] As used herein, an "increase" in the yield of a product (e.g., steviol glycosides) from a reaction occurs when a particular component (e.g., a GH enzyme) present in the reaction causes more product to be produced compared to a reaction carried out under the same conditions in the absence of the component of interest but with the same substrates and other substituents.

[0123] As used herein, "hydrolysis" of cellulose or other polysaccharides occurs when at least a portion of the glycosidic bonds between two monosaccharides present in the substrate are hydrolyzed, thereby dissociating the two previously linked monomers from one another.

[0124] A reaction is "substantially free" of a particular enzyme if the amount of that enzyme is less than about 2%, about 1%, or about 0.1% (wt / wt) relative to other enzymes involved in catalyzing the reaction.

[0125] As used herein, "fractionating" a liquid (e.g., a culture broth) means applying a separation process (e.g., salting out precipitation, column chromatography, size exclusion, filtration) or a combination of such processes to obtain a solution in which the desired proteins (e.g., rebaudioside) constitute a higher percentage of total protein in solution than in the initial liquid product.

[0126] As used herein, "starting composition" refers to any composition that includes at least one substrate. In some embodiments, the starting composition includes any cellulosic substrate.

[0127] In some alternative embodiments, the term "starting composition" refers to any composition that includes at least one steviol glycoside, where one or more of the steviol glycosides act as a substrate for biotransformation. In some embodiments, the starting composition is provided as an aqueous solution. In some embodiments, the starting composition comprises at least one glycoside of steviol selected from stevioside, steviol monoside, steviolbioside, rubusoside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside G, rebaudioside C, rebaudioside F, rebaudioside A, rebaudioside I, rebaudioside E, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M (also called rebaudioside X), rebaudioside D, rebaudioside N, rebaudioside O, and synthetic steviol glycosides (e.g., enzymatically glucosylated steviol glycosides). In some embodiments, the starting composition comprises two or more steviol glycosides. In some embodiments, the starting composition is an extract obtained from purification of Stevia rebaudiana plant material (e.g., leaves). In some alternative embodiments, the starting composition comprises a commercially available stevia extract. Additional starting compositions include by-products of processes used to isolate and purify steviol glycosides. In some embodiments, the starting composition comprises a purified or partially purified steviol glycoside substrate. In some embodiments, the starting composition comprises greater than about 99% by weight of a particular steviol glycoside.

[0128] In some embodiments, the starting composition includes at least one glycoside and a cellulosic component as substrates for producing at least one steviol glycoside (e.g., rebaudioside A, D, etc.).

[0129] As used herein, "product" in the context of an enzymatic conversion process refers to a compound or molecule resulting from the action of an enzyme polypeptide on a substrate. As used herein, in some embodiments, the term refers to a compound or molecule resulting from the action of a glycosyltransferase polypeptide on a substrate. In some embodiments, the product provided by the present invention is a steviol glycoside. In some embodiments, the product comprises at least one steviol glycoside selected from stevioside, steviol monoside, steviolbioside, rubusoside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside G, rebaudioside C, rebaudioside F, rebaudioside A, rebaudioside I, rebaudioside E, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M (also called rebaudioside X), rebaudioside D, rebaudioside N, rebaudioside O, and synthetic steviol glycosides (e.g., enzymatically glucosylated steviol glycosides).

[0130] As used herein, the term "culturing" refers to growing a population of microbial cells under any suitable conditions (e.g., using liquid, gel, or solid media).

[0131] Recombinant polypeptides can be produced using any suitable method known in the art. A gene encoding a wild-type polypeptide of interest can be cloned into a vector, such as a plasmid, and expressed in a desired host, such as E. coli. Variants of recombinant polypeptides can be generated by a variety of methods known in the art. Indeed, there are a wide variety of different mutagenesis techniques well known to those skilled in the art. In addition, mutagenesis kits are also available from many commercial molecular biology suppliers. Methods are available that perform specific substitutions at defined amino acids (site-directed), specific or random mutations in local regions of a gene (position-directed), or random mutagenesis throughout a gene (e.g., saturation mutagenesis). A large number of 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 methodology finds use in the present invention and is well known in the art (e.g., U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, 5,837,458, 5,928,905, 6,096,548, 6,117,679, 6,132,970, 6,165,793, 6,180,406, 6,251,674, 6,265,201, 6,277,638, 6,287, ,861, 6,287,862, 6,291,242, 6,297,053, 6,303,344, 6,309,883, 6,319,713, 6,319,714, 6,323,030, 6,326,204, 6,335,160, 6,335,198, 6,344,356, 6,352,859, 6,355,484, 6,358,740, 6,358,742, 6,365,377, 6,365,408, 6,368,861, 6,372,497, 6,337,186, 6,376,246, 6,379,964, 6,387,702, 6,391,552, 6,391,640, 6,395,547, 6,406,855, 6,406,910, 6,413,745, 6,413,774, 6,420,175 ,6,423,542, 6,426,224, 6,436,675, 6,444,468, 6,455,253, 6,479,652, 6,482,647, 6,483,011, 6,484,105, 6,489,146, 6,500,617, 6,500,639, 6,5 06,602, 6,506,603, 6,518,065, 6,519,065, 6,521,453, 6,528,311, 6,537,746, 6,573,098, 6,576,467, 6,579,678, 6,586,182, 6,602,986, 6,605, 430, 6,613,514, 6,653,072, 6,686,515, 6,703,240, 6,716,631, 6,825,001, 6,902,922, 6,917,882, 6,946,296, 6,961,664, 6,995,017, 7,024,312, 7,058,515, 7,105,297, 7,148,054, 7,220,566, 7,288,375, 7,384,387, 7,421,347, 7,430,477, 7,462,469, 7,534,564, 7,620,500, 7,620,502, 7,6 29,170, 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, 7,873,4 99, 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, 9,665,694, 9,684,771, and all related U.S. patents, as well as PCT and non-U.S. counterparts; Ling et al.,Anal. Biochem., 254(2):157-78

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[1994] ; WO95 / 22625; WO97 / 0078; WO97 / 35966; WO98 / 27230; WO00 / 42651; WO01 / 75767; and WO2009 / 152336, all of which are incorporated by reference herein.

[0132] In some embodiments, the enzyme clones obtained after the mutagenesis treatment are screened by subjecting the enzyme preparation to a predetermined temperature (or other assay conditions) and measuring the amount of enzyme activity remaining after heat treatment or other suitable assay conditions. Clones containing the polynucleotides encoding the polypeptides are then isolated from the gene, sequenced to identify nucleotide sequence changes (if any), and used to express the enzyme in a host cell. Measurement of 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).

[0133] After the variants are produced, they can be screened for any desired properties (e.g., high or increased activity or low or decreased activity, increased thermal activity, increased thermostability, and / or acidic pH stability, etc.). In some embodiments, "recombinant glycosyltransferase polypeptides" (also referred to herein as "engineered glycosyltransferase polypeptides," "variant glycosyltransferase enzymes," "glycosyltransferase variants," and "glycosyltransferase combination variants") are used. In some embodiments, "recombinant sucrose synthase polypeptides" (also referred to herein as "engineered sucrose synthase polypeptides," "variant sucrose synthase enzymes," "sucrose synthase variants," and "sucrose synthase combination variants") are used.

[0134] As used herein, a "vector" is a DNA construct for introducing a DNA sequence into a cell. In some embodiments, the vector is an expression vector that is operably linked to a suitable control sequence that can effect the expression of the polypeptide encoded by the DNA sequence in a suitable host. In some embodiments, an "expression vector" has a promoter sequence operably linked to a DNA sequence (e.g., a transgene) for driving expression in a host cell, and in some embodiments, also includes a transcription terminator sequence.

[0135] As used herein, the term "expression" includes any step involved in the production of a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from the cell.

[0136] As used herein, the term "produce" refers to the production of a protein and / or other compound by a cell. The term is intended to encompass any step involved in the production of a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses the secretion of a polypeptide from a cell.

[0137] As used herein, an amino acid or nucleotide sequence (e.g., a promoter sequence, a signal peptide, a terminator sequence, etc.) is "heterologous" to another sequence to which it is operably linked if the two sequences are not naturally associated. For example, a "heterologous polynucleotide" is any polynucleotide that is introduced into a host cell by laboratory techniques, including polynucleotides that have been removed from the host cell, subjected to laboratory manipulation, and then reintroduced into the host cell.

[0138] As used herein, the terms "host cell" and "host strain" refer to a suitable host for an expression vector containing DNA provided herein (e.g., a polynucleotide encoding a glycosyltransferase variant). In some embodiments, a host cell is a eukaryotic or prokaryotic cell that has been transformed or transfected with a vector constructed using recombinant DNA techniques known in the art.

[0139] The term "analog" refers to a polypeptide having greater than 70% sequence identity, but less than 100% sequence identity (e.g., greater than 75%, 78%, 80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) with a reference polypeptide. In some embodiments, an analog refers to a polypeptide that contains not only naturally occurring amino acids, but also one or more non-naturally occurring amino acids, including, but not limited to, homoarginine, ornithine, and norvaline. In some embodiments, an analog also includes one or more D-amino acid residues, and a non-peptide linkage between two or more amino acid residues.

[0140] The term "effective amount" means an amount sufficient to produce a desired result. One of ordinary skill in the art can determine what an effective amount is by using routine experimentation.

[0141] The terms "isolated" and "purified" are used to refer to a molecule (e.g., an isolated nucleic acid, polypeptide, etc.) or other component that has been removed from at least one other component with which it is naturally associated. The term "purified" does not require absolute purity, but rather is intended as a relative definition.

[0142] As used herein, "stereoselectivity" refers to the preferential formation of one stereoisomer over another in a chemical or enzymatic reaction. Stereoselectivity can be partial, when one stereoisomer is preferred over the other, or complete, when only one stereoisomer is formed. When the stereoisomers are enantiomers, stereoselectivity is referred to as enantioselectivity, which is the fraction (typically reported as a percentage) of one enantiomer in the sum of both enantiomers. This is generally instead reported in the art as the enantiomeric excess ("ee") (typically as a percentage), calculated therefrom according to the formula [major enantiomer-minor enantiomer] / [major enantiomer+minor enantiomer]. When the stereoisomers are diastereoisomers, the stereoselectivity is called diastereoselectivity, which is the fraction (typically reported as a percentage) of one diastereomer in a mixture of two diastereomers, and is commonly reported instead as diastereomeric excess ("de"). Enantiomeric excess and diastereomeric excess are types of stereoisomeric excess.

[0143] As used herein, "regioselectivity" and "regioselective reaction" refer to a reaction in which one direction of bond making or cleavage occurs in preference to all other possible directions. A reaction can be completely (100%) regioselective, where discrimination is complete and substantially regioselective (at least 75%), or partially regioselective (x%, where the percentage is set depending on the reaction of interest), where the product of the reaction at one site predominates over the product of the reaction at the other site.

[0144] As used herein, "thermostable" refers to a glycosyltransferase polypeptide that, after exposure to an elevated temperature (e.g., 40-80° C.) for a period of time (e.g., 0.5-24 hours), maintains similar activity (e.g., 60%-80% greater) compared to a wild-type enzyme exposed to the same elevated temperature.

[0145] As used herein, "solvent stable" refers to a glycosyltransferase polypeptide that maintains similar activity (e.g., 60%-80% greater) after exposure to various concentrations (e.g., 5-99%) of a solvent (ethanol, isopropyl alcohol, dimethyl sulfoxide [DMSO], tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene, butyl acetate, methyl tert-butyl ether, etc.) for a period of time (e.g., 0.5-24 hours) compared to a wild-type enzyme exposed to the same solvent at the same concentration.

[0146] As used herein, "thermo- and solvent stable" refers to a glycosyltransferase polypeptide that is both thermostable and solvent stable.

[0147] As used herein, a "reducing agent" is defined as Fe +3 Fe +2 An exemplary reducing agent is ascorbic acid, which is commonly in the form of L-ascorbic acid.

[0148] As used herein, "optionally" and "optionally" mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances in which the event or circumstance occurs and instances in which it does not occur. For any molecule described as containing one or more optional substituents, those of skill in the art will understand that only sterically realistic and / or synthetically feasible compounds are intended to be included. "Optionally substituted" refers to all subsequent modifiers in a chemical group term or set of chemical groups.

[0149] Glycosylation Glycosylation can alter many properties of natural and synthetic products, including stability, pharmacodynamics, solubility and membrane transport. The present invention provides compositions, methods and enzymes suitable for the production of new glycosylated compounds from various aglycones and glycosylated substrates. In some embodiments, the present invention provides a means for the efficient production of known glycosylated compounds from readily available precursors. In some cases, glycosylation is accomplished by chemical synthesis methods. However, these methods generally require undesirable chemicals and processes and can result in mixed products (e.g., with bonds in the wrong position and / or with undesirable anomeric configurations). Furthermore, carbohydrate chemistry requires multiple protection and deprotection steps.

[0150] In contrast, glycosylation enzymes can be active under mild conditions and can confer high regioselectivity and stereospecificity in a single step. Many naturally occurring glycosylated metabolites are generated in vivo using glycosyltransferases that transfer sugar moieties from various sugar nucleosides. Many molecules, including many secondary metabolites with antibacterial, antitumor, natural sweet properties, etc., contain nonribosomal peptide, polyketide or terpenoid backbones modified with β-glycosidic bonds. Many of the diterpene glycosides extracted from the plant, Stevia rebaudiana Bertoni, contain β-linked glucose molecules. Naturally, these molecules are glycosylated in vivo using UDP-glucose-dependent glycosyltransferase enzymes. The present invention provides methods (see FIG. 1 ) that use new engineered glycosyltransferases to transfer a glucose moiety from a nucleoside diphosphoglucose to a substrate (e.g., rebaudioside D or stevioside) to produce one or more β-glucose linked products (e.g., rebaudioside M, rebaudioside A, or rebaudioside I). However, when used in vitro, UDP-glucose can be prohibitively expensive and / or unavailable. In some additional embodiments, a synthase (e.g., sucrose synthase or trehalose synthase) works in the reverse direction to form a nucleoside diphosphoglucose compound from a nucleoside diphosphate and a glucose donor (e.g., sucrose, trehalose, or starch).

[0151] Thus, glycosylation is used to produce natural sweeteners, such as those derived from the aromatic herb, Stevia rebaudiana Bertoni. As mentioned above, this plant produces several diterpene glycosides that are characterized by high-intensity sweetness and sensory properties superior to many other strong sweeteners. The sweet glycosides mentioned above have a common aglycone (i.e., steviol), but differ in the number and type of carbohydrate residues at the C13 and C19 positions. Steviol glycosides differ from each other not only in their molecular structures but also in their taste properties. Generally, stevioside is reported to be 89-143 times sweeter than sucrose, while rebaudioside A is reported to be between 85-242 times sweeter than sucrose (see, for example, Kasai et al., Nippon Kagaku Kaishi, 1981:726-735

[1981] ). Of these common compounds, rebaudioside A is the least astringent, the least bitter, and has the least lingering aftertaste. Thus, it has the most favorable sensory attributes of the major steviol glycosides, and rebaudioside A has been commercialized. However, rebaudioside A constitutes only a small portion (about 20%) of the total glycosides isolated from Stevia rebaudiana Bertoni, with stevioside (about 70%) and minor steviol glycosides making up the remainder (e.g., FAO, Chemical and Technical Assessment, 63 rd (See JECFA, Steviol Glycosides

[2004] ). A naturally occurring but even less abundant compound, rebaudioside M, also known as rebaudioside X, is 200-350 times sweeter than sucrose and has a reduced aftertaste compared to rebaudioside A (See, e.g., Prakash et al., Food, 3:162-175

[2014] ). Thus, there is interest in commercializing rebaudioside M, for example as a natural sweetener, but there is currently no viable commercial synthetic route for this compound.

[0152] Engineered glycosyltransferase polypeptides The present invention provides glycosyltransferase polypeptides, polynucleotides encoding the polypeptides, methods of preparing the polypeptides, and methods of using the polypeptides. It is understood that where a description refers to a polypeptide, the description also describes the polynucleotide encoding the polypeptide. In some embodiments, the present invention provides engineered, non-naturally occurring GT enzymes with improved properties compared to wild-type GT enzymes. Any suitable reaction conditions are used in the present invention. In some embodiments, the method is used to analyze the improved properties of engineered polypeptides for performing transferase reactions. In some embodiments, the reaction conditions are improved with respect to conditions including concentration or amount of polypeptide, substrate, co-substrate, buffer, co-solvent, pH, temperature and reaction time, and / or conditions associated with the polypeptide immobilized on a solid support, as further described below and in the Examples.

[0153] In some embodiments, the engineered GT polypeptides described herein have improved properties compared to wild-type GT enzymes, e.g., improved properties in terms of conversion of steviol glycosides to further glycosylated steviol glycosides (e.g., stevioside to rebaudioside A, or rebaudioside D to rebaudioside M) and in terms of usage of adenine diphosphoglucose or other nucleoside diphosphates.

[0154] In some embodiments, additional reaction components or techniques are utilized to supplement the reaction conditions, including taking measures to stabilize enzymes or prevent enzyme inactivation, to reduce product inhibition, or to shift the equilibrium of the reaction toward glycosylated product formation.

[0155] In some further embodiments, any of the above processes for converting substrate compounds to product compounds may further comprise one or more steps selected from: extraction of product compounds; isolation; purification; and crystallization, filtration or lyophilization.Methods, techniques and protocols for extracting, isolating, purifying and / or crystallizing glycosylation products from the biocatalytic reaction mixture produced by the above-disclosed process are known to those skilled in the art and / or obtained by routine experimentation.In addition, illustrative methods are provided in the following examples.

[0156] Engineered sucrose synthase polypeptides The present invention provides engineered sucrose synthase (SuS) polypeptides, polynucleotides encoding the polypeptides, methods of preparing the polypeptides, and methods of using the polypeptides. It is understood that where a description refers to a polypeptide, the description also describes the polynucleotide encoding the polypeptide. In some embodiments, the present invention provides engineered, non-naturally occurring SuS enzymes with improved properties compared to wild-type SuS enzymes. Any suitable reaction conditions are used in the present invention. In some embodiments, the method is used to analyze the improved properties of the engineered polypeptides for performing synthase reactions. In some embodiments, the reaction conditions are improved with respect to conditions including the concentration or amount of engineered SuS, substrates, buffers, solvents, pH, temperature and reaction time, and / or conditions associated with the engineered SuS polypeptide immobilized on a solid support, as further described below and in the Examples.

[0157] In some embodiments, the engineered SuS polypeptides described herein have improved properties compared to wild-type SuS enzymes, e.g., improved properties in terms of the reactions described herein.

[0158] In some embodiments, additional reaction components or techniques are utilized to supplement the reaction conditions, including taking measures to stabilize enzymes or prevent enzyme inactivation, to reduce product inhibition, or to shift the equilibrium of the reaction toward glycosylated product formation.

[0159] In some further embodiments, any of the above processes for conversion of substrate compounds to product compounds may further comprise one or more steps selected from extraction, isolation, purification, crystallization, filtration and / or lyophilization of the product compounds. Methods, techniques and protocols for extracting, isolating, purifying and / or crystallizing products (e.g., rebaudioside) from biocatalytic reaction mixtures produced by the processes provided herein are known to those of skill in the art and / or obtained by routine experimentation. Additionally, illustrative methods are provided in the Examples below.

[0160] Polynucleotides encoding engineered polypeptides, expression vectors and host cells The present invention provides a polynucleotide that encodes the engineered enzyme polypeptide described herein.In some embodiments, the polynucleotide is operably linked to one or more heterologous regulatory sequences that control gene expression to produce a recombinant polynucleotide that can express the polypeptide.In some embodiments, the expression construct that contains at least one heterologous polynucleotide that encodes the engineered enzyme polypeptide is introduced into a suitable host cell to express the corresponding enzyme polypeptide.

[0161] As will be apparent to those skilled in the art, the availability of a protein sequence and knowledge of the codons corresponding to various amino acids provides a description of all polynucleotides that can code for a polypeptide of interest. The degeneracy of the genetic code, in which the same amino acid is coded for by alternative or synonymous codons, allows for the creation of a very large number of nucleic acids, all of which code for an engineered enzyme (e.g., GT or SuS) polypeptide. Thus, the present invention provides methods and compositions for the production of any and all possible variations of enzyme polynucleotides that can be made to code for the enzyme polypeptides described herein by selecting combinations based on possible codon choices, and all such variations are contemplated and specifically disclosed for any polypeptide described herein, including the amino acid sequences presented in the examples (e.g., in the various tables).

[0162] In some embodiments, codons are preferably optimized for use by the selected host cell for protein production. For example, the preferred codons used in bacteria are typically used for expression in bacteria. Thus, the codon-optimized polynucleotide encoding the engineered enzyme polypeptide contains preferred codons at about 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% of the codon positions in the full-length coding region.

[0163] In some embodiments, the enzyme polynucleotide encodes an engineered polypeptide having the enzymatic activity and properties disclosed herein, and comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a reference sequence selected from the SEQ ID NOs provided herein, or the amino acid sequence of any variant (e.g., those provided in the Examples) and one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residue positions) residue differences compared to the amino acid sequence of the reference polynucleotide or any variant disclosed in the Examples.

[0164] In some embodiments, the polynucleotide can hybridize under highly stringent conditions to a reference polynucleotide sequence selected from any of the polynucleotide sequences provided herein, or its complementary sequence, or to a polynucleotide sequence encoding any of the variant enzyme polypeptides provided herein. In some embodiments, the polynucleotide that can hybridize under highly stringent conditions encodes an enzyme polypeptide that includes an amino acid sequence that has one or more residue differences compared to the reference sequence.

[0165] In some embodiments, the isolated polynucleotide encoding any of the engineered enzyme polypeptides herein is engineered in various ways to facilitate expression of the enzyme polypeptide. In some embodiments, the polynucleotide encoding the enzyme polypeptide comprises an expression vector in which one or more control sequences are present to regulate the expression of the enzyme polynucleotide and / or polypeptide. Manipulation of the isolated polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector utilized. Techniques for modifying polynucleotides and nucleic acid sequences using recombinant DNA methods are well known in the art. In some embodiments, the control sequences include, among others, promoters, leader sequences, polyadenylation sequences, propeptide sequences, signal peptide sequences, and transcription terminators. In some embodiments, a suitable promoter is selected based on the host cell selection. For bacterial host cells, suitable promoters for directing transcription of the nucleic acid constructs of the disclosure include, but are not limited to, promoters obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and prokaryotic beta-lactamase genes (e.g., Villa-Kamaroff et al., Proc. Natl Acad. Sci. USA 75: 3727-3731).

[1978] ), as well as the tac promoter (see, e.g., DeBoer et al., Proc. Natl Acad. Sci. USA 80: 21-25

[1983] ).Exemplary promoters for filamentous fungal host cells include, but are not limited to, promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid-stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (see, e.g., WO 96 / 00787), as well as the NA2-tpi promoter (a promoter from the gene for Aspergillus niger neutral alpha-amylase and the Aspergillus oryzae triosephosphate isomerase gene), as well as mutants, truncations and hybrid promoters thereof. Exemplary yeast cell promoters can be derived from the 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, for example, Romanos et al., Yeast 8:423-488

[1992] ).

[0166] In some embodiments, the control sequence is also a suitable transcription terminator sequence (i.e., a sequence recognized by a host cell to terminate transcription). In some embodiments, the terminator sequence is operably linked to the 3' end of the nucleic acid sequence encoding the enzyme polypeptide. Any suitable terminator that is functional in the host cell of choice can be used in the present invention. Exemplary transcription terminators for filamentous fungal host cells can be obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease. Exemplary terminators for yeast host cells can be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are known in the art (see, for example, Romanos et al., supra).

[0167] In some embodiments, the control sequence is also a suitable leader sequence (i.e., a non-translated region of an mRNA that is important for translation by the host cell). In some embodiments, the leader sequence is operably linked to the 5' end of the nucleic acid sequence encoding the enzyme polypeptide. Any suitable leader sequence that is functional in the host cell of choice finds use in the present invention. Exemplary leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, and Aspergillus nidulans triose phosphate isomerase. Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0168] In some embodiments, the control sequence is also a polyadenylation sequence (i.e., a sequence operably linked to the 3' end of a nucleic acid sequence, which, when transcribed, is recognized by a host cell as a signal for the addition of polyadenosine residues to the transcribed mRNA). Any suitable polyadenylation sequence that is functional in the host cell of choice can be used in the present invention. Exemplary polyadenylation sequences for filamentous fungal host cells include, but are not limited to, the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase. Useful polyadenylation sequences for yeast host cells are known (see, e.g., Guo and Sherman, Mol. Cell. Biol., 15:5983-5990

[1995] ).

[0169] In some embodiments, the control sequence is also a signal peptide (i.e., a coding region that encodes an amino acid sequence linked to the amino terminus of a polypeptide that directs the encoded polypeptide into the secretory pathway of a cell). In some embodiments, the 5' end of the coding sequence of the nucleic acid sequence inherently contains a signal peptide coding region that is naturally linked in translation reading frame with the segment of the coding region that encodes the polypeptide to be secreted. Alternatively, in some embodiments, the 5' end of the coding sequence contains a signal peptide coding region that is foreign to the coding sequence. Any suitable signal peptide coding region that directs the expressed polypeptide into the secretory pathway of a chosen host cell is used to express the engineered polypeptide. Effective signal peptide coding regions for bacterial host cells include those encoding Bacillus NCIB 11837 maltogenic amylase, Bacillus Examples of signal peptide coding regions that may be used include, but are not limited to, those from the genes of Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM), and Bacillus subtilis prsA. Additional signal peptides are known in the art (see, e.g., Simonen and Palva, Microbiol. Rev., 57:109-137

[1993] ). In some embodiments, useful signal peptide coding regions for filamentous fungal host cells include, but are not limited to, the signal peptide coding regions obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase. Useful signal peptides for yeast host cells include, but are not limited to, those from the genes for Saccharomyces cerevisiae alpha factor and Saccharomyces cerevisiae invertase.

[0170] In some embodiments, the control sequence is also a propeptide coding region that codes for an amino acid sequence located at the amino terminus of a polypeptide. The resulting polypeptide is referred to as a "proenzyme," "propolypeptide," or "zymogen." A propolypeptide can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding region can be obtained from any suitable source, including, but not limited to, the genes of Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae alpha factor, Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophila lactase (see, for example, WO95 / 33836). When both the signal peptide region and the propeptide region are present at the amino terminus of a polypeptide, the propeptide region is located adjacent to the amino terminus of the polypeptide, and the signal peptide region is located adjacent to the amino terminus of the propeptide region.

[0171] In some embodiments, regulatory sequences are also utilized. These sequences aid in regulating the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those that cause gene expression to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. In prokaryotic host cells, suitable regulatory sequences include, but are not limited to, the lac, tac, and trp operator systems. In yeast host cells, suitable regulatory sequences include, but are not limited to, the ADH2 system or the GAL1 system. In filamentous fungi, suitable regulatory sequences include, but are not limited to, the TAKA alpha-amylase promoter, the Aspergillus niger glucoamylase promoter, and the Aspergillus oryzae glucoamylase promoter.

[0172] In another aspect, the present invention relates to a recombinant expression vector comprising a polynucleotide encoding an engineered enzyme polypeptide and one or more expression control regions, such as a promoter and terminator, an origin of replication, etc., depending on the type of host to be introduced. In some embodiments, the various nucleic acids and control sequences described herein are linked together to generate a recombinant expression vector that comprises one or more convenient restriction sites to allow for the insertion or replacement of the nucleic acid sequence encoding the 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 comprising the sequence into an appropriate vector for expression. In some embodiments, including the creation of an expression vector, the coding sequence is located within the vector such that the coding sequence is operably linked to the appropriate control sequence for expression.

[0173] The recombinant expression vector can be any suitable vector (e.g., plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can cause the expression of the enzyme polynucleotide sequence.The selection of the vector typically depends on the compatibility of the vector with the host cell into which the vector will be introduced.The vector can be a linear plasmid or a closed circular plasmid.

[0174] In some embodiments, the expression vector is a self-replicating vector (i.e., a vector that exists as an extrachromosomal entity and whose replication is not dependent on chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome). The vector may contain any means to ensure self-replication. In some alternative embodiments, the vector may be one that, when introduced into a host cell, integrates into the genome and replicates together with the chromosome into which it is integrated. Furthermore, in some embodiments, a single vector or plasmid, or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell and / or into a transposon, are utilized.

[0175] In some embodiments, the expression vector contains one or more selectable markers that allow easy selection of transformed cells. A "selectable marker" is a gene whose product confers biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. 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. orzyae), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), and equivalents thereof. In another aspect, the invention provides a host cell comprising at least one polynucleotide encoding at least one engineered enzyme polypeptide of the invention, wherein the polynucleotide is operably linked to one or more control sequences for expression of the engineered enzyme(s) in the host cell.Suitable host cells for use in expressing the polypeptides encoded by the expression vectors of the invention are well known in the art and include, but are not limited to, bacterial cells, such as E. coli, Vibrio fluvialis, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178)); insect cells, such as Drosophila S2 and Spodoptera Sf9 cells; animal cells, such as CHO, COS, BHK, 293 and Bowes melanoma cells; and plant cells. Exemplary host cells also include various Escherichia coli strains (e.g., W3110 (ΔfhuA) and BL21).

[0176] Thus, in another aspect, the invention provides a method for producing an engineered enzyme polypeptide, the method comprising culturing a host cell capable of expressing a polynucleotide encoding the engineered enzyme polypeptide under conditions suitable for expression of the polypeptide, in some embodiments the method further comprises isolating and / or purifying the enzyme polypeptide as described herein.

[0177] Suitable culture medium and growth conditions for host cells are well known in the art.Any suitable method for introducing polynucleotide into cells for expression of enzyme polypeptide is contemplated to be used in the present invention.Suitable techniques include, but are not limited to, electroporation, biolistic particle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion.

[0178] Various features and embodiments of the present invention are illustrated in the following representative examples, which are intended to be illustrative and not limiting. EXAMPLES

[0179] experiment The following examples, including the experiments performed and the results, are provided for illustrative purposes only and should not be construed as limiting the invention. Indeed, there are a variety of suitable sources for many of the reagents and equipment listed below. It is not intended that the invention be limited to any particular source for any reagent or equipment item.

[0180] In the experiments disclosed below, the following abbreviations apply: M (molar); mM (millimolar), uM and μM (micromolar); nM (nanomolar); mol (mole); gm and g (grams); mg (milligrams); ug and μg (micrograms); L and l (liters); ml and mL (milliliters); cm (centimeters); mm (millimeters); um and μm (micrometers); sec. (seconds); min (minutes); h and hr (hours); U (units); MW (molecular weight); rpm (revolutions per minute) );psi and PSI (pounds per square inch);℃ (degrees Celsius);RT and rt (room temperature);CV (coefficient of variation);CAM and cam (chloramphenicol);PMBS (polymyxin B sulfate);IPTG (isopropyl β-D-1-thiogalactopyranoside);LB (Luria Broth);TB (Terrific Broth);SFP (Shake Flask Powder);CDS (coding sequence);DNA (deoxyribonucleic acid);RNA (ribonucleic acid);nt (nucleotide; polynucleotide);aa (amino acid; polypeptide);E. coli W3110 (a commonly used laboratory strain of E. coli available from the Coli Genetic Stock Center [CGSC], New Haven, CT).coli strains); AcSus (Acidithiobacillus caldus sucrose synthase); SUS, SuS and SuSy (sucrose synthase, also known as sucrose synthetase); NDP (nucleoside diphosphate); adenosine diphosphate (ADP); cytidine diphosphate (CDP); guanosine diphosphate (GDP); thymidine diphosphate (TDP); uridine diphosphate (UDP); inosine diphosphate (IDP); GT (glycosyltransferase); UGT (UDP-glucose-dependent glycosyltransferase); NGT (NDP-nucleoside diphosphate-dependent glycosyltransferase); AGT (ADP-glucose-dependent glycosyltransferase); P-glucose-dependent glycosyltransferase;CGT (CDP-glucose-dependent glycosyltransferase);GGT (GDP-glucose-dependent glycosyltransferase);TGT (TDP-glucose-dependent glycosyltransferase);IGT (IDP-glucose-dependent glycosyltransferase);UGT (UDP-glucose-dependent glycosyltransferase);reb (rebaudioside);rebA (rebaudioside A);rebD (rebaudioside D);rebI (rebaudioside I);rebM (rebaudioside M);"Reb A 60" is an approximately 1:2 mixture of stevioside and rebaudioside A, respectively; HTP (high throughput); HPLC (high pressure liquid chromatography); HPLC-UV (HPLC-ultraviolet-visible detector); 1H NMR (proton nuclear magnetic resonance spectroscopy); HSQC NMR (heteronuclear single quantum coherence spectroscopy NMR); COSY NMR (homonuclear correlation spectroscopy NMR); Acorn (Acorn NMR, Livermore, CA); FIOPC (fold improvement over positive control); Sigma and Sigma-Aldrich (Sigma-Aldrich, St.Louis, MO; Difco (Difco Laboratories, BD Diagnostic Systems, Detroit, MI); Microfluidics (Microfluidics, Westwood, MA); ChromaDex (ChromaDex, Inc., Irvine, CA); Life Technologies (Life Technologies, Fisher Amresco (Amresco, LLC, Solon, OH); Carbosynth (Carbosynth, Ltd., Berkshire, UK); Varian (Varian Medical Systems, Palo Alto, CA); Agilent (Agilent Technologies, Inc., Santa Clara, CA); .

[0181] Example 1 Synthesis, optimization and assay This example describes the methods used to synthesize, optimize, and assay UGT enzymes with glucosylation activity.

[0182] Gene synthesis and optimization: A polynucleotide sequence (SEQ ID NO:1) encoding a wild-type Stevia rebaudiana polypeptide (SEQ ID NO:2), which has been reported to glucosylate steviolbioside to rebaudioside B and stevioside to rebaudioside A (see, e.g., Richman et al., Plant J., 2002). 41:56-67

[2005] ) was codon optimized and synthesized as the gene of SEQ ID NO:3. This synthetic gene (SEQ ID NO:3) was cloned into the pCK110900 vector system (see, e.g., U.S. Patent Application Publication No. 2006 / 0195947, which is hereby incorporated by reference), and then expressed in E. coli W3110(ΔfhuA). The E. coli strain W3110 expressed the UGT enzyme under the control of the lac promoter. Evolution rounds were performed as described in the Examples below.

[0183] Production of shake flask powder (SFP): A shake flask procedure was used to generate glycosyltransferase polypeptide shake flask powders (SFPs) for characterization assays used in the biocatalytic processes described herein. Shake flask powder (SFP) preparation of enzymes yields more purified preparations of enzymes (e.g., up to >30% total protein) compared to cell lysates used in HTP assays and also allows for the use of more concentrated enzyme solutions. A single colony of E. coli containing a plasmid encoding an engineered polypeptide of interest was inoculated into 5 mL of Luria-Bertani broth containing 30 μg / mL chloramphenicol and 1% glucose. Cells were grown overnight (at least 16 hours) in a 30° C. incubator with shaking at 250 rpm. The culture was diluted into 250 mL of Terrific Broth (12 g / L bacto-tryptone, 24 g / L yeast extract, 4 mL / L glycerol, 65 mM potassium phosphate, pH 7.0, 1 mM MgSO4) containing 30 μg / ml CAM in a 1 L flask to an optical density at 600 nm (OD600) of 0.2 and grown at 30°C.

[0184] When the culture reached an OD600 of 0.6-0.8, expression of the glycosyltransferase genes was induced by adding IPTG to a final concentration of 1 mM. Incubation was then continued overnight (at least 16 hours). Cells were harvested by centrifugation (5000 rpm, 15 min, 4°C) and the supernatant was discarded. The cell pellet was resuspended in 2 volumes of 25 mM triethanolamine buffer, pH 7.5, and passed through a MICROFLUIDIZER® high pressure homogenizer (Microfluidics) set for standard E. coli lysis and maintained at 4°C. Cell debris was removed by centrifugation (10,000 rpm, 45 min, 4°C). The clarified lysate supernatant was collected, frozen at -80°C, and then either His affinity purified and dialyzed to yield purified protein or lyophilized to yield a dry shake flask powder of crude protein.

[0185] Assay of SFP for stevioside glucosylation: SFP was reconstituted to give a 20 g / L powder. 50 μL of these stocks were then diluted in a total reaction volume of 200 μL of 50 mM Tris-HCl buffer, pH 7.5, with 3 mM MgSO4 and 1 mM stevioside (ChromaDex, purity >94%) and 2 mM uridine diphosphoglucose. Reactions were carried out at 30°C for 16-18 hours with shaking at 300 RPM in a THERMOTRON® titer plate shaker.

[0186] HPLC-MS / MS analysis: The reaction was quenched with 0.5 vol / vol acetonitrile with 0.2% formic acid and precipitated by centrifugation. Glycosylated stevioside products were detected in the supernatant by LC-MS / MS using the following instruments and parameters: [Table 1]

[0187] Activity was detected for SEQ ID NO: 4. A high degree of conversion of stevioside to rebaudioside A (i.e., >95%) was observed in LC-MS / MS analysis of the above assay samples.

[0188] Example 2 GT variant of SEQ ID NO:4 This example describes an experiment in the evolution and screening of GT polypeptides derived from SEQ ID NO:4 for improved glucosylation of steviol glycosides using ADP-glucose. Directed evolution of GT encoded by SEQ ID NO:3 (i.e., SEQ ID NO:4) was performed by constructing libraries of variant genes with mutagenesis at positions related to certain structural features of the enzyme. These libraries were then plated, grown, and screened using high-throughput (HTP) assays described below to obtain first-round ("round 1") engineered GT variant polypeptides with glucosyltransferase activity to ADP-glucose and steviol glycosides. Many additional rounds of evolution were performed by constructing libraries of variant genes that recombined the mutations associated with activity improvement in round 1 and then with mutations associated with activity improvement in subsequent evolution rounds. These libraries were then plated, grown, and screened using high-throughput (HTP) growth, expression, lysate preparation, and assays described below.

[0189] HTP growth, expression and lysate preparation Cells were harvested in 96-well plates and grown overnight at 30° C., 200 rpm, and 85% humidity in LB medium containing 1% glucose and 30 μg / mL CAM. 20 μL of the overnight growth was then transferred to a deep-well plate containing 380 μL TB growth medium containing 30 μg / mL CAM, induced with 1 mM IPTG, and incubated for 18 hours at 30° C., 200 rpm, and 85% humidity. The cell culture was centrifuged at 4000 rpm and 4° C. for 10 minutes and the medium was discarded. The cell pellet thus obtained was frozen at −80° C. and lysed in 250 μL of lysis buffer (0.5 g / L lysozyme and 0.5 g / L PMBS in 20 mM Tris-HCl buffer, pH 7.5) with slow shaking at room temperature for 2 hours on a titer plate shaker. The plates were then centrifuged at 4000 rpm and 4° C. for 20 minutes and the clarified lysate supernatants were used in the HTP assay reactions described below.

[0190] HTP assay for glucose transfer from ADP-glucose to stevioside The assay was performed using 96-well plates of clarified E. coli culture lysates expressing SEQ ID NO:3 variants with a lysate load of 50 μL lysate in a 200 μL reaction, and a substrate load of 1 mM stevioside (ChromaDex, purity >94%) from a 20 mM stock solution in 50% ethanol, and a co-substrate load of 0.5 mM ADP-glucose (Sigma, purity >93%). The following reaction conditions were used: 50 mM Tris-HCl buffer, pH 7.5, 3 mM MgCl, 18 hours with shaking at 300 RPM in a THERMOTRON® titer plate shaker. 2 , 30° C. Reactions were quenched with 100 μL / well of acetonitrile with 0.2% formic acid, centrifuged for 10 min at 4° C., and the supernatants were analyzed by HPLC-MS / MS as described in Example 1, Table 1.1.

[0191] The formation of rebaudioside A from stevioside in the presence of wild-type UGT76G1 (SEQ ID NO: 4) and ADP-glucose was indistinguishable from the no enzyme control. In contrast to the wild-type enzyme of SEQ ID NO: 4, glycosyltransferase variant polypeptides were identified that produced rebaudioside A from stevioside and ADP-glucose. These engineered polypeptides are listed in Table 2.1. The parent and variant constructs contained an N-terminal histidine tag for affinity purification, but for clarity, mutations are numbered relative to the untagged reference sequence. Shake flask scale cultures of variants produced during multiple evolution rounds were grown for protein purification as described in Example 1.

[0192] Example 3 Synthesis, optimization, and assay of glycosyltransferase enzymes with glucosylation activity This example describes the methods used to synthesize, optimize, and assay UGT enzymes with glucosylation activity.

[0193] Gene synthesis and optimization Polynucleotide sequences encoding the beta-1,2-glycosyltransferase Solanum tuberosum were codon-optimized and synthesized as the gene of SEQ ID NO: 11. These synthetic genes were cloned into the pCK110900 vector system (see, e.g., U.S. Pat. No. 9,714,437, which is hereby incorporated by reference) and then expressed in E. coli W3110 (ΔfhuA). The E. coli strain W3110 expressed the UGT enzyme under the control of the lac promoter.

[0194] Production of shake flask powder (SFP) A shake flask procedure was used to generate glycosyltransferase polypeptide shake flask powders (SFPs) for characterization assays used in the biocatalytic processes described herein. Shake flask powder (SFP) preparation of enzymes results in more purified preparations of enzymes (e.g., up to >30% total protein) compared to cell lysates used in HTP assays and also allows for the use of more concentrated enzyme solutions. A single colony of E. coli containing a plasmid encoding an engineered polypeptide of interest was inoculated into 5 mL of Luria-Bertani broth containing 30 μg / ml chloramphenicol and 1% glucose. Cells were grown overnight (at least 16 hours) in a 30° C. incubator with shaking at 250 rpm. 250 mL of Terrific Broth (12 g / L Bacto-tryptone, 24 g / L Yeast Extract, 4 mL / L Glycerol, 65 mM Potassium Phosphate, pH 7.0, 1 mM MgSO 4 ) containing 30 μg / ml CAM in a 1 L flask. 4 ) and diluted the culture to an optical density at 600 nm (OD600) of 0.2 and grown at 30°C.

[0195] When the culture reached an OD600 of 0.6-0.8, expression of the glycosyltransferase genes was induced by adding IPTG to a final concentration of 1 mM. Incubation was then continued overnight (at least 16 hours). Cells were harvested by centrifugation (5000 rpm, 15 min, 4°C) and the supernatant was discarded. The cell pellet was resuspended in 2 volumes of 25 mM triethanolamine buffer, pH 7.5, and passed through a MICROFLUIDIZER® high pressure homogenizer (Microfluidics) set for standard E. coli lysis and maintained at 4°C. Cell debris was removed by centrifugation (10,000 rpm, 45 min, 4°C). The clarified lysate supernatant was collected, frozen at -80°C, and then either His affinity purified and dialyzed to yield purified protein or lyophilized to yield a dry shake flask powder of crude protein.

[0196] Assay for Rebaudioside A Glucosylation in Purified Proteins First, 50 μL of purified protein was diluted in a total reaction volume of 200 μL consisting of 50 mM Tris-HCl buffer pH 7.5, 3 mM magnesium chloride, 1 mM rebaudioside A, and 0.5 mM uridine diphosphoglucose. The reaction was carried out for 18 hours at 30° C. with shaking at 300 RPM in a THERMOTRON® titer plate shaker. A boiled enzyme reaction was used as a negative control. 10 μL of the reaction was quenched with 90 μL of acetonitrile with 0.2% formic acid and precipitated by centrifugation. Glycosylated rebaudioside A products were detected in the supernatant by LC-MS / MS as described in Example 1, Table 1.1. Despite poor soluble expression, SEQ ID NO: 12 demonstrated high specific activity and good selectivity for the generation of β-1,2-glucose linkages in steviol glycoside substrates.

[0197] Assay for rebaudioside A glucosylation in shake flask powder The lyophilized shake flask powders were reconstituted to 20 mg / mL. 10 μL of these stocks were then added to 10 mL of 3 mM MgCl 2 and diluted in 50 mM potassium phosphate (KPhos) buffer, pH 7 with 1 mM rebaudioside A (purity >97%) and 2 mM uridine diphosphoglucose (UDP-glucose) in a total reaction volume of 100 μL. The reaction was carried out for 16-18 hours at 40° C. with shaking at 300 RPM in a THERMOTRON® titer plate shaker. Higher activity than the negative control was detected for SEQ ID NO: 12. Low conversion (i.e., <10%) of rebaudioside A to rebaudioside D was observed by LC-MS / MS analysis.

[0198] Example 4 GT variant of SEQ ID NO: 12 This example describes an experiment in evolving and screening GT polypeptides derived from SEQ ID NO:12 for improved glucosylation of steviol glycosides. Directed evolution of GT encoded by SEQ ID NO:11 (i.e., SEQ ID NO:12) was performed by constructing combinatorial libraries of variant genes with mutagenesis at positions relevant to the surface residues of the enzyme. These libraries were then plated, grown, and screened using the high-throughput (HTP) assays described below to obtain a first round ("round 1") of engineered GT variant polypeptides with β-1,2-glucosyltransferase activity to steviol glycosides. Many additional rounds of evolution were performed by constructing libraries of variant genes that recombined the mutations associated with activity improvement in round 1 and then with mutations associated with activity improvement in subsequent evolution rounds. These libraries were then plated, grown, and screened using the high-throughput (HTP) growth, expression, lysate preparation, and assays described below.

[0199] HTP growth, expression and lysate preparation Cells were harvested in 96-well plates and grown overnight at 30° C., 200 rpm, and 85% humidity in LB medium containing 1% glucose and 30 μg / mL CAM. 20 μL of the overnight growth was then transferred to a deep-well plate containing 380 μL TB growth medium containing 30 μg / mL CAM, induced with 1 mM IPTG, and incubated for 18 hours at 30° C., 200 rpm, and 85% humidity. The cell culture was centrifuged at 4000 rpm and 4° C. for 10 minutes and the medium was discarded. The cell pellet thus obtained was frozen at −80° C. and lysed in 250 μL of lysis buffer (0.5 g / L lysozyme and 0.5 g / L PMBS in 20 mM Tris-HCl buffer, pH 7.5) with slow shaking at room temperature for 2 hours on a titer plate shaker. The plates were then centrifuged at 4000 rpm and 4° C. for 20 minutes and the clarified lysate supernatants were used in the HTP assay reactions described below.

[0200] HTP Assay for Rebaudioside A Glucosylation Assays were performed using 96-well plates of clarified E. coli culture lysates expressing the enzyme variants, with a lysate load of 25 μL lysate in a 100 μL reaction, and a substrate load of 1 mM rebaudioside A (Sigma, purity >96%) from a 20 mM stock solution in 50% ethanol, and a co-substrate load of 0.5 mM UDP-glucose (Sigma, purity >98%). The following reaction conditions were used: 4 hours with shaking at 300 RPM in a THERMOTRON® titer plate shaker, 50 mM Tris-HCl buffer, pH 7.5, 3 mM MgCl 2 , 30°C. Reactions were quenched with 0.5 vol / vol acetonitrile with 0.2% formic acid and precipitated by centrifugation for 10 min at 4°C. After 1:20 dilution with water, glycosylation products were detected in the supernatant by LC-MS / MS using the instrument and parameters described in Example 1, Table 1.1. Glycosyltransferase variant polypeptides producing rebaudioside D from rebaudioside A in greater amounts than the reference backbone sequence were identified and used for subsequent evolution rounds. For analysis of variants, shake flask scale cultures for lyophilized powder production were grown as described in Example 1.

[0201] Shake flask lysate characterization assay and analysis of rebaudioside A glucosylation First, 250 mL shake flask cultures were grown, induced, and lysed. Cell debris was removed by centrifugation as described in Example 1, and the clarified lysate supernatant was collected. 10 μL of the lysate was then diluted with 50 mM Tris-HCl buffer, pH 7.5 and 3 mM MgCl 2and 1 mM Rebaudioside A (Sigma, purity >96%) and 2 mM UDP-glucose (Sigma, purity >98%) in a total reaction volume of 100 μL. The reaction was carried out for 0-18 hours at 30° C. with shaking at 300 RPM in a THERMOTRON® titer plate shaker. The above reaction was quenched with 0.5 vol / vol acetonitrile with 0.2% formic acid and precipitated by centrifugation. After 1:20 dilution with water, glycosylation products were detected in the supernatant by LC-MS / MS using the instrument and parameters described in Example 1, Table 1.1.

[0202] Example 5 Sucrose synthase variant of SEQ ID NO:6 This example describes an evolution and screening experiment of sucrose synthase (SuS) polypeptides derived from the codon-optimized Acidothiobacillus caldus sucrose synthase polynucleotide (SEQ ID NO:5) for improved production of ADP-glucose from sucrose and ADP. Directed evolution of SuS encoded by SEQ ID NO:5 (i.e., SEQ ID NO:6) was performed by constructing libraries of variant genes that were subjected to saturation mutagenesis at positions related to certain structural features of the enzyme and recombined with diversity from homologs in public databases. These libraries were then plated, grown, and screened using high-throughput (HTP) assays described below to obtain first-round ("round 1") engineered SuS variant polypeptides with improved activity towards the synthesis of ADP-glucose. Many additional rounds of evolution were performed by constructing libraries of variant genes that recombined the mutations associated with activity improvement in round 1 and then with mutations associated with activity improvement in subsequent evolution rounds. These libraries were then plated, grown, and screened using high-throughput (HTP) growth, expression, lysate preparation, and assays described below.

[0203] HTP assay for glucose transfer from sucrose to ADP The assay was performed using 96-well plates of clarified E. coli culture lysates expressing SEQ ID NO:5 variants (i.e., variants of SEQ ID NO:6) with a lysate load of 25 μL lysate in a 100 μL reaction, and a substrate load of 30% w / v sucrose (Sigma) from a 60% stock solution in water, and a co-substrate load of 2 mM ADP (Sigma, purity >95%). The following reaction conditions were used: 2 hours in a Thermocycler, 50 mM Tris-HCl buffer, pH 7.5, 3 mM MgCl 2 , 30°C. Reactions were heat quenched at 95°C for 10 min and then analyzed by a colorimetric D-fructose dehydrogenase assay adapted from the literature (see, e.g., Ameyama et al., J. Bacteriol., 145:814-823

[1981] ; and Ameyama, Meth. Enzymol., 89:20-29

[1982] ). Briefly, overnight enzyme-linked assays were performed in 96-well plates with 20 μL of sample diluted to a fructose concentration of <1 g / L, 20 μL of 100 mM potassium ferricyanide (Sigma P-8131), and 160 μL of 0.8 units / mL fructose dehydrogenase (Sigma F4892) dissolved in pH 4.6 McIlvain's buffer with 0.1% Triton® X-100. This reaction converts fructose to K 4 Fe(CN) 6 therefore, 67 μL of the overnight reaction was added to 33 μL of stop solution (0.3 w / v% sodium dodecyl sulfate, Sigma L-4509, 8.1 v / v% phosphoric acid, Sigma P-6560, and 0.5 w / v% ferric sulfate, Sigma F-1135) and shaken for 20 min to obtain K 4 Fe(CN) 6 The K was determined by allowing complete conversion of Prussian blue to Prussian blue and reading the absorbance at 690 nm using a plate reader. 4 Fe(CN) 6After the primary assay, engineered sucrose synthase (SuS) variant polypeptides with higher fructose-forming activity, and therefore higher stoichiometric ADP-glucose-forming activity, than SEQ ID NO:6 were screened in triplicate at a lower substrate load of 2 w / v% sucrose (Sigma) and a co-substrate load of 1 mM ADP (Sigma, >95%).

[0204] HTP binding assay for glucose transfer from sucrose to ADP and then to rebaudioside D The library was screened using the following HTP enzyme-linked assay: Pelleted E. coli culture was lysed in 250 μL of Tris-HCl, pH 7.5 with 1 mM magnesium sulfate and 0.5 mg / mL lysozyme and polymyxin B sulfate (PMBS) and clarified by centrifugation. The lysate was diluted 20-fold by adding Tris-HCl, pH 7.5. 10 μL of the diluted SuS lysate and 2 g / L of GT SEQ ID NO:8 were then combined with a substrate load of approximately 1 mM rebaudioside D and a co-substrate load of 1 mM ADP (Sigma, >95%) and 10 mM sucrose (Sigma) in a 100 μL reaction volume. The following reaction conditions were used: 50 mM potassium phosphate buffer, pH 7, 3 mM MgCl for 2 hours with shaking at 300 RPM in a THERMOTRON® titer plate shaker. 2 , 50° C. The reactions were quenched by adding 10 μL assay mixture to 90 μL acetonitrile with 0.2% formic acid and precipitated by centrifugation. The supernatant was diluted 10-fold with water and steviol glycoside products were detected by RapidFire SPE-MS / MS using the instrument and parameters listed in Table 5.1. [Table 5]

[0205] Production of shake flask powder (SFP) A shake flask procedure was used to generate glycosyltransferase polypeptide shake flask powders (SFPs) for characterization assays used in the biocatalytic processes described herein. Shake flask powder (SFP) preparation of enzymes results in more purified preparations of enzymes (e.g., up to >30% total protein) compared to cell lysates used in HTP assays and also allows for the use of more concentrated enzyme solutions. A single colony of E. coli containing a plasmid encoding an engineered polypeptide of interest was inoculated into 5 mL of Luria-Bertani broth containing 30 μg / ml chloramphenicol and 1% glucose. Cells were grown overnight (at least 16 hours) in a 30° C. incubator with shaking at 250 rpm. 250 mL of Terrific Broth (12 g / L Bacto-tryptone, 24 g / L Yeast Extract, 4 mL / L Glycerol, 65 mM Potassium Phosphate, pH 7.0, 1 mM MgSO 4 ) containing 30 μg / ml CAM in a 1 L flask. 4 ) and diluted the culture to an optical density at 600 nm (OD600) of 0.2 and grown at 30°C.

[0206] Shake flask powder characterization assay and analysis of glucosyl transfer from sucrose to rebaudioside D Experiments were conducted to characterize the activity of engineered SuS variants towards sucrose and ADP to promote the formation of rebaudioside M from rebaudioside D. Shake flask powder (SFP) was added at a concentration of 0.125 g / L to a total reaction volume of 100 μL containing 50 mM potassium phosphate buffer, pH 7, 3 mM magnesium chloride, 1 g / L rebaudioside D, 10 mM sucrose, 1 mM ADP, and 2 g / L GT SEQ ID NO:10. The reaction was carried out at 50° C. for 1 hour with shaking at 300 RPM in a THERMOTRON® titer plate shaker. The reaction was quenched by adding 10 μL of the reaction mixture to 90 μL acetonitrile with 0.2% formic acid and precipitated by centrifugation. The supernatant was diluted 10-fold with water and analyzed for steviol glycosides by RapidFire SPE-MS / MS using the instrument and parameters listed in Table 5.1. Based on these results, additional rounds of evolution were performed to optimize the enzyme.

[0207] Example 6 Sucrose synthase variants The directed evolution of the sucrose synthase enzyme was continued by constructing libraries of variant genes that recombined the mutations associated with the improved activity in the previous evolutionary rounds. These libraries were then plated, grown, and screened using the high-throughput (HTP) assay described below to obtain additional iterative rounds of engineered SuS variant polypeptides with activity towards the production of ADP-glucose.

[0208] HTP binding assay for glucose transfer from sucrose to ADP and then to rebaudioside A The following HTP enzyme binding assay was used to screen the combinatorial library: Pelleted E. coli culture was lysed in 400 μL of Tris-HCl, pH 7.5 with 1 mM magnesium sulfate and 0.5 mg / mL lysozyme and polymyxin B sulfate (PMBS) and clarified by centrifugation. The lysate was diluted approximately 90-fold by adding Tris-HCl, pH 7.5. 10 μL of diluted SuS lysate and 1 g / L GT SEQ ID NO:14 were then combined with a substrate load of 4.5-7.5 mM rebaudioside A97 and a co-substrate load of 0.2-0.25 mM ADP (Sigma, >95%) and 30 mM sucrose (Sigma) in a 100 μL reaction volume. The following reaction conditions were used: 50 mM potassium phosphate buffer, pH 6, 55° C. for 2 hours with shaking at 300 RPM in a THERMOTRON® titer plate shaker. The above reactions were solubilized by adding 10 μL of assay to 90-190 μL of water, quenched by adding 10 μL of solubilized assay to 90 μL acetonitrile with 0.2% formic acid, and precipitated by centrifugation. The supernatants were diluted 4.4-6.7 times with water and steviol glycoside products were detected by RapidFire SPE-MS / MS using the instrument and parameters listed in Table 5.1. After analysis, engineered SuS variant polypeptides were identified that conjugated with GT and showed improved activity towards rebaudioside A.

[0209] Shake flask powder characterization assay and analysis of glucosyl transfer from sucrose to rebaudioside A Experiments were performed to characterize the activity of engineered SuS variants towards sucrose and ADP to promote the formation of rebaudioside D from rebaudioside A. Shake flask powder (SFP) was added at a concentration of 0.02 g / L to a total reaction volume of 100 μL containing 50 mM potassium phosphate buffer, pH 6, 7.5 mM rebaudioside A97, 30 mM sucrose, 0.2 mM ADP, and 1 g / L GT SEQ ID NO:14. The reaction was carried out at 50° C. for 1 hour with shaking at 300 RPM in a THERMOTRON® titer plate shaker. The reaction was solubilized by adding water to dilute 20-fold, quenched by adding 10 μL of the diluted reaction to 90 μL acetonitrile with 0.2% formic acid, and precipitated by centrifugation. The supernatant was diluted 4.4-fold with water and analyzed for steviol glycosides by RapidFire SPE-MS / MS using the instrument and parameters listed in Table 5.1. Variants with higher activity than the reference sequence and higher levels of rebaudioside D produced from rebaudioside A were selected for further directed evolution for catalysis of the salvage reaction transferring glucose from sucrose to ADP.

[0210] Example 7 Beta-1,3-ADP-glycosyltransferase variant of SEQ ID NO: 20 This example describes an evolution and screening experiment of β1,3-glycosyltransferase (β1,3GT) polypeptides derived from SEQ ID NO:8598 for improved glucosylation of steviol glycosides using in situ synthesized ADP-glucose. Directed evolution of GT encoded by SEQ ID NO:19 (i.e., SEQ ID NO:20) was performed by constructing libraries of variant genes that were engineered with mutations associated with improved production identified during the development of the present invention and that were subjected to saturation mutagenesis of certain structural features. These libraries were then plated, grown, and screened using a high-throughput (HTP) assay described below to obtain another round of 72 engineered GT variant polypeptides with glucosyltransferase activity toward ADP-glucose and steviol glycosides.

[0211] HTP assay for glucose transfer from sucrose to ADP, followed by glucose transfer from ADP-glucose to rebaudioside A60 The assay was performed using 96-well plates of clarified E. coli culture lysates expressing SEQ ID NO:19 variants (i.e., variants of SEQ ID NO:20). The pellets were lysed and the lysates were clarified as described in Example 6, then diluted 10-fold into 50 mM potassium phosphate buffer, pH 6. To heat challenge the lysates, they were pre-incubated at 75° C. for 1 hour with shaking at 300 RPM in a Thermotron® titer plate shaker. The assay was performed using 10 μL of pre-incubated lysate in a 100 μL reaction with 20 g / L rebaudioside A 60% substrate, 0.025 g / L ADP (Amresco, Ultra Pure Grade) co-substrate, 0.05 g / L SUS SFP SEQ ID NO:18, 0.12 g / L β-1,2-glycosyltransferase (β12GT) SEQ ID NO:16, and 40 g / L sucrose. The following reaction conditions were used: 50 mM potassium phosphate buffer, pH 6, 60° C., 16 hours with shaking at 300 RPM in a THERMOTRON® titer plate shaker. The reaction was solubilized by 20-fold dilution with water, quenched by 5-fold dilution with acetonitrile with 0.2% formic acid, and diluted 20-fold with water for analysis. Samples were analyzed by RapidFire-MS / MS as described in Example 5, Table 5.1. Glycosyltransferase variant polypeptides were identified that produce greater amounts of rebaudioside M from rebaudioside A60 using in situ synthesized ADP-glucose than SEQ ID NO: 20. These engineered polypeptides are listed in Tables 7.1 and 7.2. For analysis of the variants shown in Table 7.3 compared to SEQ ID NO: 20, shake flask scale cultures were grown, lysed, clarified, and lyophilized to a powder as described in Example 1. [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4]

[0212] SFP characterization assay and analysis of glucosyl transfer from sucrose to ADP and then ADP-glucose to stevioside and rebaudioside D Shake flask powder (SFP) was reconstituted to a concentration of 20 g / L and diluted to 0.005-0.15 g / L of SFP in a total reaction volume of 100 μL with 50 mM potassium phosphate buffer, pH 6, 15 mM stevioside (purity >95%) or rebaudioside D, 0.02 g / L ADP (Amresco, Ultra Pure Grade) cosubstrate, 0.05 g / L SUS SFP SEQ ID NO: 18, and 37.5 mM sucrose. Reactions were incubated at 60°C for 4 hours with shaking at 300 RPM in a Thermotron® titer plate shaker. Reactions were solubilized by 20-fold dilution in water, quenched by 5-fold dilution in acetonitrile with 0.2% formic acid, clarified by centrifugation, and diluted 15-fold in water for analysis. Glycosylation products were detected by SPE-QQQ as described in Example 5, Table 5.1. One-pot reactions were performed using 0.01-0.3 g / L SFP in a total reaction volume of 100 μL with 50 mM potassium phosphate buffer, pH 6, 20 g / L RebA60, 0.02 g / L ADP (Amresco, Ultra Pure Grade) cosubstrate, 0.05 g / L SUS SFP SEQ ID NO:18, 0.12 g / L β1,2GT SFP SEQ ID NO:16, and 30 g / L sucrose. Reactions were incubated at 60°C for 16 hours with shaking at 300 RPM in a THERMOTRON® titer plate shaker. Reactions were solubilized by 20-fold dilution with water, quenched by 5-fold dilution with acetonitrile with 0.2% formic acid, clarified by centrifugation, and diluted 20-fold with water for analysis. A similar one-pot reaction was also performed using 10 μL of crude shake flask lysate after 20-fold dilution in 50 mM potassium phosphate buffer, pH 6, and incubation at 75.1° C. for 24 hours. [Table 7-5]

[0213] In these experiments, at least four variants in Table 7.3 produced more rebaudioside M from rebaudioside A60 than SEQ ID NO: 20, and three variants also produced more rebaudioside A from stevioside, and rebaudioside M from rebaudioside D. All variants were less thermostable than SEQ ID NO: 20. SEQ ID NO: 36 was selected as the starting point for further enzyme engineering due to its superior performance in the one-pot assay and slight reduction in thermostability.

[0214] Example 8 Beta-1,3-ADP-glycosyltransferase variant of SEQ ID NO: 36 This example describes an evolution and screening experiment of β1,3-glycosyltransferase (β1,3GT) polypeptides derived from SEQ ID NO:36 for improved glucosylation of steviol glycosides using in situ synthesized ADP-glucose. Directed evolution of GT encoded by SEQ ID NO:35 (i.e., SEQ ID NO:36) was performed by constructing libraries of variant genes engineered with mutations associated with improved production identified during the development of the present invention and targeted to the N-terminal coding region. These libraries were then plated, grown, and screened using a high-throughput (HTP) assay described below to obtain another round of 58 engineered GT variant polypeptides with glucosyltransferase activity towards ADP-glucose and steviol glycosides.

[0215] HTP assay for glucose transfer from sucrose to ADP, followed by glucose transfer from ADP-glucose to rebaudioside A60 The assay was performed using 96-well plates of clarified E. coli culture lysates expressing SEQ ID NO:35 (i.e., a variant of SEQ ID NO:36). The pellets were lysed and the lysates were clarified as described in Example 6, then diluted 20-fold into 50 mM potassium phosphate buffer, pH 6. To heat challenge the lysates, they were pre-incubated at 75° C. for 2 hours with shaking at 300 RPM in a THERMOTRON® titer plate shaker. The assay was performed using 10 μL of pre-incubated lysate in a 100 μL reaction with 20 g / L rebaudioside A 60% substrate, 0.02 g / L ADP (Amresco, Ultra Pure Grade) co-substrate, 0.03 g / L SUS SFP SEQ ID NO:22, 0.08 g / L β-1,2-glycosyltransferase (β12GT) SEQ ID NO:858, and 30 g / L sucrose. The following reaction conditions were used: 50 mM potassium phosphate buffer, pH 6, 60° C., 16 hours with shaking at 300 RPM in a THERMOTRON® titer plate shaker. The reaction was solubilized by 20-fold dilution with water, quenched by 5-fold dilution with acetonitrile with 0.2% formic acid, and diluted 20-fold with water for analysis. Samples were analyzed by RapidFire-MS / MS as described in Example 5, Table 5.1. Glycosyltransferase variant polypeptides were identified that produce greater amounts of rebaudioside M from rebaudioside A60 using in situ synthesized ADP-glucose than SEQ ID NO:36. These engineered polypeptides are listed in Table 8.1. Four additional variants were also identified with modified N-terminal DNA coding sequences but no amino acid mutations, SEQ ID NOs:277 / 278, 279 / 280, 281 / 282, and 283 / 284. For analysis of the variants shown in Table 8.2 compared to SEQ ID NO:36, shake flask scale cultures were grown, lysed, clarified, and lyophilized to a powder as described in Example 1. [Table 8-1] [Table 8-2]

[0216] SFP characterization assay and analysis of glucosyl transfer from sucrose to ADP and then ADP-glucose to stevioside and rebaudioside D Shake flask powder (SFP) was reconstituted to a concentration of 20 g / L and diluted to 0.005-0.15 g / L of SFP in a total reaction volume of 100 μL with 50 mM potassium phosphate buffer, pH 6, 15 mM stevioside (purity >95%) or rebaudioside D, 0.02 g / L ADP (Amresco, Ultra Pure Grade) cosubstrate, 0.05 g / L SUS SFP SEQ ID NO: 22, and 37.5 mM sucrose. Reactions were incubated at 60°C for 4 hours with shaking at 300 RPM in a Thermotron® titer plate shaker. Reactions were solubilized by 20-fold dilution in water, quenched by 5-fold dilution in acetonitrile with 0.2% formic acid, clarified by centrifugation, and diluted 15-fold in water for analysis. Glycosylation products were detected by SPE-QQQ as described in Example 5, Table 5.1. One-pot reactions were performed using 0.01-0.3 g / L SFP in a total reaction volume of 100 μL with 50 mM potassium phosphate buffer, pH 6, 20 g / L RebA60, 0.02 g / L ADP (Amresco, Ultra Pure Grade) cosubstrate, 0.03 g / L SUS SFP SEQ ID NO:22, 0.08 g / L β1,2GT SFP SEQ ID NO:858, and 30 g / L sucrose. Reactions were incubated at 60°C for 16 hours with shaking at 300 RPM in a THERMOTRON® titer plate shaker. Reactions were solubilized by 20-fold dilution with water, quenched by 5-fold dilution with acetonitrile with 0.2% formic acid, clarified by centrifugation, and diluted 20-fold with water for analysis. A similar one-pot reaction was also performed using 10 μL of crude shake flask lysate after 20-fold dilution in 50 mM potassium phosphate buffer, pH 6, and incubation at 75.2° C. for 24 hours. [Table 8-3] [Table 8-4]

[0217] In these experiments, the two N-terminal modified sequences in Table 8.2 performed the same as SEQ ID NO: 36, and the six combination variants performed better with all three substrates. SEQ ID NO: 174 was selected as the starting point for further enzyme engineering due to its superior performance with all three substrates.

[0218] Example 9 Beta-1,3-ADP-glycosyltransferase variant of SEQ ID NO: 174 This example describes an evolution and screening experiment of β1,3-glycosyltransferase (β1,3GT) polypeptides derived from SEQ ID NO: 174 for improved glucosylation of steviol glycosides using in situ synthesized ADP-glucose. Directed evolution of GT encoded by SEQ ID NO: 173 (i.e., SEQ ID NO: 174) was performed by constructing libraries of variant genes that were engineered with mutations associated with improved production identified during the development of the present invention and that were subjected to saturation mutagenesis of certain structural features. These libraries were then plated, grown, and screened using a high-throughput (HTP) assay described below to obtain another round of 60 engineered GT variant polypeptides with glucosyltransferase activity toward ADP-glucose and steviol glycosides.

[0219] HTP assay for glucose transfer from sucrose to ADP, followed by glucose transfer from ADP-glucose to rebaudioside A60 The assay was performed using 96-well plates of clarified E. coli culture lysates expressing SEQ ID NO: 173 variants (i.e., variants of SEQ ID NO: 174). The pellets were lysed and the lysates were clarified as described in Example 6, then diluted 25-33.3 fold into 50 mM potassium phosphate buffer, pH 6. To heat challenge the lysates, they were pre-incubated at 75°C for 2 hours with shaking at 300 RPM in a THERMOTRON® titer plate shaker. The assay was performed using 10 μL of pre-incubated lysate in a 100 μL reaction with 20 g / L rebaudioside A 60% substrate, 0.02 g / L ADP (Amresco, Ultra Pure Grade) co-substrate, 0.03 g / L SUS SFP SEQ ID NO: 1652, 0.08 g / L β-1,2-glycosyltransferase (β12GT) SEQ ID NO: 858, and 30 g / L sucrose. The following reaction conditions were used: 50 mM potassium phosphate buffer, pH 6, 60° C., 16 hours with shaking at 300 RPM in a THERMOTRON® titer plate shaker. The reaction was solubilized by 20-fold dilution with water, quenched by 5-fold dilution with acetonitrile with 0.2% formic acid, and diluted 20-fold with water for analysis. Samples were analyzed by RapidFire-MS / MS as described in Example 5, Table 5.1. Glycosyltransferase variant polypeptides were identified that produce higher amounts of rebaudioside M from rebaudioside A60 using in situ synthesized ADP-glucose than SEQ ID NO: 174. These engineered polypeptides are listed in Tables 9.1 and 9.2. In addition, the saturation mutagenesis variants listed in Table 9.2 were assayed under four additional conditions. One was the same as the assay above except that they were incubated at 70° C. for 16 hours. Another was the same as above except that 40 g / L of RebA60 was included in the potassium phosphate dilution buffer preincubation.The other two were single substrate reaction conditions in which the clarified lysate was diluted 25-fold into 50 mM potassium phosphate buffer, pH 6, pre-incubated at 75° C. for 2 hours, and then diluted 10-fold into 100 μL reactions containing 15 mM steviol glycosides 95% or rebaudioside D, 30 g / L sucrose, 0.02 g / L ADP, 50 mM potassium phosphate pH 6, and 0.03 g / L SUS SFP SEQ ID NO: 1652. These reactions were incubated at 60° C. for 16 hours, solubilized by 20-fold dilution with water, quenched by 5-fold dilution by addition to acetonitrile with 0.2% formic acid, and precipitated by low-temperature centrifugation. The supernatant was then diluted 15-fold with water and analyzed by RapidFire-MS / MS. For analysis of the variants shown in Table 9.3 compared to SEQ ID NO: 174, shake flask scale cultures were grown, lysed, clarified and lyophilized to a powder as described in Example 1. [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4]

[0220] SFP characterization assay and analysis of glucosyl transfer from sucrose to ADP and then ADP-glucose to stevioside and rebaudioside D Shake flask powder (SFP) was reconstituted to a concentration of 20 g / L and diluted to 0.005-0.15 g / L SFP in a total reaction volume of 100 μL with 50 mM potassium phosphate buffer, pH 6, 15 mM stevioside (purity >95%) or rebaudioside D, 0.02 g / L ADP (Amresco, Ultra Pure Grade) cosubstrate, 0.03 g / L SUS SFP SEQ ID NO: 1652, and 37.5 mM sucrose. Reactions were incubated at 60°C for 4 hours with shaking at 300 RPM in a THERMOTRON® titer plate shaker. Reactions were solubilized by 20-fold dilution with water, quenched by 5-fold dilution with acetonitrile with 0.2% formic acid, clarified by centrifugation, and diluted 15-fold with water for analysis. Glycosylation products were detected by SPE-QQQ as described in Example 5, Table 5.1. One-pot reactions were performed using 50 mM potassium phosphate buffer, pH 6, 20 g / L RebA60, 0.02 g / L ADP (Amresco, Ultra Pure Grade) cosubstrate, 0.03 g / L SUS SFP SEQ ID NO: 1652, 0.08 g / L β1,2GT SFP SEQ ID NO: 858, and 30 g / L sucrose at 0.01-0.3 g / L in a total reaction volume of 100 μL. Reactions were incubated at 60° C. for 16 hours with shaking at 300 RPM in a THERMOTRON® titer plate shaker. Reactions were solubilized by 20-fold dilution in water, quenched by 5-fold dilution in acetonitrile with 0.2% formic acid, clarified by centrifugation, and diluted 20-fold in water for analysis. A similar one-pot reaction was also performed using 10 μL of crude shake flask lysate after 20-fold dilution in 50 mM potassium phosphate buffer, pH 6, and incubation at 75.2° C. for 24 hours. [Table 9-5] [Table 9-6]

[0221] In these experiments, five variants in Table 9.3 produced more rebaudioside M from rebaudioside A60 than SEQ ID NO:174, four variants also produced more rebaudioside A from stevioside, and three of these also produced more rebaudioside M from rebaudioside D. Several variants were significantly more thermostable than SEQ ID NO:190. The most thermostable variant, SEQ ID NO:350, was recloned with the best N-terminal DNA sequence from Example 8 (SEQ ID NOs:279 / 280) and this enzyme, SEQ ID NO:406, was used as the starting point for further enzyme engineering.

[0222] Example 10 Beta-1,3-ADP-glycosyltransferase variant of SEQ ID NO: 406 This example describes an evolution and screening experiment of β1,3-glycosyltransferase (β1,3GT) polypeptides derived from SEQ ID NO:406 for improved glucosylation of steviol glycosides using in situ synthesized ADP-glucose. Directed evolution of GT encoded by SEQ ID NO:405 (i.e., SEQ ID NO:406) was performed by constructing libraries of variant genes engineered with mutations associated with improved production identified during the development of the present invention. These libraries were then plated, grown, and screened using a high-throughput (HTP) assay described below to obtain another round of 16 engineered GT variant polypeptides with glucosyltransferase activity towards ADP-glucose and steviol glycosides.

[0223] HTP assay for glucose transfer from sucrose to ADP, followed by glucose transfer from ADP-glucose to rebaudioside A60 The assay was performed using a 96-well plate of clarified E. coli culture lysates expressing the SEQ ID NO:405 variant (i.e., the variant of SEQ ID NO:406). The pellets were lysed and the lysates were clarified as described in Example 6, then diluted 15-fold into 50 mM potassium phosphate buffer, pH 6, and separately diluted 15-fold into the same buffer with 40 g / L of rebaudioside A 60% substrate. To heat challenge the lysates, they were pre-incubated at 75° C. for 2 hours with shaking at 300 RPM in a THERMOTRON® titer plate shaker. The assay was performed using 10 μL of pre-incubated lysate in a 100 μL reaction with 20 g / L rebaudioside A 60% substrate, 0.02 g / L ADP (Amresco, Ultra Pure Grade) co-substrate, 0.03 g / L SUS SFP SEQ ID NO: 1822, 0.06 g / L β-1,2-glycosyltransferase (β12GT) SEQ ID NO: 994, and 30 g / L sucrose. The following reaction conditions were used: 50 mM potassium phosphate buffer, pH 6, 60° C., 16 hours with shaking at 300 RPM in a THERMOTRON® titer plate shaker. The reaction was solubilized by 20-fold dilution with water, quenched by 5-fold dilution with acetonitrile with 0.2% formic acid, and diluted 20-fold with water for analysis. Samples were analyzed by RapidFire-MS / MS as described in Example 5, Table 5.1. Glycosyltransferase variant polypeptides were identified that produce higher amounts of rebaudioside M from rebaudioside A60 using in situ synthesized ADP-glucose than SEQ ID NO: 406. These engineered polypeptides are listed in Table 10.1. [Table 10]

[0224] SEQ ID NO:408 from the variants listed in Table 10.1 was selected as the starting point for further enzyme engineering based on its improved stability upon pre-incubation in potassium phosphate buffer with and without 60% rebaudioside A, as well as the benefits associated with the two mutations A85V and E259T.

[0225] Example 11 Beta-1,3-ADP-glycosyltransferase variant of SEQ ID NO: 408 This example describes an evolution and screening experiment of β1,3-glycosyltransferase (β1,3GT) polypeptides derived from SEQ ID NO:408 for improved glucosylation of steviol glycosides using in situ synthesized ADP-glucose. Directed evolution of GT encoded by SEQ ID NO:407 (i.e., SEQ ID NO:408) was performed by constructing libraries of variant genes that were engineered with mutations associated with improved production identified during the development of the present invention and that were subjected to saturation mutagenesis of certain structural features. These libraries were then plated, grown, and screened using a high-throughput (HTP) assay described below to obtain another round of 39 engineered GT variant polypeptides with glucosyltransferase activity toward ADP-glucose and steviol glycosides.

[0226] HTP assay for glucose transfer from sucrose to ADP, followed by glucose transfer from ADP-glucose to rebaudioside A60 The assay was performed using 96-well plates of clarified E. coli culture lysates expressing the SEQ ID NO:407 variant (i.e., the variant of SEQ ID NO:408). The pellets were lysed and the lysates were clarified as described in Example 6 and then diluted 20-fold into 50 mM potassium phosphate buffer, pH 6 with 60 g / L rebaudioside A 60%. To heat challenge the lysates, they were pre-incubated at 75° C. for 2 hours with shaking at 300 RPM in a THERMOTRON® titer plate shaker. The assay was performed using 10 μL of pre-incubated lysate in a 100 μL reaction with 20 g / L rebaudioside A 60% substrate, 0.02 g / L ADP (Amresco, Ultra Pure Grade) co-substrate, 0.03 g / L SUS SFP SEQ ID NO: 1822, 0.06 g / L β-1,2-glycosyltransferase (β12GT) SEQ ID NO: 1080, and 30 g / L sucrose. The following reaction conditions were used: 50 mM potassium phosphate buffer, pH 6, 60° C., 16 hours with shaking at 300 RPM in a THERMOTRON® titer plate shaker. The reaction was solubilized by 20-fold dilution with water, quenched by 5-fold dilution with acetonitrile with 0.2% formic acid, and diluted 20-fold with water for analysis. Samples were analyzed by RapidFire-MS / MS as described in Example 5, Table 5.1. Glycosyltransferase variant polypeptides were identified that produce greater amounts of rebaudioside M from rebaudioside A60 using in situ synthesized ADP-glucose than SEQ ID NO:408. These engineered polypeptides are listed in Tables 11.1 and 11.2. For analysis of the variants shown in Table 11.3 compared to SEQ ID NO:408, shake flask scale cultures were grown, lysed, clarified, and lyophilized to a powder as described in Example 1. [Table 11-1] [Table 11-2] [Table 11-3]

[0227] SFP characterization assay and analysis of glucosyl transfer from sucrose to ADP and then ADP-glucose to stevioside and rebaudioside D Shake flask powder (SFP) was reconstituted to a concentration of 20 g / L and diluted to 0.005-0.15 g / L SFP in a total reaction volume of 100 μL with 50 mM potassium phosphate buffer, pH 6, 15 mM stevioside (purity >95%) or rebaudioside D, 0.02 g / L ADP (Amresco, Ultra Pure Grade) cosubstrate, 0.03 g / L SUS SFP SEQ ID NO: 1822, and 37.5 mM sucrose. Reactions were incubated at 60°C for 4 hours with shaking at 300 RPM in a THERMOTRON® titer plate shaker. Reactions were solubilized by 20-fold dilution with water, quenched by 5-fold dilution with acetonitrile with 0.2% formic acid, clarified by centrifugation, and diluted 15-fold with water for analysis. Glycosylation products were detected by SPE-QQQ as described in Example 5, Table 5.1. One-pot reactions were performed with 0.01-0.3 g / L SFPs in a total reaction volume of 100 μL with 50 mM potassium phosphate buffer, pH 6, 20 g / L RebA60, 0.02 g / L ADP (Amresco, Ultra Pure Grade) cosubstrate, 0.03 g / L SUS SFP SEQ ID NO: 1822, 0.06 g / L β1,2GT SFP SEQ ID NO: 1080, and 30 g / L sucrose. One set of SFP dilutions was preincubated in 50 mM potassium phosphate buffer, pH 6 for 2 hours at 75°C, while the other set was not preincubated. Reactions were incubated at 60°C for 16 hours with shaking at 300 RPM in a THERMOTRON® titer plate shaker. Reactions were solubilized by 20-fold dilution in water, quenched by 5-fold dilution in acetonitrile with 0.2% formic acid, clarified by centrifugation, and diluted 20-fold in water for analysis. A similar one-pot reaction was also performed using 10 μL of crude shake flask lysate after 20-fold dilution in 50 mM potassium phosphate buffer, pH 6, and incubation at 75.2° C. for 24 hours. [Table 11-4]

[0228] In these experiments, all four variants in Table 11.3 produced more rebaudioside M from rebaudioside A60, more rebaudioside A from stevioside, and more rebaudioside M from rebaudioside D than SEQ ID NO:408. The most thermostable variant, SEQ ID NO:440, was used as the starting point for further enzymatic engineering.

[0229] Example 12 Beta-1,3-ADP-glycosyltransferase variant of SEQ ID NO: 440 This example describes an evolution and screening experiment of β1,3-glycosyltransferase (β1,3GT) polypeptides derived from SEQ ID NO:440 for improved glucosylation of steviol glycosides using in situ synthesized ADP-glucose. Directed evolution of GT encoded by SEQ ID NO:439 (i.e., SEQ ID NO:440) was performed by constructing libraries of variant genes that were engineered with mutations associated with improved production identified during the development of the present invention and that were subjected to saturation mutagenesis of certain structural features. These libraries were then plated, grown, and screened using the high-throughput (HTP) assay described below to obtain another round of 36 engineered GT variant polypeptides with glucosyltransferase activity toward ADP-glucose and steviol glycosides.

[0230] HTP assay for glucose transfer from sucrose to ADP, followed by glucose transfer from ADP-glucose to rebaudioside A60 The assay was performed using 96-well plates of clarified E. coli culture lysates expressing SEQ ID NO: 440 variants. The pellets were lysed and the lysates were clarified as described in Example 6, then diluted 20-fold into 50 mM potassium phosphate buffer, pH 6 with 80 g / L rebaudioside A 60%. To heat challenge the lysates, they were pre-incubated at 75° C. for 2 hours with shaking at 300 RPM in a THERMOTRON® titer plate shaker. The assay was performed using 10 μL of pre-incubated lysate in a 100 μL reaction with 20 g / L rebaudioside A 60% substrate, 0.02 g / L ADP (Amresco, Ultra Pure Grade) co-substrate, 0.03 g / L SUS SFP SEQ ID NO: 1822, 0.06 g / L β-1,2-glycosyltransferase (β12GT) SEQ ID NO: 1080, and 30 g / L sucrose. The following reaction conditions were used: 50 mM potassium phosphate buffer, pH 6, 60° C., 16 hours with shaking at 300 RPM in a THERMOTRON® titer plate shaker. The reaction was solubilized by 20-fold dilution with water, quenched by 5-fold dilution with acetonitrile with 0.2% formic acid, and diluted 20-fold with water for analysis. Samples were analyzed by RapidFire-MS / MS as described in Example 5, Table 5.1. Glycosyltransferase variant polypeptides were identified that produce higher amounts of rebaudioside M from rebaudioside A60 using in situ synthesized ADP-glucose than SEQ ID NO: 440. These engineered polypeptides are listed in Tables 12.1 and 12.2. For analysis of the variants shown in Table 12.3 compared to SEQ ID NO: 440, shake flask scale cultures were grown, lysed, clarified, and lyophilized to a powder as described in Example 1. [Table 12-1] [Table 12-2] [Table 12-3] [Table 12-4]

[0231] SFP characterization assay and analysis of glucosyl transfer from sucrose to ADP and then ADP-glucose to stevioside and rebaudioside D Shake flask powder (SFP) was reconstituted to a concentration of 20 g / L and diluted to 0.005-0.15 g / L SFP in a total reaction volume of 100 μL with 50 mM potassium phosphate buffer, pH 6, 15 mM stevioside (purity >95%) or rebaudioside D, 0.02 g / L ADP (Amresco, Ultra Pure Grade) cosubstrate, 0.03 g / L SUS SFP SEQ ID NO: 1822, and 37.5 mM sucrose. Reactions were incubated at 60°C for 4 hours with shaking at 300 RPM in a Thermotron® titer plate shaker. Reactions were solubilized by 20-fold dilution with water, quenched by 5-fold dilution with acetonitrile with 0.2% formic acid, clarified by centrifugation, and diluted 15-fold with water for analysis. Glycosylation products were detected by SPE-QQQ as described in Example 5, Table 5.1. One-pot reactions were performed with 0.01-0.3 g / L SFP in a total reaction volume of 100 μL with 50 mM potassium phosphate buffer, pH 6, 20 g / L RebA60, 0.02 g / L ADP (Amresco, Ultra Pure Grade) cosubstrate, 0.03 g / L SUS SFP SEQ ID NO: 1822, 0.06 g / L β1,2GT SFP SEQ ID NO: 1080, and 30 g / L sucrose. One set of SFP dilutions was preincubated with 80 g / L rebaudioside A60% in 50 mM potassium phosphate buffer, pH 6 for 2 hours at 75°C, while the other set was not preincubated. Reactions were incubated at 60° C. for 16 hours with shaking at 300 RPM in a THERMOTRON® titer plate shaker. Reactions were solubilized by 20-fold dilution with water, quenched by 5-fold dilution with acetonitrile with 0.2% formic acid, clarified by centrifugation, and diluted 20-fold with water for analysis. A similar one-pot reaction was also performed using 10 μL of 1.5 mg / mL shake flask powder in 50 mM potassium phosphate buffer, pH 6, incubated at 78.5° C. for 24 hours. [Table 12-5] [Table 12-6]

[0232] In these experiments, all eight variants in Table 12.3 produced more rebaudioside M from rebaudioside A60 and from rebaudioside D than SEQ ID NO:440. All of the variants were also less active at catalyzing the glucosylation of stevioside to form rebaudioside A. The most active variant on the three substrates, SEQ ID NO:520, was used as the starting point for further enzyme engineering.

[0233] Example 13 Beta-1,3-ADP-glycosyltransferase variant of SEQ ID NO: 520 This example describes an evolution and screening experiment of β1,3-glycosyltransferase (β1,3GT) polypeptides derived from SEQ ID NO:520 for improved glucosylation of steviol glycosides using in situ synthesized ADP-glucose. Directed evolution of GT encoded by SEQ ID NO:519 (i.e., SEQ ID NO:520) was performed by constructing libraries of variant genes that were engineered with mutations associated with improved production identified during the development of the present invention and that were subjected to saturation mutagenesis of certain structural features. These libraries were then plated, grown, and screened using a high-throughput (HTP) assay described below to obtain another round of 44 engineered GT variant polypeptides with glucosyltransferase activity toward ADP-glucose and steviol glycosides.

[0234] HTP assay for glucose transfer from sucrose to ADP, followed by glucose transfer from ADP-glucose to rebaudioside A60 The assay was performed using 96-well plates of clarified E. coli culture lysates expressing SEQ ID NO:519 variants (i.e., variants of SEQ ID NO:520). The pellets were lysed and the lysates were clarified as described in Example 6 and then diluted 49-fold into 50 mM potassium phosphate buffer, pH 6 with 100 g / L rebaudioside A 60%. To heat challenge the lysates, they were pre-incubated at 75° C. for 2 hours with shaking at 300 RPM in a THERMOTRON® titer plate shaker. The assay was performed using 10 μL of pre-incubated lysate in a 100 μL reaction with 40 g / L rebaudioside A 60% substrate, 0.02 g / L ADP (Amresco, Ultra Pure Grade) co-substrate, 0.06 g / L SUS SFP SEQ ID NO: 2182, 0.12 g / L β-1,2-glycosyltransferase (β12GT) SEQ ID NO: 1216, and 60 g / L sucrose. The following reaction conditions were used: 50 mM potassium phosphate buffer, pH 6, 60° C., 16 hours with shaking at 300 RPM in a THERMOTRON® titer plate shaker. The reaction was solubilized by 40-fold dilution with water, quenched by 5-fold dilution with acetonitrile with 0.2% formic acid, and diluted 20-fold with water for analysis. Samples were analyzed by RapidFire-MS / MS as described in Example 5, Table 5.1. Glycosyltransferase variant polypeptides were identified that produce higher amounts of rebaudioside M from rebaudioside A60 using in situ synthesized ADP-glucose than SEQ ID NO:520. These engineered polypeptides are listed in Tables 13.1 and 13.2. For analysis of the variants shown in Table 13.3 compared to SEQ ID NO:520, shake flask scale cultures were grown, lysed, clarified, and lyophilized to a powder as described in Example 1. [Table 13-1] [Table 13-2] [Table 13-3]

[0235] SFP characterization assay and analysis of glucosyl transfer from sucrose to ADP and then ADP-glucose to stevioside and rebaudioside D Shake flask powder (SFP) was reconstituted to a concentration of 20 g / L and diluted to 0.005-0.15 g / L SFP in a total reaction volume of 100 μL with 50 mM potassium phosphate buffer, pH 6, 15 mM stevioside (purity >95%) or rebaudioside D, 0.02 g / L ADP (Amresco, Ultra Pure Grade) cosubstrate, 0.03 g / L SUS SFP SEQ ID NO: 2182, and 37.5 mM sucrose. Reactions were incubated at 60°C for 4 hours with shaking at 300 RPM in a THERMOTRON® titer plate shaker. Reactions were solubilized by 20-fold dilution with water, quenched by 5-fold dilution with acetonitrile with 0.2% formic acid, clarified by centrifugation, and diluted 15-fold with water for analysis. Glycosylation products were detected by SPE-QQQ as described in Example 5, Table 5.1. One-pot reactions were performed using 0.01-0.3 g / L SFPs in a total reaction volume of 100 μL with 50 mM potassium phosphate buffer, pH 6, 40 g / L RebA60, 0.02 g / L ADP (Amresco, Ultra Pure Grade) cosubstrate, 0.03 g / L SUS SFP SEQ ID NO: 2182, 0.12 g / L β1,2GT SFP SEQ ID NO: 1216, and 60 g / L sucrose. One set of SFP dilutions was preincubated with 100 g / L rebaudioside A60% in 50 mM potassium phosphate buffer, pH 6 for 2 hours at 75°C, while the other set was not preincubated. Reactions were incubated at 60° C. for 16 hours with shaking at 300 RPM in a THERMOTRON® titer plate shaker. Reactions were solubilized by 40-fold dilution with water, quenched by 5-fold dilution with acetonitrile with 0.2% formic acid, clarified by centrifugation, and diluted 20-fold with water for analysis. A similar one-pot reaction was also performed using 10 μL of crude clarified lysate diluted 25-fold in 50 mM potassium phosphate buffer, pH 6 with 100 g / L rebaudioside A 60% and incubated at 75.2° C. for 24 hours. [Table 13-4] [Table 13-5]

[0236] In these experiments, all seven variants in Table 13.3 produced more rebaudioside M from rebaudioside A60 and from rebaudioside D, and more rebaudioside A from stevioside, than SEQ ID NO: 520. Four variants had similar thermal stability as SEQ ID NO: 520. The variant with the highest activity against stevioside and RebA60 without preincubation, SEQ ID NO: 626, was used as the starting point for further enzyme engineering.

[0237] Example 14 Beta-1,3-ADP-glycosyltransferase variant of SEQ ID NO: 626 This example describes an evolution and screening experiment of β1,3-glycosyltransferase (β1,3GT) polypeptides derived from SEQ ID NO:626 for improved glucosylation of steviol glycosides using in situ synthesized ADP-glucose. Directed evolution of GT encoded by SEQ ID NO:625 (i.e., SEQ ID NO:626) was performed by constructing libraries of variant genes that were engineered with mutations associated with improved production identified during the development of the present invention and that were subjected to saturation mutagenesis of certain structural features. These libraries were then plated, grown, and screened using a high-throughput (HTP) assay described below to obtain another round of 43 engineered GT variant polypeptides with glucosyltransferase activity toward ADP-glucose and steviol glycosides.

[0238] HTP assay for glucose transfer from sucrose to ADP, followed by glucose transfer from ADP-glucose to rebaudioside A60 The assay was performed using 96-well plates of clarified E. coli culture lysates expressing the SEQ ID NO:625 variant (i.e., the variant of SEQ ID NO:626). The pellets were lysed and the lysates were clarified as described in Example 6 and then diluted 100-fold into 50 mM potassium phosphate buffer, pH 6 with 100 g / L rebaudioside A 60%. To heat challenge the lysates, they were pre-incubated at 75° C. for 2 hours with shaking at 300 RPM in a THERMOTRON® titer plate shaker. The assay was performed using 10 μL of pre-incubated lysate in a 100 μL reaction with 40 g / L rebaudioside A 60% substrate, 0.02 g / L ADP (Amresco, Ultra Pure Grade) co-substrate, 0.06 g / L SUS SFP SEQ ID NO: 2182, 0.09 g / L β-1,2-glycosyltransferase (β12GT) SEQ ID NO: 1488, and 60 g / L sucrose. The following reaction conditions were used: 50 mM potassium phosphate buffer, pH 6, 60° C., 16 hours with shaking at 300 RPM in a THERMOTRON® titer plate shaker. The reaction was solubilized by 40-fold dilution with water, quenched by 5-fold dilution with acetonitrile with 0.2% formic acid, and diluted 20-fold with water for analysis. Samples were analyzed by RapidFire-MS / MS as described in Example 5, Table 5.1. Glycosyltransferase variant polypeptides were identified that produce higher amounts of rebaudioside M from rebaudioside A60 using in situ synthesized ADP-glucose than SEQ ID NO:626. These engineered polypeptides are listed in Tables 14.1 and 14.2. For analysis of the variants shown in Table 14.3 compared to SEQ ID NO:626, shake flask scale cultures were grown, lysed, clarified, and lyophilized to a powder as described in Example 1. [Table 14-1] [Table 14-2] [Table 14-3] [Table 14-4]

[0239] SFP characterization assay and analysis of glucosyl transfer from sucrose to ADP and then ADP-glucose to stevioside and rebaudioside D Shake flask powder (SFP) was reconstituted to a concentration of 20 g / L and diluted to 0.005-0.15 g / L SFP in a total reaction volume of 100 μL with 50 mM potassium phosphate buffer, pH 6, 15 mM stevioside (purity >95%) or rebaudioside D, 0.02 g / L ADP (Amresco, Ultra Pure Grade) cosubstrate, 0.03 g / L SUS SFP SEQ ID NO: 2182, and 37.5 mM sucrose. Reactions were incubated at 60°C for 4 hours with shaking at 300 RPM in a THERMOTRON® titer plate shaker. Reactions were solubilized by 20-fold dilution with water, quenched by 5-fold dilution with acetonitrile with 0.2% formic acid, clarified by centrifugation, and diluted 15-fold with water for analysis. Glycosylation products were detected by SPE-QQQ as described in Example 5, Table 5.1. One-pot reactions were performed using 0.01-0.3 g / L SFPs in a total reaction volume of 100 μL with 50 mM potassium phosphate buffer, pH 6, 50 g / L RebA60, 0.025 g / L ADP (Amresco, Ultra Pure Grade) cosubstrate, 0.075 g / L SUS SFP SEQ ID NO: 2182, 0.09 g / L β1,2GT SFP SEQ ID NO: 1488, and 75 g / L sucrose. One set of SFP dilutions was preincubated with 100 g / L rebaudioside A60% in 50 mM potassium phosphate buffer, pH 6 for 2 hours at 75°C, while the other set was not preincubated. Reactions were incubated at 60° C. for 16 hours with shaking at 300 RPM in a THERMOTRON® titer plate shaker. Reactions were solubilized by 50-fold dilution in water, quenched by 5-fold dilution in acetonitrile with 0.2% formic acid, clarified by centrifugation, and diluted 20-fold in water for analysis. A similar one-pot reaction was also performed using 10 μL of crude clarified lysate diluted 25-fold in 50 mM potassium phosphate buffer, pH 6 with 100 g / L rebaudioside A 60% and incubated at 75.2° C. for 24 hours. [Table 14-5] [Table 14-6]

[0240] In these experiments, five variants in Table 14.3 produced more rebaudioside M from rebaudioside A60 than SEQ ID NO: 626. Of these, one variant, SEQ ID NO: 678, also had improved catalysis of stevioside to rebaudioside A and rebaudioside D to rebaudioside M and was more thermostable than SEQ ID NO: 626. This active and thermostable variant, SEQ ID NO: 678, was used as the starting point for further enzyme engineering.

[0241] Example 15 Beta-1,3-ADP-glycosyltransferase variant of SEQ ID NO: 678 This example describes an experiment of evolving and screening β1,3-glycosyltransferase (β1,3GT) polypeptides derived from SEQ ID NO:678 for improved glucosylation of steviol glycosides using in situ synthesized ADP-glucose. Directed evolution of GT encoded by SEQ ID NO:677 (i.e., SEQ ID NO:678) was performed by constructing libraries of variant genes engineered with mutations associated with improved production identified during the development of the present invention. These libraries were then plated, grown, and screened using a high-throughput (HTP) assay described below to obtain another round of 47 engineered GT variant polypeptides with glucosyltransferase activity toward ADP-glucose and steviol glycosides.

[0242] HTP assay for glucose transfer from sucrose to ADP, followed by glucose transfer from ADP-glucose to rebaudioside A60 The assay was performed using 96-well plates of clarified E. coli culture lysates expressing the SEQ ID NO:677 variant (i.e., the variant of SEQ ID NO:678). The pellets were lysed and the lysates were clarified as described in Example 6 and then diluted 70-fold into 50 mM potassium phosphate buffer, pH 6 with 100 g / L rebaudioside A 60%. To heat challenge the lysates, they were pre-incubated at 75° C. for 2 hours with shaking at 300 RPM in a THERMOTRON® titer plate shaker. The assay was performed using 10 μL of pre-incubated lysate in a 100 μL reaction with 50 g / L rebaudioside A 60% substrate, 0.025 g / L ADP (Amresco, Ultra Pure Grade) co-substrate, 0.075 g / L SUS SFP SEQ ID NO: 2322, 0.09 g / L β-1,2-glycosyltransferase (β12GT) SEQ ID NO: 1488, and 75 g / L sucrose. The following reaction conditions were used: 50 mM potassium phosphate buffer, pH 6, 60° C., 16 hours with shaking at 300 RPM in a THERMOTRON® titer plate shaker. The reaction was solubilized by 50-fold dilution with water, quenched by 5-fold dilution with acetonitrile with 0.2% formic acid, and diluted 20-fold with water for analysis. Samples were analyzed by RapidFire-MS / MS as described in Example 5, Table 5.1. Glycosyltransferase variant polypeptides were identified that produce greater amounts of rebaudioside M from rebaudioside A60 using in situ synthesized ADP-glucose than SEQ ID NO: 678. These engineered polypeptides are listed in Table 15.1. For analysis of the variants shown in Table 15.2 compared to SEQ ID NO: 678, shake flask scale cultures were grown, lysed, clarified, and lyophilized to a powder as described in Example 1. [Table 15-1] [Table 15-2] [Table 15-3]

[0243] SFP characterization assay and analysis of glucosyl transfer from sucrose to ADP and then ADP-glucose to stevioside and rebaudioside D Shake flask powder (SFP) was reconstituted to a concentration of 20 g / L and diluted from 0.0125 to 0.2 g / L of SFP in a total reaction volume of 100 μL with 50 mM potassium phosphate buffer, pH 6, 15 mM stevioside (purity >95%) or rebaudioside D or rebaudioside E (prepared in-house from stevioside treated with β1,2GT), 0.02 g / L ADP (Amresco, Ultra Pure Grade) cosubstrate, 0.025 g / L SUS SFP SEQ ID NO: 2322, and 37.5 mM sucrose. Reactions were incubated at 60 °C for 4 hours with shaking at 300 RPM in a THERMOTRON® titer plate shaker. Reactions were solubilized by 20-fold dilution in water, quenched by 5-fold dilution in acetonitrile with 0.2% formic acid, clarified by centrifugation, and diluted 15-fold in water for analysis. Glycosylation products were detected by SPE-QQQ as described in Example 5, Table 5.1. 50 mM potassium phosphate buffer, pH 6, 100 g / L RebA60, 0.05 g / L ADP (Amresco, Ultra Pure Grade) cosubstrate, 0.2 g / L SUS SFP SEQ ID NO: 2322, and 0.2 g / L β1,2GT One-pot reactions were performed using SFP SEQ ID NO: 1516 and 150 g / L sucrose at 0.0125-0.2 g / L in a total reaction volume of 100 μL. One set of SFP dilutions was preincubated with 100 g / L rebaudioside A 60% in 50 mM potassium phosphate buffer, pH 6 for 2 hours at 75°C, while the other set was not preincubated. Reactions were incubated at 60°C for 16 hours with shaking at 300 RPM in a THERMOTRON® titer plate shaker. Reactions were solubilized by 100-fold dilution in water, quenched by 5-fold dilution in acetonitrile with 0.2% formic acid, clarified by centrifugation, and diluted 20-fold in water for analysis. A similar one-pot reaction was also performed using 10 μL of crude clarified lysate diluted 25-fold in 50 mM potassium phosphate buffer, pH 6 with 100 g / L rebaudioside A 60% and incubated at 75.2° C. for 24 hours. [Table 15-4]

[0244] In these experiments, the three variants in Table 15.2 produced more rebaudioside M from rebaudioside A60 than SEQ ID NO: 678. Of these, one variant, SEQ ID NO: 768, was also at least similar in thermostability to SEQ ID NO: 678. Therefore, SEQ ID NO: 768 was selected as the best enzyme for catalyzing the glycosyl transfer from ADP-glucose to stevioside and rebaudioside D to form rebaudioside A and rebaudioside M, respectively.

[0245] Example 16 Beta-1,2-ADP-glycosyltransferase variant of SEQ ID NO: 24 This example describes an experiment of evolution and screening of GT polypeptides derived from SEQ ID NO:24 for improved glucosylation of steviol glycosides using ADP-glucose. Directed evolution of GT encoded by SEQ ID NO:23 was performed by constructing a library of variant genes. The libraries were recombined with mutations associated with improved production identified during the development of the invention and subjected to saturation mutagenesis of certain structural features of the enzyme. These libraries were then plated, grown, and screened using the HTP assay described below to obtain another round of engineered GT variant polypeptides with glucosyltransferase activity towards ADP-glucose and steviol glycosides. Sixteen engineered variants were identified from the combinatorial library (Table 16.1) and 51 from the saturation mutagenesis library (Table 16.2).

[0246] HTP binding assay for glucose transfer from sucrose to ADP and then to rebaudioside A60 The assay was performed using a 96-well plate of clarified E. coli culture lysate expressing the SEQ ID NO:23 variant (i.e., the variant of SEQ ID NO:24). The volume of the lysis buffer was 400 μL, and the lysate was diluted 150-fold in 50 mM potassium phosphate, pH 6.0, and pre-incubated at 75° C. for 1 hour. The assay was then performed using 10 μL of the diluted lysate, 0.03 g / L SUS SFP SEQ ID NO:22, and 0.1 g / L β-1,3-glycosyltransferase (β1,3GT) SFP SEQ ID NO:20 in a reaction volume of 100 μL with 20 g / L rebaudioside A 60% (RebA60) substrate, 0.02 g / L ADP (Amresco, Ultra Pure Grade) co-substrate, and 30 g / L sucrose. The following reaction conditions were used: 50 mM potassium phosphate buffer, pH 6, 60°C, 16-18 hours with shaking at 300 RPM in a THERMOTRON® titer plate shaker. Reactions were solubilized by 20-fold dilution by addition to water, quenched by 5-fold dilution by addition to acetonitrile with 0.2% formic acid, precipitated by centrifugation, and diluted 20-fold by addition to water for analysis as described above. The resulting engineered variants with glucosyltransferase activity towards RebA60 are listed in Tables 16.1 and 16.2. For the variants listed in Table 16.3, shake flask scale cultures were grown, lysed, and lyophilized to powder. [Table 16-1] [Table 16-2] [Table 16-3] [Table 16-4] [Table 16-5]

[0247] Shake flask powder characterization assay and analysis of glucosyl transfer from sucrose to ADP and then to rebaudioside A60 Shake flask powder (SFP) was reconstituted to a concentration of 4 g / L and diluted to 0.0013-0.04 g / L in a total reaction volume of 100 μL containing 50 mM potassium phosphate buffer, pH 6, 20 g / L RebA97 (single substrate) or RebA60 (one-pot), 0.02 g / L ADP, 20 (single substrate) or 30 g / L (one-pot) sucrose, 0.04 g / L SUS SFP SEQ ID NO: 22, and for the one-pot reaction only, 0.15 g / L β-1,3-glycosyltransferase (β1,3GT) SFP SEQ ID NO: 36. Reactions were run at 60 °C in a THERMOTRON® titer plate shaker at 300 RPM for 4 hours (single substrate) or 16-18 hours (one-pot). One set of SFP dilutions was pre-incubated in 50 mM potassium phosphate buffer, pH 6 for 1 hour at 75° C., while the other set was not. Reactions were solubilized by 20-fold dilution in water, quenched by 5-fold dilution in acetonitrile with 0.2% formic acid, clarified by centrifugation, and diluted 20-fold in water for analysis. Glycosylation products were detected by SPE-QQQ as described in Example 5, Table 5.1. To assess thermal stability, a similar single substrate rebaudioside A reaction was also performed using 10 μL of crude clarified lysate diluted 200-fold in 50 mM potassium phosphate buffer, pH 6 and incubated at 66.2° C. for 24 hours. Thermal stability results and production levels of rebaudioside D in single substrate and rebaudioside M in one-pot reaction by these variants at 0.01 g / L SFP loading are shown in Table 16.3. [Table 16-6] [Table 16-7]

[0248] In these experiments, three variants in Table 16.3 showed improved catalysis of the glucosylation of rebaudioside A to rebaudioside D and the β1,2-glucosylation involved in the conversion of rebaudioside A 60% to rebaudioside M compared to SEQ ID NO: 24. Two of these variants were also more thermostable than SEQ ID NO: 24. Of these, the variant with the most improvement upon pre-incubation, SEQ ID NO: 858, was selected as the starting point for further enzyme engineering.

[0249] (Example 17) Beta-1,2-ADP-glycosyltransferase variant of SEQ ID NO: 858 This example describes an experiment of evolving and screening GT polypeptides derived from SEQ ID NO:858 for improved glucosylation of steviol glycosides using ADP-glucose. Directed evolution of GT encoded by SEQ ID NO:857 was performed by constructing a library of variant genes. The library recombined mutations associated with improved production identified during the development of the present invention. These libraries were then plated, grown, and screened using the HTP assay described below to obtain another round of engineered GT variant polypeptides with glucosyltransferase activity towards ADP-glucose and steviol glycosides. Forty-four engineered variants were identified from the combinatorial library (Table 17.1).

[0250] HTP binding assay for glucose transfer from sucrose to ADP and then to rebaudioside A60 The assay was performed using a 96-well plate of clarified E. coli culture lysate expressing the SEQ ID NO:857 variant (i.e., the variant of SEQ ID NO:858). The volume of the lysis buffer was 400 μL, and the lysate was diluted 200-fold in 50 mM potassium phosphate, pH 6.0 and pre-incubated at 75° C. for 2 hours. The assay was then performed using 10 μL of the diluted lysate, 0.03 g / L SUS SFP SEQ ID NO:1652 and 0.1 g / L β-1,3-glycosyltransferase (β1,3GT) SFP SEQ ID NO:174 in a reaction volume of 100 μL with 20 g / L rebaudioside A 60% (RebA60) substrate, 0.02 g / L ADP (Amresco, Ultra Pure Grade) co-substrate, and 30 g / L sucrose. The following reaction conditions were used: 50 mM potassium phosphate buffer, pH 6, 60°C, 16-18 hours with shaking at 300 RPM in a THERMOTRON® titer plate shaker. Reactions were solubilized by 20-fold dilution by addition to water, quenched by 5-fold dilution by addition to acetonitrile with 0.2% formic acid, precipitated by centrifugation, and diluted 20-fold by addition to water for analysis as described above. The resulting engineered variants with glucosyltransferase activity towards RebA60 are listed in Table 17.1. For the variants listed in Table 17.2, shake flask scale cultures were grown, lysed, and lyophilized to powder. [Table 17-1] [Table 17-2]

[0251] Shake flask powder characterization assay and analysis of glucosyl transfer from sucrose to ADP and then to rebaudioside A60 Shake flask powder (SFP) was reconstituted to a concentration of 4 g / L and diluted to 0.0013-0.04 g / L in a total reaction volume of 100 μL containing 50 mM potassium phosphate buffer, pH 6, 20 g / L RebA97 (single substrate) or RebA60 (one-pot), 0.02 g / L ADP, 20 (single substrate) or 30 g / L (one-pot) sucrose, 0.03 g / L SUS SFP SEQ ID NO: 1652, and for the one-pot reaction only, 0.1 g / L β-1,3-glycosyltransferase (β1,3GT) SFP SEQ ID NO: 174. Reactions were run at 60 °C in a THERMOTRON® titer plate shaker at 300 RPM for 4 hours (single substrate) or 16-18 hours (one-pot). One set of SFP dilutions was pre-incubated in 50 mM potassium phosphate buffer, pH 6 for 2 hours at 75° C., while the other set was not. Reactions were solubilized by 20-fold dilution in water, quenched by 5-fold dilution in acetonitrile with 0.2% formic acid, clarified by centrifugation, and diluted 20-fold in water for analysis. Glycosylation products were detected by SPE-QQQ as described in Example 5, Table 5.1. To assess thermal stability, a similar single substrate rebaudioside A reaction was also performed using 10 μL of crude clarified lysate diluted 70-fold in 50 mM potassium phosphate buffer, pH 6 and incubated at 71.8° C. for 24 hours. Thermal stability results and production levels of rebaudioside D in single substrate and rebaudioside M in one-pot reaction by these variants at 0.005 g / L SFP loading are shown in Table 17.2. [Table 17-3]

[0252] In these experiments, all eight variants in Table 17.2 showed improved catalysis of the glucosylation of rebaudioside A to rebaudioside D and the β1,2-glucosylation involved in the conversion of rebaudioside A 60% to rebaudioside M, as compared to SEQ ID NO: 858. The most active variant for both substrates, SEQ ID NO: 994, was selected as the starting point for further enzyme engineering.

[0253] (Example 18) Beta-1,2-ADP-glycosyltransferase variant of SEQ ID NO: 994 This example describes an experiment of evolution and screening of GT polypeptides derived from SEQ ID NO:994 for improved glucosylation of steviol glycosides using ADP-glucose. Directed evolution of GT encoded by SEQ ID NO:994 was performed by constructing a library of variant genes. The libraries were recombined with mutations associated with improved production identified during the development of the invention and subjected to saturation mutagenesis of certain structural features of the enzyme. These libraries were then plated, grown, and screened using the HTP assay described below to obtain another round of engineered GT variant polypeptides with glucosyltransferase activity towards ADP-glucose and steviol glycosides. Nineteen engineered variants were identified from the combinatorial library (Table 18.1) and 25 from the saturation mutagenesis library (Table 18.2).

[0254] HTP binding assay for glucose transfer from sucrose to ADP and then to rebaudioside A60 The assay was performed using 96-well plates of clarified E. coli culture lysates expressing SEQ ID NO: 994 variants (i.e., variants of SEQ ID NO: 994). The volume of lysis buffer was 400 μL and lysates were diluted 100-fold into 50 mM potassium phosphate, pH 6.0 with or without 20 g / L (combinatorial library) or 30 g / L (saturation mutagenesis library) Rebaudioside A 60% and pre-incubated at 75° C. for 2 hours. The assay was then performed using 10 μL of diluted lysate, 0.03 g / L SUS SFP SEQ ID NO:1822 and 0.1 g / L β-1,3-glycosyltransferase (β1,3GT) SFP SEQ ID NO:350 in a 100 μL reaction volume with 20 g / L rebaudioside A 60% (RebA60) substrate, 0.02 g / L ADP (Amresco, Ultra Pure Grade) co-substrate and 30 g / L sucrose. The following reaction conditions were used: 50 mM potassium phosphate buffer, pH 6, 60°C, 16-18 hours with shaking at 300 RPM in a THERMOTRON® titer plate shaker. The reaction was solubilized by 20-fold dilution by addition to water, quenched by 5-fold dilution by addition to acetonitrile with 0.2% formic acid, precipitated by centrifugation and 20-fold dilution by addition to water for analysis as described above. The resulting engineered variants with glucosyltransferase activity towards RebA60 are listed in Tables 18.1 and 90.2. For the variants listed in Table 18.3, shake flask scale cultures were grown, lysed and lyophilized to a powder. [Table 18-1] [Table 18-2] [Table 18-3] [Table 18-4]

[0255] Shake flask powder characterization assay and analysis of glucosyl transfer from sucrose to ADP and then to rebaudioside A60 Shake flask powder (SFP) was reconstituted to a concentration of 4 g / L and diluted to 0.0013-0.04 g / L in a total reaction volume of 100 μL containing 50 mM potassium phosphate buffer, pH 6, 20 g / L RebA97 (single substrate) or RebA60 (one-pot), 0.02 g / L ADP, 20 (single substrate) or 30 g / L (one-pot) sucrose, 0.03 g / L SUS SFP SEQ ID NO: 1822, and for the one-pot reaction only, 0.1 g / L β-1,3-glycosyltransferase (β1,3GT) SFP SEQ ID NO: 350. Reactions were run at 60 °C in a THERMOTRON® titer plate shaker at 300 RPM for 4 hours (single substrate) or 16-18 hours (one-pot). One set of SFP dilutions was preincubated in 50 mM potassium phosphate buffer, pH 6 with 10 g / L rebaudioside A60% for 2 hours at 75°C, while the other set was not preincubated. Reactions were solubilized by 20-fold dilution in water, quenched by 5-fold dilution in acetonitrile with 0.2% formic acid, clarified by centrifugation, and diluted 20-fold in water for analysis. Glycosylation products were detected by SPE-QQQ as described in Example 5, Table 5.1. To assess thermal stability, a similar one-pot RebA60 reaction was also performed using 10 μL of crude clarified lysate diluted 70-fold in 50 mM potassium phosphate buffer, pH 6 with 10 g / L RebA60 and incubated at 71.8°C for 24 hours. The thermostability results and production levels of rebaudioside D with single substrate and rebaudioside M in one-pot reaction by these variants at 0.005 g / L SFP loading are shown in Table 18.3. [Table 18-5]

[0256] In these experiments, three variants in Table 18.3 showed significantly improved catalysis of glucosylation of rebaudioside A to rebaudioside D after preincubation compared to SEQ ID NO: 994. The most thermostable variant, SEQ ID NO: 1080, was selected as the starting point for further enzyme engineering.

[0257] (Example 19) Beta-1,2-ADP-glycosyltransferase variant of SEQ ID NO: 1080 This example describes an experiment of evolution and screening of GT polypeptides derived from SEQ ID NO: 1080 for improved glucosylation of steviol glycosides using ADP-glucose. Directed evolution of GT encoded by SEQ ID NO: 1079 was performed by constructing a library of variant genes. The libraries were recombined with mutations associated with improved production identified during the development of the invention and subjected to saturation mutagenesis of certain structural features of the enzyme. These libraries were then plated, grown, and screened using the HTP assay described below to obtain another round of engineered GT variant polypeptides with glucosyltransferase activity towards ADP-glucose and steviol glycosides. Thirty-three engineered variants were identified from the combinatorial library (Table 19.1) and 38 from the saturation mutagenesis library (Table 19.2).

[0258] HTP binding assay for glucose transfer from sucrose to ADP and then to rebaudioside A60 The assay was performed using 96-well plates of clarified E. coli culture lysates expressing SEQ ID NO: 1079 variants (i.e., variants of SEQ ID NO: 1080). The volume of lysis buffer was 400 μL and lysates were diluted 100-fold into 50 mM potassium phosphate, pH 6.0 with 30 g / L (combinatorial library) or 10 g / L (saturation mutagenesis retest) rebaudioside A 60% and pre-incubated at 75° C. for 2 hours. The assay was then performed using 10 μL of diluted lysate, 0.03 g / L SUS SFP SEQ ID NO:1822 and 0.1 g / L β-1,3-glycosyltransferase (β1,3GT) SFP SEQ ID NO:350 in a 100 μL reaction volume with 20 g / L rebaudioside A 60% (RebA60) substrate, 0.02 g / L ADP (Amresco, Ultra Pure Grade) co-substrate and 30 g / L sucrose. The following reaction conditions were used: 50 mM potassium phosphate buffer, pH 6, 60°C, 16-18 hours with shaking at 300 RPM in a THERMOTRON® titer plate shaker. The reaction was solubilized by 20-fold dilution by addition to water, quenched by 5-fold dilution by addition to acetonitrile with 0.2% formic acid, precipitated by centrifugati...

Claims

[Claim 1] An invention described in part in this specification.

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