Compositions and methods for producing rebaudioside D

Abstract

The present disclosure provides enzymes and methods for the transfer of sugar moieties to substrate steviol glycosides, specifically, the conversion of stevioside and Reb A to Reb E and Reb D using engineered β-1,2-glycosyltransferase and sucrose synthase in a one-pot reaction.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 150,515, filed February 17, 2021, the entire contents of which are incorporated herein by reference.

[0002] Incorporating sequence tables The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety. A computer-readable copy of the sequence listing (file name: ARZE_034_01WO_SeqList_ST25.txt, data recording date: February 17, 2022, file size approximately 6.84 MB).

[0003] The present disclosure relates to enzymes and biocatalytic processes for producing steviol glycosides. In particular, the disclosure relates to the use of glycosyltransferases that can transfer a glucose moiety from an ADP-glucose sugar donor to a steviol glycoside. [Background technology]

[0004] Excessive sugar intake is linked to global health epidemics such as diabetes and heart disease, and the exorbitant costs associated with treating these diseases in healthcare systems. Replacing added sugar in foods with low-calorie, high-intensity sweeteners would have major health and economic impacts.

[0005] Stevia rebaudiana is widely cultivated for its sweet leaves and is traditionally used as a sweetener. Stevia extracts are 200-300 times sweeter than sugar and are used commercially as high-intensity sweeteners. The main glycosidic components of Stevia leaves are stevioside and rebaudioside. More than 10 different steviol glycosides are present in the leaves in significant amounts. The main sweet compounds are stevioside and rebaudioside A. Rebaudioside A (Reb A) is considered to have a higher value compared to stevioside due to its increased sweetness and decreased bitterness.

[0006] The sweetness and bitterness profile of rebaudioside D (Reb D) is improved compared to Reb A, but Reb D is present in very small amounts in Stevia leaves. Reb D can be made by adding a single glucose molecule to Reb A. The native glycosyltransferase that makes Reb D uses UDP-glucose as the glucose source for transfer to Reb A. Summary of the Invention

[0007] The present disclosure provides enzymes, particularly unnatural enzymes, and methods for the transfer of sugar moieties to substrate steviol glycosides (also referred to herein as "SG") by those enzymes. Specifically, β-1,2-glycosyltransferase (also referred to herein as "B12GT") and sucrose synthase (also referred to herein as "SuSy") are used in a one-pot reaction to convert stevioside and Reb A to rebaudioside E (Reb E) and Reb D, respectively.

[0008] In contrast to native glycosyltransferases, the disclosure provides glycosyltransferase polypeptides that can utilize ADP-glucose as a sugar donor to convert Reb A to Reb D. The disclosure provides glycosyltransferase polypeptides that comprise an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 6-882 and 1333-1466. The glycosyltransferase polypeptides can comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NOs: 6-882 and 1333-1466. The polypeptides can include one or more peptide tags used for lysis, expression, and / or purification, such as a polyhistidine tag of 4-10 histidine residues, preferably 6 histidine residues. Other suitable tags include, but are not limited to, glutathione S-transferase (GST), FLAG, maltose binding protein (MBP), calmodulin binding peptide (CBP), and Myc tags. Suitable linkers include, but are not limited to, polypeptides composed of glycine and serine, such as GSGS, polyglycine linkers, repeats of EAAAK, and sequences containing cleavage sites for enzymes such as factor Xa, enterokinase, and thrombin.

[0009] Nucleotide sugar donors, including both UDP-glucose and ADP-glucose, are expensive co-substrates, adding significant costs to processes that utilize the compounds. Sucrose synthase (SuSy, EC 2.4.1.13) catalyzes the chemical reaction of nucleotide diphosphate (NDP) with sucrose to form NDP-glucose and fructose. Thus, sucrose synthase can be used to convert NDP to NDP-glucose (an exemplary glycosyltransferase) required for B12GT. The present disclosure provides a sucrose synthase polypeptide, the sucrose synthase polypeptide comprising an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 890-1227 and 1231-1332. Specifically, the disclosed sucrose synthase can convert ADP to the ADP-glucose cofactor required for the disclosed B12GT.

[0010] The disclosure further provides a method for converting Reb A and / or stevioside to Reb D and Reb E, respectively, by a SuSy ADP-glucose recycle system in combination with a B12GT polypeptide in a one-pot reaction. In some embodiments, the method includes contacting a Stevia leaf extract purified to contain greater than 50% Reb A (RA50), ADP, and sucrose with a B1,2 glycosyltransferase and sucrose synthase to produce Reb D and / or Reb E.

[0011] In order that the present disclosure may be further understood, the accompanying drawings are provided which illustrate embodiments of the present disclosure and, together with the description, serve to explain the principles of embodiments of the present disclosure. [Brief description of the drawings]

[0012] [Figure 1]1 shows the conversion of rebaudioside A (Reb A) to rebaudioside D (Reb D) (glycosylation). [Diagram 2] The three native UDP-glucose B12GTs show measured Reb A to Reb D activity when either ADP-glucose or GDP-glucose was used as the glycosyl donor. [Diagram 3] The measured abilities of native sucrose synthase to convert ADP to ADP-glucose (top), GDP to GDP-glucose (middle), and UDP to UDP-glucose (bottom) are shown. [Figure 4] (Top) shows the measured conversion of Reb A to Reb D to Reb M2 in a one-pot reaction of B12GT pA10143 and one of the seven native sucrose synthases. (Bottom) shows the measured conversion of Reb A to Reb D in a one-pot reaction of B12GT pA12549 and one of the seven native sucrose synthases. [Diagram 5] The top designs of pA10143 from a site-saturation mutagenesis library of the active site are shown. The parent enzyme pA10143 is shown in grey. [Figure 6] Measured Reb A to Reb D conversion by mutated residues for all enzymes from the pA10143 active site SSM library is shown. [Figure 7] LCMS chromatograms of reaction products produced by large-scale one-pot reaction of pA21841 and pA29798 are shown. [Figure 8] LCMS chromatograms of reaction products generated from a large-scale one-pot reaction of pA21841 and pA29646 are shown. [Figure 9] 1 shows an SDS-PAGE gel of engineered B12GT purified from Pichia pastoris expression. [Figure 10]A shows an SDS-PAGE gel of two engineered B12GTs purified from 1 L Pichia pastoris fermentation (from left to right: pA29798 (B12GT-1), ladder, pA32946 (B12GT-2)). B shows an SDS-PAGE gel of two engineered SuSys purified from 1 L Pichia pastoris fermentation (from left to right: ladder, pA34103 (SuSy-1), ladder, pA32691 (SuSy-2)). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The disclosure provides enzymatic and biocatalytic processes for preparing a composition comprising a target steviol glycoside by contacting a feedstock composition comprising a substrate steviol glycoside, sucrose, and NDP with an NDP-glucosyltransferase polypeptide and a sucrose synthase, ultimately producing a composition comprising a target steviol glycoside that contains one or more additional glucose units than the substrate steviol glycoside.

[0014] As used herein, "biocatalysis" or "biocatalytic" refers to the use of natural catalysts, such as protein enzymes, to perform chemical transformations in organic compounds. Biocatalysis is also known as biotransformation or biosynthesis. Both isolated and whole cell biocatalytic methods are known in the art. Biocatalytic protein enzymes can be naturally occurring or recombinant proteins.

[0015] As used herein, the term “steviol glycoside(s)” refers to naturally occurring steviol glycosides, (e.g., steviol-13-O-glucoside, steviol-19-O-glucoside, rubusoside, steviol-1,2-bioside, steviol-1,3-bioside, rubusoside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside G, rebaudioside H, rebaudioside I, rebaudioside J, rebaudioside K, rebaudioside N ... Rebaudioside F, Rebaudioside A, Rebaudioside I, Rebaudioside E, Rebaudioside H, Rebaudioside L, Rebaudioside K, Rebaudioside J, Rebaudioside M, Rebaudioside D, Rebaudioside N, Rebaudioside O, Rebaudioside Q), synthetic steviol glycosides (e.g., enzymatically glucosylated steviol glycosides), and combinations thereof.

[0016] As used herein, the term "feedstock composition" refers to any composition (generally an aqueous solution) that contains one or more steviol glycosides, which serve as substrates for biotransformation.

[0017] As used herein, the terms "polynucleotide" or "nucleic acid" are used interchangeably, unless otherwise indicated by context, to refer to any polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, typically DNA.

[0018] As used herein, "expression" refers, depending on the context, to either or both steps in the two-step process by which a polynucleotide is transcribed into mRNA, which is then translated into a polypeptide.

[0019] "Under transcriptional control" means that transcription of a polynucleotide, usually a DNA sequence, is dependent on being operably linked to elements that promote transcription.

[0020] "Operably linked" means that a polynucleotide element is positioned so that it can function within a cell, typically to produce a polypeptide within the cell; for example, the disclosure provides a promoter operably linked to a downstream sequence that encodes a polypeptide.

[0021] The term "encoding" refers to the ability of a polynucleotide to produce an mRNA or a polypeptide, if the polynucleotide can be transcribed to produce an mRNA and then translated to produce a polypeptide or a fragment thereof. In each case, the polynucleotides are referred to as those that encode the mRNA and those that encode the polypeptide. The antisense strand is the complementary strand of such a nucleic acid, from which a coding sequence can be deduced. Similarly, "coding sequence" refers to the region of a nucleic acid that encodes an mRNA or a polypeptide.

[0022] The term "promoter" as used herein refers to a control sequence that is a portion of a polynucleotide sequence that controls the initiation and rate of transcription of a coding sequence. An "enhancer" is a regulatory element that increases the expression of a target sequence. A "promoter / enhancer" is a polynucleotide that has sequences that provide both promoter and enhancer functions.

[0023] Regulatory elements such as enhancers and promoters can be "homologous" or "heterologous". A "homologous" regulatory element is one that is naturally linked to a particular polynucleotide in the genome, for example, it can be a promoter found in nature in an organism upstream of the encoded polypeptide. A "heterologous" regulatory element is one that is juxtaposed to a polynucleotide by recombinant molecular biology techniques, but is not in a combination found in nature. In many cases, promoters, enhancers and other regulatory elements are heterologous to facilitate expression of a polypeptide in a host cell other than the host cell in which the polypeptide naturally occurs. Thus, as used herein, "heterologous expression" refers to producing mRNA and / or a polypeptide in a host cell, such as a microorganism, where the polynucleotide is not found in nature or one or more regulatory elements operably linked to the polynucleotide in the host cell are not found in nature.

[0024] The term "polypeptide" is used herein to refer to a molecule of two or more subunits of amino acids linked by peptide bonds. Typically, but not always, a polypeptide contains several hundred amino acids, e.g., about 400 to about 800 amino acids.

[0025] A "plasmid" is a DNA molecule that is usually separated from chromosomal DNA and can replicate independently of chromosomal DNA. It is often circular and double stranded. Plasmid vectors often exist as extrachromosomal circular DNA molecules, but it is known in the art that plasmid vectors can also be designed to stably integrate into host chromosomes in a random or targeted manner. Many plasmids are commercially available for various applications. The gene to be replicated is inserted into a copy of the plasmid, which contains a gene that makes the cell resistant to a particular antibiotic, and a multiple cloning site (MCS, or polylinker), a short region that contains several commonly used restriction sites that allow easy insertion of DNA fragments at this location. Typically, the polypeptides disclosed herein are expressed from a plasmid.

[0026] The term "about" or "approximately" immediately preceding a numerical value refers to a range (e.g., ±10% of that value). For example, unless otherwise indicated in the context of the disclosure or inconsistent with such an interpretation, "about 50" can refer to 45 to 55, "about 25,000" can refer to 22,500 to 27,500, etc. For example, a list of numerical values ​​such as "about 49, about 50, about 55, ...", "about 50" refers to a range that spans less than half the interval(s) between the preceding and succeeding values, e.g., from greater than 49.5 to less than 52.5. Furthermore, the phrase "less than" an "about" value or "greater than" an "about" value should be understood in light of the definition of the term "about" provided herein. Similarly, the term "about" preceding a series of numerical values ​​or a range of values ​​(e.g., "about 10, 20, 30" or "about 10 to 30") refers to all values ​​in the series, or to the end points of the range, respectively.

[0027] As used herein, the terms "microorganisms" or "microbes" should be interpreted broadly. They are used interchangeably and include, but are not limited to, the two prokaryotic domains, bacteria and archaea, as well as certain eukaryotic fungi and protists. In some embodiments, the present disclosure refers to "microorganisms" or "microbes" in the lists and figures present in this disclosure. Characterization can refer not only to the identified taxonomic genus, but also to the identified taxonomic species, as well as various novel and newly identified or designed strains of any organism in said tables or figures. The same characterization applies to the explanation of those terms in other parts of the specification, such as the Examples.

[0028] When referring to nucleic acid or protein sequences, the term "identity" is used to indicate the similarity between two sequences. Sequence identity or similarity can be determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the sequence identity alignment algorithm of Needleman & Wunsch, J Mol. Biol. 48, 443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85, 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, WI), by the Best Fit sequence program described by Devereux et al., Nucl. Acid Res. 12, 387-395 (1984), or by inspection. Another example of a suitable algorithm is the BLAST algorithm described in Altschul et al., J Mol. Biol. 215, 403-410, (1990) and Karlin et al., Proc. Natl. Acad. Sci. USA 90, 5873-5787 (1993). An exemplary BLAST program is the WU-BLAST-2 program, taken from Altschul et al., Methods in Enzymology, 266, 460-480 (1996), blast.wustl / edu / blast / README.html. WU-BLAST-2 uses several search parameters, which are preferably set to default values. The parameters are dynamic values, established by the program itself depending on the composition of the particular sequence and the composition of the particular database in which the sequence of interest is searched, but the values ​​can also be adjusted to increase sensitivity.Another algorithm is Gapped BLAST, as reported by Altschul et al, (1997) Nucleic Acids Res. 25, 3389-3402. Other algorithms can also be described herein.

[0029] The present disclosure provides a non-naturally occurring engineered β-1,2-ADP glycosyltransferase (B12GT) that can use an ADP-glucose sugar donor to convert stevioside to Reb E and Reb A to Reb D. In certain embodiments, the glucosyltransferase polypeptide is one of SEQ ID NOs: 6-882 and 1333-1466. In another embodiment, the glucosyltransferase polypeptide is a polypeptide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one of SEQ ID NOs: 6-882 and 1333-1466.

[0030] In bioinformatics, several methods are developed to find and determine related polypeptide sequences. For example, sequence identity percentage, position specific scoring matrix (PSSM), and hidden Markov model (HMM) are all widely used to find sequences similar to a specific query sequence. Sequence identity percentage calculates the number of amino acids shared between two sequences. Sequence identity percentage is calculated based on a specific alignment between two sequences. Identity percentage can be calculated using alignment program Clustal Omega (available at / www.ebi.ac.uk / Tools / msa / clustalo / ) with default settings. The default transition matrix is ​​Gonnet, gap opening penalty is 6 bits, and gap extension is 1 bit. Clustal Omega uses the HHalign algorithm and its default settings as the core alignment engine. The algorithm is described in Soding, J. (2005) 'Protein homology detection by HMM-HMM comparison'. Bioinformatics 21, 951-960.

[0031] A position-specific scoring matrix (PSSM) is a concise way of representing many related sequences. PSSMs are often generated by multiple sequence alignments. The sequence search tool PSI-BLAST generates PSSMs and uses them to search for related polypeptide sequences. A PSSM used to score a polypeptide sequence is a matrix (i.e., a table) consisting of 21 columns by N rows, where N is the length of the related sequence. Each row corresponds to a position in the polypeptide sequence, and each column represents a different amino acid (or gap) that that residue position can take. Each entry in the PSSM represents the score of a particular amino acid at a particular position in the polypeptide sequence. To score a sequence using a PSSM, first align the sequence to a reference sequence, then calculate the sum of:

number

[0032] The present disclosure also provides a non-naturally occurring engineered sucrose synthase (SuSys) that can use a sucrose sugar donor to convert ADP to ADP-glucose. In certain embodiments, the SuSy polypeptide is one of SEQ ID NOs: 890-1227 and 1231-1332. In another embodiment, the SuSy polypeptide is a polypeptide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one of SEQ ID NOs: 890-1227 and 1231-1332.

[0033] In some embodiments, the glucosyltransferase and / or sucrose synthase polypeptides are prepared by expression in a host microorganism. Suitable host microorganisms include, but are not limited to, E. coli, Saccharomyces sp., Aspergillus sp., Pichia sp., and Bacillus sp. In certain embodiments, the glucosyltransferase and sucrose synthase are expressed in E. coli. In certain embodiments, the glucosyltransferase and sucrose synthase are expressed in Pichia pastoris.

[0034] The B12GT and / or SuSy polypeptides may be provided in any suitable form, including free, immobilized, or whole cell systems. The glucosyltransferase polypeptide may be of varying purity, for example, provided as a crude enzyme preparation, a semi-purified enzyme preparation, or a purified enzyme preparation. In one embodiment, the glycosyltransferase polypeptide is free. In another embodiment, the glycosyltransferase polypeptide is immobilized on a solid support, for example, an inorganic or organic support. In some embodiments, the solid support is derivatized cellulose, glass, ceramic, methacrylate, styrene, acrylic, metal oxide, or membrane. In some embodiments, the glucosyltransferase polypeptide is immobilized on the solid support by covalent bonding, adsorption, crosslinking, entrapment, or encapsulation.

[0035] Furthermore, in another embodiment, the B12GT and / or SuSy polypeptides are provided in the form of a whole cell system, for example as live fermenting microbial cells, or as killed and stabilized microbial cells, or in the form of a cell lysate.

[0036] The present disclosure provides a biocatalytic process for preparing a composition comprising a target steviol glycoside from a feed composition comprising a substrate steviol glycoside, the target steviol glycoside comprising one or more additional glucose units than the substrate steviol glycoside. The biocatalytic process comprises contacting B12GT and SuSy with a feed composition comprising one or more steviol glycosides, a non-UDP nucleotide diphosphate, and sucrose. In another embodiment, the biocatalytic process comprises contacting modified B12GT and SuSy with a feed composition comprising one or more steviol glycosides, a non-UDP nucleotide diphosphate, and sucrose. In another embodiment, the biocatalytic process comprises contacting modified B12GT and modified SuSy with a feed composition comprising one or more steviol glycosides, a non-UDP nucleotide diphosphate, and sucrose. In some embodiments, the method comprises contacting RA50, ADP, and sucrose with modified B1,2 glycosyltransferase and sucrose synthase to produce Reb D and Reb E.

[0037] In one embodiment, the B12GT polypeptide is one of SEQ ID NOs: 1-882 and 1333-1466. In another embodiment, the glucosyltransferase polypeptide is a polypeptide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one of SEQ ID NOs: 1-882. In another embodiment, the glucosyltransferase polypeptide is a polypeptide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one of SEQ ID NOs:6-882 and 1333-1466. Preferably, the catalytic domain of the B12GT polypeptide comprises residues corresponding to H at position 15, D at position 114, D at position 357, and Q at position 358, numbered according to SEQ ID NO:5.

[0038] In one embodiment, the sucrose synthase is any polypeptide having sucrose synthase activity. In another embodiment, the sucrose synthase is from an organism in the Bacteria domain. In another embodiment, the sucrose synthase is from an organism in the Plantae kingdom. In another embodiment, the sucrose synthase is from an organism in the Plantae kingdom. In another embodiment, the sucrose synthase is from an organism belonging to the Proteobacteria, Deferibacteria, or Cyanobacteria phylum. In another embodiment, the sucrose synthase is from Acidithiobacillus caldus, Nitrosomonas europaea, Denitrovibrio acetiphilus, Thermosynechococcus elongatus, Oryza sativa, Arabidopsis thaliana, or Coffea arabica. In one embodiment, the sucrose synthase is one of SEQ ID NOs: 883-1227 and 1231-1332. In another embodiment, the sucrose synthase is an engineered sucrose synthase having a polypeptide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to one of SEQ ID NOs:883-1227. In another embodiment, the sucrose synthase is an engineered sucrose synthase having a polypeptide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to one of SEQ ID NOs: 890-1227 and 1231-1332. Preferably, the catalytic domain of the SuSy polypeptide comprises residues corresponding to H at position 425, R at position 567, K at position 572, and E at position 663, numbered according to SEQ ID NO:885, or residues corresponding to H at position 436, R at position 578, K at position 583, and E at position 674, numbered according to SEQ ID NO:888.

[0039] In some embodiments, the glucosyltransferase and / or sucrose synthase polypeptides are prepared by expression in a host microorganism. Suitable host microorganisms include, but are not limited to, E. coli, Saccharomyces sp., Aspergillus sp., Pichia sp., and Bacillus sp. In certain embodiments, the glucosyltransferase and sucrose synthase are expressed in E. coli. In another embodiment, the glucosyltransferase and sucrose synthase are expressed in Pichia pastoris. In another embodiment, the glucosyltransferase and / or sucrose synthase polypeptides are prepared by cell-free expression.

[0040] The B12GT and sucrose synthase polypeptides may be provided in any suitable form, including free, immobilized, or whole cell systems. The purity of the polypeptides may vary, and they may be provided, for example, as crude enzyme preparations, semi-purified enzyme preparations, or purified enzyme preparations. In one embodiment, the B12GT and / or SuSy polypeptides are free. In another embodiment, the B12GT and / or SuSy polypeptides are immobilized on a solid support, for example, an inorganic or organic support. In some embodiments, the solid support is derivatized cellulose, glass, ceramic, methacrylate, styrene, acrylic, metal oxide, or membrane. In some embodiments, the B12GT and / or SuSy polypeptides are immobilized on the solid support by covalent bonding, adsorption, crosslinking, entrapment, or encapsulation.

[0041] Furthermore, in another embodiment, the B12GT and / or SuSy polypeptides are provided in the form of a whole cell system, for example as live fermenting microbial cells, or as killed and stabilized microbial cells, or in the form of a cell lysate.

[0042] The steviol glycoside component(s) of the feedstock composition serve as substrate(s) for the production of target steviol glycoside(s), as described herein, where the target(s) of the target steviol glycoside differ chemically from the corresponding substrate steviol glycoside by one or more additional glucose units.

[0043] The raw steviol glycoside composition may include at least one substrate steviol glycoside. In one embodiment, the substrate steviol glycoside is steviol, steviol-13-O-glucoside, steviol-19-O-glucoside, rubusoside, steviol-1,2-bioside, steviol-1,3-bioside, rubusoside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside G, rebaudioside H, rebaudioside I, rebaudioside J, rebaudioside K, rebaudioside N, rebaudioside N, rebaudioside N, rebaudioside N, rebaudioside N, rebaudioside N, rebaudioside N, rebaudioside N, rebaudioside N, rebaudioside N, rebaudioside B, rebaudioside B, rebaudioside N ... The raw steviol glycoside composition is selected from the group consisting of stevioside F, rebaudioside A, rebaudioside I, rebaudioside E, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M, rebaudioside D, rebaudioside N, rebaudioside O, rebaudioside Q, isomers thereof, synthetic steviol glycosides, or combinations thereof. In another embodiment, the raw steviol glycoside composition is comprised of stevioside and Reb A. In another embodiment, the raw steviol glycoside composition is comprised of stevioside. In yet another embodiment, the raw steviol glycoside composition is comprised of Reb A.

[0044] The raw steviol glycoside composition can be synthetic or purified (partially or completely), commercially available, or prepared. One example of a raw composition useful in the methods of the present disclosure is an extract obtained from the purification of Stevia rebaudiana plant material (e.g., leaves). Another example of a raw composition is a commercially available stevia extract in solution with a solvent. Yet another example of a raw composition is a commercially available mixture of steviol glycosides in solution with a solvent. Other suitable raw compositions include by-products of processes for isolating and purifying steviol glycosides.

[0045] In one embodiment, the feedstock composition comprises purified substrate steviol glycosides. For example, the feedstock composition may comprise, on an anhydrous basis, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, greater than about 99%, or greater than about 99.6% by weight of one or more substrate steviol glycosides.

[0046] In another embodiment, the feedstock composition comprises a partially purified substrate steviol glycoside composition, e.g., the feedstock composition contains greater than about 0.5%, greater than about 1%, greater than about 2%, greater than about 3%, greater than about 4%, greater than about 5%, greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, or greater than about 50% by weight of one or more substrate steviol glycosides on an anhydrous basis.

[0047] In another embodiment, the substrate steviol glycoside is purified rebaudioside A or an isomer thereof. In certain embodiments, the substrate steviol glycoside comprises greater than 99% by weight rebaudioside A or an isomer thereof on an anhydrous basis. In another embodiment, the substrate steviol glycoside comprises partially purified rebaudioside A. In certain embodiments, the substrate steviol glycoside contains greater than about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% rebaudioside A by weight on an anhydrous basis.

[0048] Additionally, in another embodiment, the substrate steviol glycoside comprises purified stevioside or an isomer thereof. In certain embodiments, the substrate steviol glycoside comprises greater than 99% by weight stevioside or an isomer thereof on an anhydrous basis. In another embodiment, the substrate steviol glycoside comprises partially purified stevioside. In certain embodiments, the substrate steviol glycoside comprises greater than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% by weight stevioside on an anhydrous basis.

[0049] Additionally, in another embodiment, the substrate steviol glycoside is a combination of stevioside and rebaudioside A. In certain embodiments, the substrate steviol glycoside may be greater than about 5% by weight stevioside and greater than about 5% by weight Reb A, greater than about 10% by weight stevioside and greater than about 10% by weight Reb A, greater than about 20% by weight stevioside and greater than about 20% by weight Reb A, greater than about 30% by weight stevioside and greater than about 30% by weight Reb A, greater than about 40% by weight stevioside and greater than about 40% by weight Reb A, greater than about 45% by weight stevioside and greater than about 45% by weight Reb A, greater than about 40% by weight stevioside and greater than about 50% by weight Reb A, greater than about 30% by weight stevioside and greater than about 60% by weight Reb A, greater than about 20% by weight stevioside and greater than about 70% by weight Reb A, or greater than about 100% by weight stevioside. A, greater than about 10% by weight stevioside and greater than about 80% by weight Reb A, greater than about 5% by weight stevioside and greater than about 90% by weight Reb A, greater than about 50% by weight stevioside and greater than about 40% by weight Reb A, greater than about 60% by weight stevioside and greater than about 30% by weight Reb A, greater than about 70% by weight stevioside and greater than about 20% by weight Reb A, greater than about 80% by weight stevioside and greater than about 10% by weight Reb A, or greater than about 90% by weight stevioside and greater than about 5% Reb A.

[0050] In another embodiment, the substrate steviol glycoside is derived from stevia leaf extract. In one embodiment, RA50 (stevia leaf extract purified to contain 50% or more Reb A) is used as the steviol glycoside substrate. In one embodiment, RA50 is used at a concentration of about 1 to 800 mg / mL. In another embodiment, RA50 is used at a concentration of about 100 mg / mL.

[0051] The one-pot reaction can be carried out with a nucleotide cofactor that can be converted to NDP-glucose by sucrose synthase. In some embodiments, the nucleotide can be a non-UDP nucleotide (i.e., ADP-glucose, GDP-glucose, CDP-glucose, or TDP-glucose). In another embodiment, the nucleotide is ADP. In certain embodiments, the one-pot reaction can be carried out with ADP at a concentration of about 0.01 to 10 mM, e.g., 0.01 mM to 0.05 mM, 0.05 mM to 0.1 mM, 0.1 mM to 0.5 mM, 0.5 mM to 1 mM, 1 mM to 5 mM, or 5 mM to 10 mM. In certain embodiments, ADP is used at a concentration of 0.5 mM.

[0052] The one-pot reaction can be carried out with sucrose at a concentration of about 10 mM to 2 M, for example, greater than 10 mM, greater than 50 mM, greater than 100 mM, greater than 250 mM, greater than 500 mM, greater than 1 M, greater than 1.5 M, and greater than 2 M. In certain embodiments, sucrose is used at a concentration of 250 mM.

[0053] In one embodiment, the reaction is carried out at any temperature. In another embodiment, the one-pot reaction is carried out at a temperature of about 10° C. to 80° C., such as 10° C. to 20° C., 20° C. to 30° C., 30° C. to 40° C., 40° C. to 50° C., 50° C. to 60° C., 60° C. to 70° C., 70° C. to 80° C., or 80° C. In a particular embodiment, the one-pot reaction is carried out at 60° C.

[0054] The reaction medium for the conversion is typically aqueous, such as purified water, a buffer, or a combination thereof. In certain embodiments, the reaction medium is a buffer. Suitable buffers include, but are not limited to, acetate buffer, citrate buffer, HEPES, and phosphate buffer. In certain embodiments, the reaction medium is a phosphate buffer. The reaction medium can have a pH of about 4 to 10. In certain embodiments, the pH of the reaction medium is 6. Alternatively, the reaction medium can be an organic solvent.

[0055] The step of contacting the feedstock composition with the glycosyltransferase and sucrose synthase polypeptides can be carried out for about 1 hour to 1 week, e.g., 30 minutes to 1 hour, 1 hour to 4 hours, 4 hours to 6 hours, 6 hours to 12 hours, 12 hours to 24 hours, 1 day to 2 days, 2 days to 3 days, 3 days to 4 days, 4 days to 5 days, 6 days to 7 days, etc. In certain embodiments, the reaction is carried out for 24 hours.

[0056] Reactions may be monitored by any appropriate method, including but not limited to, HPLC, LCMS, TLC, IR or NMR.

[0057] The target steviol glycoside can be any steviol glycoside. In one embodiment, the target steviol glycoside is steviol, steviol-13-O-glucoside, steviol-19-O-glucoside, rubusoside, steviol-1,2-bioside, steviol-1,3-bioside, rubusoside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F, rebaudioside A, rebaudioside I. , rebaudioside E, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M, rebaudioside D, rebaudioside N, rebaudioside O, rebaudioside Q, rebaudiosides having seven covalently linked glucose units (e.g., rebaudioside M+1 glucose unit), synthetic steviol glycosides, isomers thereof, and / or steviol glycoside compositions. In another embodiment, the target steviol glycoside is rebaudioside E or an isomer thereof. In another embodiment, the target steviol glycoside is rebaudioside D or an isomer thereof. Yet, in another embodiment, the target steviol glycoside is Reb D and Reb E.

[0058] In one embodiment, the conversion of Reb A to Reb D and / or Reb D isomer(s) is at least about 2% complete, as determined by any of the methods described above. In certain embodiments, the conversion of Reb A to Reb D and / or Reb D isomer(s) is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% complete. In certain embodiments, the conversion of reb A to reb D and / or reb D isomer(s) is at least about 95% complete. In some embodiments, at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the Reb A in the feedstock composition is converted to Reb D and / or Reb D isomer(s).

[0059] In one embodiment, the conversion of stevioside to Reb E and / or Reb E isomer(s) is at least about 2% complete as determined by any of the methods described above. In certain embodiments, the conversion of stevioside to Reb E and / or Reb E isomer(s) is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% complete. In certain embodiments, the conversion of stevioside to Reb E and / or Reb E isomer(s) is at least about 95% complete. In some embodiments, at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the stevioside in the feedstock composition is converted to Reb E and / or Reb E isomer(s).

[0060] The target steviol glycoside(s) may be in any polymorphic or amorphous form, including hydrates, solvates, anhydrates, or combinations thereof.

[0061] Optionally, the methods of the present disclosure further include isolating the target steviol glycoside from the target composition. The target steviol glycoside(s) can be isolated by any suitable method, such as, for example, crystallization, membrane separation, centrifugation, extraction, chromatographic separation, or a combination of such methods.

[0062] In one embodiment, separation of the target steviol glycoside(s) produces a composition comprising greater than about 80% by weight of the target steviol glycoside on an anhydrous basis, i.e., a highly purified steviol glycoside composition. In another embodiment, the separation produces a composition that comprises greater than about 0.5%, greater than about 1%, greater than about 2%, greater than about 3%, greater than about 4%, greater than about 5%, greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, greater than about 99%, or greater than about 99.6% by weight of the target steviol glycoside. In certain embodiments, the composition comprises greater than about 95% by weight of the target steviol glycoside(s).

[0063] The purified target steviol glycosides can be used as sweeteners in consumer products. Suitable consumer products include, but are not limited to, foods, beverages, pharmaceutical compositions, tobacco products, dietary supplement compositions, oral hygiene compositions, and cosmetic compositions.

[0064] Plasmids containing nucleic acids encoding the enzymes having SEQ ID NOs: 1-1227 and 1231-1466 are set forth in Table 1 below.

[0065] [Table 1-1] [Table 1-2] [Table 1-3]

Table 1-4

Table 1-5

Table 1-6

Table 1-7

Table 1-8

Table 1-9

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Example

[0066] Example 1: In vivo production of native β-1,2-glycosyltransferase (B12GT) Polynucleotides encoding the amino acid sequences of known β-1,2-UDP-glycosyltransferases from five different organisms (Table 1.1) were synthesized (Twist Bioscience) and inserted into the pARZ4 expression vector. The polynucleotides were ordered as full-length genes or ordered as gene fragments and then assembled using Gibson assembly. The recombinant vectors were used to transform E. coli HMS174(DE3) (Novagen) by the heat shock method, thereby preparing recombinant microorganisms.

[0067] Each transformed recombinant microorganism was inoculated into 1 ml of LB-kanamycin medium and grown overnight at 37°C with shaking. The cultures were inoculated into 5 ml of TB-kanamycin medium and grown at 37°C for 2 hours followed by 25°C for 1 hour. The cultures were induced with 50 μL of 50 mM IPTG and grown overnight. Finally, the cultures were centrifuged at maximum speed for 5 minutes and stored at -80°C.

[0068] [Table 2]

[0069] Example 2: Purification of β-1,2-glycosyltransferase (B12GT) The microorganisms prepared in Example 1 were lysed in lysis buffer (lysozyme, DNAseI, Bugbuster, 300 mL of 20 mM HEPES pH 7.5, 500 mM NaCl, and 20 mM imidazole). Two to three glass beads were added to each well and disrupted by shaking at 25°C and 220 rpm for 30 minutes. The disruption solution was centrifuged at 2200 x g for 6 to 10 minutes. The resulting supernatant was loaded onto a Ni-NTA plate and shaken at room temperature for 10 minutes. The plate was centrifuged at 100 x g for 4 minutes, followed by washing twice with 500 μL of binding buffer (300 mL of 20 mM HEPES pH 7.5, 500 mM NaCl, 20 mM imidazole) and centrifuging (500 x g) for 2 minutes. Proteins were eluted with 150 μL elution buffer (15 mL 20 mM HEPES pH 7.5, 500 mM NaCl, 500 mM imidazole) and shaken for 1 min at 0.25 maximum shaking speed, followed by centrifugation at 500×g for 2 min. Recovered proteins were desalted in enzyme activity assay buffer (50 mM HEPES pH 7.5, 50 mM NaCl).

[0070] Example 3: Measurement of β-1,2-glycosyltransferase (B12GT) activity using ADP-glucose and GDP-glucose The wild-type β-1,2-UDP-glycosyltransferases, pA10132, pA10143, and pA12549, were assayed for activity towards ADP-glucose and GDP-glucose. The purified proteins were reacted with 0.5 mM RA99 (99% pure Reb A), 2 mM NDP-glucose (ADP-glucose or GDP-glucose) in 50 mM MOPS buffer at pH 7.8 at 30° C. for 72 hours. As shown in FIG. 1, the conversion of Reb A to Reb D was monitored by liquid chromatography mass spectrometry (LCMS) using an Agilent 6470 QQQ mass spectrometer (column: Waters ACQUITY UPLC HSS T3 column, 100 mm×2.1 mm). QQQ was performed by multiple reaction monitoring (MS / MS) to accurately quantify the steviol glycosides of the present invention. When ADP-glucose or GDP-glucose was used as the reactive sugar donor, all three wild-type B12GTs exhibited minimal Reb A to Reb D activity (Fig. 2).

[0071] Example 4: In vivo production of native sucrose synthase Polynucleotides encoding the amino acid sequences of sucrose synthase (SuSys) from seven different organisms (Table 2) were synthesized (Twist Bioscience) and inserted into the pARZ4 expression vector. The polynucleotides were ordered as full-length genes or ordered as gene fragments and then assembled using Gibson assembly. The recombinant vectors were used to transform E. coli NEBT7EL (New England Biolabs) by the heat shock method, thereby preparing recombinant microorganisms.

[0072] Each transformed recombinant microorganism was inoculated into 1 ml of LB-kanamycin medium and grown overnight at 37°C with shaking. The cultures were inoculated into 5 ml of TB-kanamycin medium and grown at 37°C for 2 hours followed by 25°C for 1 hour. The cultures were induced with 50 μL of 50 mM IPTG and grown overnight. Finally, the cultures were centrifuged at maximum speed for 5 minutes and stored at -80°C.

[0073] [Table 3]

[0074] Example 5: Purification of native sucrose synthase The microorganisms prepared in Example 4 were lysed in lysis buffer (lysozyme, DNAseI, Bugbuster, 300 mL of 20 mM HEPES pH 7.5, 500 mM NaCl, and 20 mM imidazole). Two to three glass beads were added to each well and disrupted by shaking at 25°C and 220 rpm for 30 minutes. The disruption solution was centrifuged at 2200 x g for 6 to 10 minutes. The resulting supernatant was loaded onto a Ni-NTA plate and shaken at room temperature for 10 minutes. The plate was centrifuged at 100 x g for 4 minutes, followed by washing twice with 500 μL of binding buffer (300 mL of 20 mM HEPES pH 7.5, 500 mM NaCl, 20 mM imidazole) and centrifuging (500 x g) for 2 minutes. Proteins were eluted with 150 μL elution buffer (15 mL 20 mM HEPES pH 7.5, 500 mM NaCl, 500 mM imidazole) and shaken for 1 min at 0.25 maximum shaking speed, followed by centrifugation at 500 × g for 2 min. Recovered proteins were desalted in enzyme activity assay buffer (50 mM MOPS pH 6.5, 50 mM NaCl).

[0075] Example 6: Measurement of sucrose synthase activity with UDP, GDP, and ADP The purified enzyme from Example 5 was reacted with 50 mM sucrose and 5 mM nucleotides (ADP, GDP or UDP) in 50 mM MOPS buffer (pH 6.5) and 50 mM NaCl at 60° C. for 24 hours. The conversion of NDP to NDP-glucose was monitored by liquid chromatography mass spectrometry (LCMS) using an Agilent 6545 QTOF mass spectrometer (column: Agilent HILIC-OH 150x2.1 mm). Wild-type sucrose synthase was active towards all three nucleotides (FIG. 3).

[0076] Example 7: Conversion of Reb A to Reb D in a one-pot reaction A one-pot reaction containing B12GT and SuSy was performed to demonstrate the ability to convert Reb A to Reb D using the ADP-glucose generated by SuSy. Purified B12GT (pA10143 (FIG. 4(top)) or pA12549 (FIG. 4(bottom))) and purified SuSy (pA10142, pA12546, pA21838, pA21839, pA21840, pA21841, or pA21842) were reacted with 0.5 mg / mL RA99, 50 mM sucrose, and 5 mM ADP in 50 mM MOPS buffer (pH 6.5), 3.0 mM MgCl2, and 50 mM NaCl at 30° C. for 24 hours. The product rebaudioside was monitored by LCMS as in Example 3. All one-pot reactions were able to produce Reb D from Reb A (Figure 4). The produced Reb D was further converted to Reb M2 by the one-pot reaction containing pA10143.

[0077] Example 8: Improving the activity of pA10143 by site-saturation mutagenesis A homology model of B12GT encoded by pA10143 was generated and used to identify active site residues of the protein. The following 20 active site residue positions were selected for site saturation mutagenesis: 81, 82, 88, 139, 178, 185, 260, 284, 317, 320, 324, 332, 336, 339, 341, 358, 359, 360, 362, 363. Gibson assembly using bridging oligos was used to generate 217 single point mutant variants of pA10143 (sequence numbers 6-222). Each B12GT variant was expressed and purified as in Example 2. Each B12GT variant was assayed in a one-pot reaction with SuSy and pA10142. Purified B12GT and SuSy were reacted with 4 mg / mL RA50, 40 mM sucrose, and 1 mM ADP in 50 mM phosphate buffer (pH 7), 3.0 mM MgCl2, and 50 mM NaCl at 30° C. for 24 hours. The product rebaudioside was monitored by LCMS as in Example 3. Several mutants showed improved activity compared to the parent pA10143 (FIG. 5). The activity of active site positions 358, 341, and 317 was most greatly improved (FIG. 6).

[0078] Example 9: Improvement of activity of pA10143 driven by computational design The homology model of B12GT encoded by pA10143 was used as input to computational design to improve pA10143. Computational design was performed to improve the stability and expression of B12GT. 93 computational designs were selected for experimental validation (SEQ ID NOs: 223-315). Expression plasmids for computational designs were constructed as in Example 1. Each B12GT variant was expressed and purified as in Example 2. Each B12GT variant was assayed in a one-pot reaction with SuSy and pA10142. Purified B12GT and SuSy were reacted with 4 mg / mL RA50, 40 mM sucrose, and 1 mM ADP in 50 mM phosphate buffer (pH 7), 3.0 mM MgCl2, and 50 mM NaCl at 30° C. for 24 hours. The product rebaudioside was monitored by LCMS as in Example 3. Several mutants showed improved expression and / or Reb D conversion compared to the parent pA10143 (18% conversion, 39 uM purified protein, Table 3).

[0079] [Table 4-1] [Table 4-2] [Table 4-3]

[0080] Computational design of pA10143 was further performed to improve the stability and expression of B12GT by co-evolutionary information. 60 computational designs were selected for experimental validation (SEQ ID NOs: 316-375). Expression plasmids for computational designs were constructed as in Example 1. Each B12GT variant was expressed and purified as in Example 2. Each B12GT variant was assayed in a one-pot reaction with SuSy and pA10142. Purified B12GT and SuSy were reacted with 4 mg / mL RA50, 40 mM sucrose, and 1 mM ADP in 50 mM phosphate buffer (pH 7), 3.0 mM MgCl2, and 50 mM NaCl at 30° C. for 24 hours. The product rebaudioside was monitored by LCMS as in Example 3. The computationally designed variants showed improved expression and / or Reb D conversion compared to the parent pA10143 (4.7% conversion, 35 uM purified protein, Table 4).

[0081] [Table 5-1] [Table 5-2]

[0082] Computational design of pA10143 was also performed to combine active site mutations. Nine computational designs were selected for experimental validation (SEQ ID NOs: 376-384). Expression plasmids for computational designs were constructed as in Example 1. Each B12GT variant was expressed and purified as in Example 2. Each B12GT variant was assayed in a one-pot reaction with SuSy and pA10142. Purified B12GT and SuSy were reacted with 4 mg / mL RA50, 40 mM sucrose, and 1 mM ADP in 50 mM phosphate buffer (pH 7), 3.0 mM MgCl2, and 50 mM NaCl at 30° C. for 24 hours. The product rebaudioside was monitored by LCMS as in Example 3. The top design, pA32576, showed activity comparable to the parent pA10143 (21% conversion, Table 5).

[0083] [Table 6]

[0084] In silico design of pA10143 was also performed to combine additional mutations. 75 in silico designs were selected for experimental validation (SEQ ID NOs: 385-459). Expression plasmids for the in silico designs were constructed as in Example 1. Each B12GT variant was expressed and purified as in Example 2. Each B12GT variant was assayed in a one-pot reaction with SuSy and pA10142. Purified B12GT and SuSy were reacted with 4 mg / mL RA50, 40 mM sucrose, and 1 mM ADP in 50 mM phosphate buffer (pH 7), 3.0 mM MgCl2, and 50 mM NaCl at 30° C. for 24 hours. The product rebaudioside was monitored by LCMS as in Example 3. The in silico designed variants showed conversion of Reb A to Reb D (Table 6).

[0085] [Table 7]

[0086] Example 10: Improved activity of pA12549 driven by computational design A homology model of B12GT encoded by pA12549 was used as input to computational design to improve pA12549. Computational design of pA12549 was performed to combine active site mutations known to be beneficial in the homologous B12GT. Eight computational designs were selected for experimental validation (SEQ ID NOs: 460-467). Expression plasmids for computational designs were constructed as in Example 1. Each B12GT mutant was expressed and purified as in Example 2. Each B12GT mutant was assayed in a one-pot reaction with SuSy and pA10142. Purified B12GT and SuSy were reacted with 0.5 mg / mL RA99, 10 mM sucrose, and 1 mM ADP in 50 mM phosphate buffer (pH 7), 3.0 mM MgCl2, and 50 mM NaCl at 30° C. for 24 hours. The product rebaudioside was monitored by LCMS as in Example 3. Several mutants showed improved expression and / or Reb D conversion compared to the parent pA12549 (Table 7).

[0087] [Table 8]

[0088] The computational design of pA12549 was performed to combine mutations known to be beneficial in the homologous B12GT. Sixty-seven computational designs were selected for experimental validation (SEQ ID NOs: 468-534). Expression plasmids for the computational designs were constructed as in Example 1. Each B12GT variant was expressed and purified as in Example 2. Each B12GT variant was assayed in a one-pot reaction with SuSy and pA10142. Purified B12GT and SuSy were reacted with 0.5 mg / mL RA99, 10 mM sucrose, and 1 mM ADP in 50 mM phosphate buffer (pH 7) and 50 mM NaCl at 30° C. for 24 hours. The product rebaudioside was monitored by LCMS as in Example 3. One computational design variant showed improved conversion of Reb A to Reb D (Table 8).

[0089] [Table 9]

[0090] Example 11: Improvement of Arabidopsis SUS1 activity and expression driven by computational design The crystal structure of SUS1 from Arabidopsis thaliana was used as input to computational design to improve pA10142. Computational design was performed to improve the stability and expression of SuSy. Thirty-five computational designs were selected for experimental validation (SEQ ID NOs: 890-924). Expression plasmids for computational designs were constructed as in Example 1. Each SuSy variant was expressed and purified as in Example 2. Each SuSy variant was assayed in a one-pot reaction with B12GT and pA10143. Purified B12GT and SuSy were reacted with 4 mg / mL RA50, 40 mM sucrose, and 1 mM ADP in 50 mM phosphate buffer (pH 7) and 50 mM NaCl at 30° C. for 24 hours. The product rebaudioside was monitored by LCMS as in Example 3. Some variants showed improved expression and / or Reb D conversion compared to the parent pA10142. The top designs showed up to a 2-fold improvement in yield and a 3-fold improvement in expression (43% conversion, 8 uM purified protein, Table 9).

[0091] [Table 10-1] [Table 10-2]

[0092] Example 12: Computational design of ADP glucose-dependent B12GT A structural model of the B12GT mutants of pA28422 was generated and used as a starting point for computational design. Computational design was performed to improve the stability and expression of B12GT. 52 computational designs are selected for experimental validation (SEQ ID NOs: 535-586). Expression plasmids for the computational designs were constructed as in Example 1. Each B12GT mutant is expressed and purified as in Example 2. Each B12GT mutant was assayed in a one-pot reaction with the SuSy mutant of pA21838. Purified B12GT and SuSy were reacted with 100 mg / mL RA50, 250 mM sucrose, and 0.5 mM ADP in 50 mM phosphate buffer (pH 6), 3 mM MgCl2, and 50 mM NaCl at 60° C. for 24 hours. The product rebaudioside was monitored by LCMS as in Example 3. Several designed enzymes were well expressed and active in converting Reb A to Reb D (Table 10).

[0093] [Table 11-1] [Table 11-2]

[0094] Computational design is further performed to improve the stability and expression of B12GT using co-evolution information. 85 computational designs were selected for experimental validation (SEQ ID NOs: 587-671). Expression plasmids for computational designs were constructed as in Example 1. Each B12GT variant was expressed and purified as in Example 2. Each B12GT variant was assayed in a one-pot reaction with the SuSy variant of pA21838. Purified B12GT and SuSy were reacted with 100 mg / mL RA50, 250 mM sucrose, and 0.5 mM ADP in 50 mM phosphate buffer (pH 6), 3 mM MgCl2, and 50 mM NaCl at 60° C. for 24 hours. The product rebaudioside was monitored by LCMS as in Example 3. Several designed enzymes expressed well and were active in converting Reb A to Reb D (Table 11).

[0095] [Table 12-1] [Table 12-2]

[0096] Computational design was further performed to improve the stability and expression of B12GT by redesigning and repacking the buried protein core. Thirty-five computational designs were selected for experimental validation (SEQ ID NOs: 672-706). Expression plasmids for computational designs were constructed as in Example 1. Each B12GT variant was expressed and purified as in Example 2. Each B12GT variant was assayed in a one-pot reaction with the SuSy variant of pA21838. Purified B12GT and SuSy were reacted with 100 mg / mL RA50, 250 mM sucrose, and 0.5 mM ADP in 50 mM phosphate buffer (pH 6), 3 mM MgCl2, and 50 mM NaCl at 60° C. for 24 hours. The product rebaudioside was monitored by LCMS as in Example 3. Several designed enzymes were well expressed and active in converting Reb A to Reb D (Table 12).

[0097] [Table 13-1] [Table 13-2]

[0098] Computational design was further performed to improve B12GT by combining mutations known to be beneficial to the homologous B12GT. Fifty-nine computational designs were selected for experimental validation (SEQ ID NOs: 707-765). Expression plasmids for computational designs were constructed as in Example 1. Each B12GT variant was expressed and purified as in Example 2. Each B12GT variant was assayed in a one-pot reaction with the SuSy variant of pA21838. Purified B12GT and SuSy were reacted with 100 mg / mL RA50, 250 mM sucrose, and 0.5 mM ADP in 50 mM phosphate buffer (pH 6) and 50 mM NaCl at 60° C. for 24 hours. The product rebaudioside was monitored by LCMS as in Example 3. Several designed enzymes expressed well and were active in converting Reb A to Reb D (Table 13). To distinguish the top designs, they were re-assayed at lower protein concentrations (Table 14).

[0099] [Table 14-1] [Table 14-2] [Table 14-3]

[0100] [Table 15]

[0101] Example 13: Computational design of ADP glucose-dependent B12GT A structural model of the second B12GT variant of pA28422 was generated and used as a starting point for computational design. Computational design was performed to improve B12GT by combining mutations known to be beneficial in the homologous B12GT. Sixty-four computational designs were selected for experimental validation (SEQ ID NOs: 766-829). Expression plasmids for the computational designs were constructed as in Example 1. Each B12GT variant was expressed and purified as in Example 2. Each B12GT variant was assayed in a one-pot reaction with the SuSy variant of pA21838. Purified B12GT and SuSy were reacted with 100 mg / mL RA50, 250 mM sucrose, and 0.5 mM ADP in 50 mM phosphate buffer (pH 6) and 50 mM NaCl at 60° C. for 24 hours. The product rebaudioside was monitored by LCMS as in Example 3. Several designed enzymes expressed well and were active in converting Reb A to Reb D (Table 15). To distinguish the top designs, they were re-assayed at lower protein concentrations (Table 16).

[0102] [Table 16]

[0103] [Table 17]

[0104] Example 14: Expression of successful B12GT designs by PSSM The successful B12GT designs from Example 12 and Example 13 were used to generate PSSMs (Table 17). PSSMs are a concise way of expressing successful designs and related sequences. Sequences with PSSM scores of more than 266.7 are considered to be related to the active computational designs described in Example 12 and Example 13. To score a sequence by PSSM, it is necessary to first align the sequence of the representative sequence SEQ ID NO:5. For example, the PSSM scores of the following successful designs, pA29646, pA32946, pA29642, and pA29798, are 287.2, 288.0, 279.2, and 276.8, while the PSSM score of wild-type B12GT pA28422 is only 257.4.

[0105] [Table 18-1] [Table 18-2] [Table 18-3] [Table 18-4] [Table 18-5] [Table 18-6] [Table 18-7] [Table 18-8] [Table 18-9] [Table 18-10] [Table 18-11] [Table 18-12] [Table 18-13] [Table 18-14]

[0106] Example 15: Computational design of ADP glucose-dependent B12GT Improved B12GTs were designed by using the designed B12GTs from Examples 12 and 13 as starting scaffolds for further design rounds. Computational design methods were used to improve the stability and expression of seven B12GTs from Examples 12 and 13. 134 computational designs were selected for experimental validation (SEQ ID NOs: 1333-1466). Expression plasmids for computational designs were constructed as in Example 1. Each B12GT variant was expressed and purified as in Example 2. Each B12GT variant was assayed in a one-pot reaction with the SuSy variant of pA21838. Purified B12GT and SuSy were reacted with 100 mg / mL RA50, 250 mM sucrose, and 0.5 mM ADP in 50 mM phosphate buffer (pH 6) and 50 mM NaCl at 60° C. for 24 hours. The product rebaudioside was monitored by LCMS as in Example 3. Several designed enzymes were well expressed and active in converting Reb A to Reb D (Table 18).

[0107] [Table 19-1] [Table 19-2] [Table 19-3] [Table 19-4]

[0108] Example 16: Computational design of ADP glucose-dependent B12GT A structural model of the third B12GT variant of pA28422 was generated and used as a starting point for computational design. Computational design was performed to improve the stability and expression of B12GT. 53 computational designs were selected for experimental validation (SEQ ID NOs: 830-882). Expression plasmids for the computational designs were constructed as in Example 1. Each B12GT variant was expressed and purified as in Example 2. Each B12GT variant was assayed in a one-pot reaction with the SuSy variant of pA21838. Purified B12GT and SuSy were reacted with 10 mg / mL RA50, 100 mM sucrose, and 10.5 mM ADP in 50 mM phosphate buffer (pH 6), 3 mM MgCl2, and 50 mM NaCl at 60°C for 24 hours. The product rebaudioside was monitored by LCMS as in Example 3. Several designed enzymes were well expressed and active in converting Reb A to Reb D (Table 19).

[0109] [Table 20]

[0110] Example 17: Computational design of ADP-dependent sucrose synthase Structural models of the two SuSy variants of pA21838 were generated and used as starting points for computational design. The design strategy used to design the ADPG-dependent B12GT was used to design the improved ADP-dependent sucrose synthase. 256 computational designs were selected for experimental validation (SEQ ID NOs: 925-1180). Expression plasmids for the computational designs were constructed as in Example 4. Each SuSy variant was expressed and purified as in Example 5. Each SuSy variant was assayed in a one-pot reaction with the B12GT variant of pA28422. Purified B12GT and SuSy were reacted with 100 mg / mL RA50, 250 mM sucrose, and 0.5 mM ADP in 50 mM phosphate buffer (pH 6), 3 mM MgCl2, and 50 mM NaCl at 60° C. for 24 hours. The product rebaudioside was monitored by LCMS as in Example 3. The relative expression and Reb A to Reb D conversion of the designed enzymes, SEQ ID NOs: 925-1048, is shown in Table 20. The relative expression and Reb A to Reb D conversion of the designed enzymes, SEQ ID NOs: 1049-1104, is shown in Table 21. The top hits from the previous two experiments were re-evaluated at more reasonable protein concentrations (Table 22). Finally, the relative expression and Reb A to Reb D conversion of the designed enzymes, SEQ ID NOs: 1105-1180, is shown in Table 23.

[0111] [Table 21-1] [Table 21-2] [Table 21-3] [Table 21-4]

[0112] [Table 22-1] [Table 22-2] [Table 22-3]

[0113] [Table 23-1] [Table 23-2] [Table 23-3]

[0114] [Table 24-1] [Table 24-2] [Table 24-3]

[0115] Example 18: Representation of successful SuSy designs with PSSM The successful SuSy designs in Example 17 were used to generate PSSMs (Table 24). PSSMs are a concise way of expressing successful designs and related sequences. Sequences with PSSM scores of more than 556 are considered to be related to the active computational designs described in Example 17. To score a sequence with the generated PSSM, it must first be aligned with representative sequence pA21838 (SEQ ID NO: 885). For example, the PSSM scores of the following successful designs, pA32853, pA32891, pA32892, pA32929, are 557.2, 558.1, 558.1, 557.8, while the PSSM score of wild-type SuSy's pA21838 is only 536.3.

[0116] [Table 25-1] [Table 25-2] [Table 25-3] [Table 25-4] [Table 25-5] [Table 25-6] [Table 25-7] [Table 25-8] [Table 25-9]

Table 25-10

Table 25-11

Table 25-12

Table 25-13

Table 25-14

Table 25-15

Table 25-16

Table 25-17

Table 25-18

Table 25-19

Table 25-20

Table 25-21

Table 25-22

Table 25-23

[0117] Example 19: Improvement of activity and expression of SUSA from Thermosynechococcus elongatus driven by computational design A homology model of sucrose synthase SUSA from Thermosynechococcus elongatus was constructed and used as input to computational design to improve pA21841. Computational design was performed to improve the stability and expression of SuSy. 47 computational designs were selected for experimental validation (SEQ ID NOs: 1181-1227). Expression plasmids for the computational designs were constructed as in Example 4. Each SuSy variant was expressed and purified as in Example 5. Each SuSy variant was assayed in a one-pot reaction with B12GT and pA29798. Purified B12GT and SuSy were reacted with 100 mg / mL RA50, 250 mM sucrose, and 0.5 mM ADP in 50 mM phosphate buffer (pH 6) and 50 mM NaCl at 60° C. for 24 hours. The product rebaudioside was monitored by LCMS as in Example 3. Several mutants showed improved expression and / or Reb D conversion compared to the parent pA21841. The top designs showed up to a 2.2-fold improvement in yield or a 5.4-fold improvement in expression (34.6% conversion, 6.8 uM purified protein, Table 25).

[0118] [Table 26-1] [Table 26-2] [Table 26-3]

[0119] Computational design was further performed to improve the stability and expression of SUSA by redesigning and repacking the buried protein core. Thirty-eight computational designs were selected for experimental validation (SEQ ID NOs: 1231-1267). Expression plasmids for computational designs were constructed as in Example 4. Each SuSy variant was expressed and purified as in Example 5. Each SuSy variant was assayed in a one-pot reaction with B12GT and pA29798. Purified B12GT and SuSy were reacted with 100 mg / mL RA50, 250 mM sucrose, and 0.5 mM ADP in 50 mM phosphate buffer (pH 6) and 50 mM NaCl at 60° C. for 24 hours. The product rebaudioside was monitored by LCMS as in Example 3. Several designed enzymes expressed well and were active in converting Reb A to Reb D (Table 26).

[0120] [Table 27-1] [Table 27-2]

[0121] Computational design was further performed to improve the stability and expression of SuSy by co-evolutionary information. 65 computational designs were selected for experimental validation (SEQ ID NOs: 1268-1332). Expression plasmids for computational designs were constructed as in Example 4. Each SuSy variant was expressed and purified as in Example 5. Each SuSy variant was assayed in a one-pot reaction with B12GT and pA29798. Purified B12GT and SuSy were reacted with 100 mg / mL RA50, 250 mM sucrose, and 0.5 mM ADP in 50 mM phosphate buffer (pH 6) and 50 mM NaCl at 60° C. for 24 hours. The product rebaudioside was monitored by LCMS as in Example 3. Several designed enzymes expressed well and were active in converting Reb A to Reb D (Table 27).

[0122] [Table 28-1] [Table 28-2] [Table 28-3]

[0123] Example 20: Representation of successful SuSy designs with PSSM The successful SuSy designs in Example 19 were used to generate PSSMs (Table 28). PSSMs are a concise way of expressing successful designs and related sequences. Sequences with PSSM scores of more than 569.5 are considered to be related to the active computational designs described in Example 19. To score a sequence with the generated PSSM, it must first be aligned with representative sequence pA21841 (SEQ ID NO: 888). For example, the PSSM scores of the following successful designs, pA34103, pA34119, and pA34099, are 576.7, 572.5, and 577.0, while the PSSM score of wild-type SuSy's pA21841 is only 565.6.

[0124] [Table 29-1] [Table 29-2] [Table 29-3] [Table 29-4] [Table 29-5] [Table 29-6] [Table 29-7] [Table 29-8] [Table 29-9]

Table 29-10

Table 29-11

Table 29-12

Table 29-13

Table 29-14

Table 29-15

Table 29-16

Table 29-17

Table 29-18

Table 29-19

Table 29-20

Table 29-21

Table 29-22

Table 29-23

[0125] Example 21: Large-scale one-pot reaction of pA21841 and pA29798 E. coli microorganisms containing SuSy (pA21841) or B12GT (pA29798) were expressed in 1 L and 10 L fermenters. Cells were harvested and lysed by French press. Expressed proteins were purified by immobilized metal affinity chromatography (IMAC) and dialyzed in desalting buffer (20 mM KPO4 (pH 6), 50 mM NaCl). One-pot reactions were used to convert Reb A and stevioside to Reb D and Reb E, respectively. pA21841 and pA29798 were reacted with 100 mg / ml RA50, 250 mM sucrose, and 0.5 mM ADP in 50 mM KPO4 (pH 6) and 50 mM NaCl. A total of ten 20 mL one-pot reactions were performed. The reactions were lyophilized and the combined reaction products were analyzed for rebaudioside content by liquid chromatography mass spectrometry (LCMS) using an Agilent 6545 QTOF mass spectrometer (column: 150x2.1mm Phenomenex C18-PS). Complete conversion of Reb A to Reb D and stevioside to Reb E was observed (Figure 7, Table 29).

[0126] [Table 30]

[0127] Example 22: Large-scale one-pot reaction of pA21841 and pA29646 E. coli microorganisms containing SuSy (pA21841) or B12GT (pA29646) were expressed in 10 L fermenters. Cells were harvested and lysed by French press. Expressed proteins were purified by immobilized metal affinity chromatography (IMAC) and dialyzed in desalting buffer (20 mM KPO4 (pH 6), 50 mM NaCl). One-pot reactions were used to convert Reb A and stevioside to Reb D and Reb E, respectively. pA21841 and pA29646 were reacted with 100 mg / ml RA50, 250 mM sucrose, and 0.5 mM ADP in 50 mM KPO4 (pH 6) and 50 mM NaCl. A total of ten 20 mL one-pot reactions were performed. The reactions were lyophilized and the combined reaction products were analyzed for rebaudioside content by liquid chromatography mass spectrometry (LCMS) using an Agilent 6545 QTOF mass spectrometer (column: 150x2.1mm phenomenex C18-PS). Complete conversion of Reb A to Reb D and stevioside to Reb E was observed (Figure 8-HMM, Table 30).

[0128] [Table 31]

[0129] Example 23: Pichia pastoris expression of engineered B12GT and SuSy Polynucleotides optimized for Pichia pastoris expression of the top designed B12GT (from Examples 12, 13, and 15) and SuSy (from Examples 17 and 19) were synthesized (Twist Bioscience) and inserted into a Pichia shuttle vector. The vectors were transformed into a commercially available Pichia pastoris strain (ATCC). The transformed microorganisms were grown in BMGY (buffered glycerol complex) medium and protein expression was induced by feeding with methanol. Pichia cells were lysed with Y-PER (yeast protein extraction reagent, Thermo Scientific) and the expressed proteins were purified by immobilized metal affinity chromatography (IMAC) and desalted with desalting buffer (20 mM KPO4 (pH 6), 50 mM NaCl). The designed B12GT and SuSy were soluble expressed and showed catalytic activity. Figure 9 shows an SDS-PAGE gel of the designed B12GT purified from expression in Pichia pastoris.

[0130] Two designed B12GTs and two designed SuSys were also expressed in 1 L fermentation. Pichia microorganisms were grown for about 24 h using glycerol as the main carbon source and then fed with methanol for about 72 h to express the desired B12GT or SuSy. Cells were harvested and lysed by French press. The expressed proteins were purified by immobilized metal affinity chromatography (IMAC) and dialyzed in desalting buffer (20 mM KPO4 (pH 6), 50 mM NaCl). Figure 10A shows an SDS-PAGE gel of two designed B12GTs, pA29798 (left, B12GT-1) and pA32946 (right, B12GT-2), purified from 1 L Pichia pastoris fermentation. Figure 10B shows an SDS-PAGE gel of two engineered SuSys, pA34103 (left, SuSy-1) and pA32691 (right, SuSy-2), purified from 1 L of Pichia pastoris fermentation. All four enzymes were successfully expressed in fermentation and showed the desired activities.

Claims

1. 1. A modified β-1,2-glycosyltransferase polypeptide comprising an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 6-882 and 1333-1466.

2. 2. The modified β-1,2-glycosyltransferase polypeptide of claim 1, comprising an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs:6-459.

3. 2. The modified β-1,2-glycosyltransferase polypeptide of claim 1, comprising an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs:460-534.

4. 2. The modified β-1,2-glycosyltransferase polypeptide of claim 1, comprising an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs:535-765.

5. 2. The modified β-1,2-glycosyltransferase of claim 1, comprising an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs:766-829.

6. 2. The modified β-1,2-glycosyltransferase of claim 1, comprising an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1333-1466.

7. A modified β-1,2-glycosyltransferase polypeptide having a score of greater than 266.7 when scored by the PSSM shown in Table 17.

8. XXXXVXMXPWLXLGHXNPXLRXAXXXAXRXXXXXXTXXXXLXXXXXRIXXXYXXXIXLXXXXLPXLPELPXXXXTTNXLPPHLNXXXLXXXXXXXPXXSKXXXXXXXLXXXD XLXXWXXKXAXXXXXPXXXXTXGXALXXYXXXXXX A polypeptide having the sequence of: XDXXDXELXXWLXXKXXXXXVXVSFGSEXFLSXEXXEEXAXGLXLSXXNXIXVXRFPKGXXXXXXXLPXGXXXRXXXRXXXXXHLVPQAXILXHXXXGGFXSHCGWNSXXEXXXFGVPIIAMPMQWDQPINARLXXEXGXAVEXXXRXXXGXXXRXIAXXXXXVXXXXXGXXLRXXVXXXXXXRXXEMXXXXXXXXXXXXLXXXXXXAXX Residue 1 is R or D or Q or P or S or T; Residue 2 is N or L or S or T; Residue 3 is Q or L or F; Residue 4 is R or Q or T; Residue 6 is A or L or T or V; Residue 8 is L or F or V; Residue 12 is A or G; Residue 16 is I or V; Residue 19 is F or Y; Residue 22 is I or L or V; Residue 24 is R or K; Residue 25 is Q or K; Residue 26 is L or M; Residue 28 is D or K; Residue 30 is N or G; Residue 31 is M or F; Residue 32 is H or I or L or S or Y or V; Residue 33 is I or V; Residue 34 is H or Y; Residue 35 is L or M or V; Residue 36 is A or C or L or V; Residue 37 is N or S; Residue 39 is A or K or M or P; Residue 40 is I or V; Residue 41 is N or Q or V; Residue 43 is N or E or K or S; Residue 44 is L or M or S; Residue 45 is A or I or L or T; Residue 46 is R or K; Residue 47 is G or H or K; Residue 50 is P or T; Residue 51 is N or E or K or Y; Residue 52 is A or K; Residue 54 is A or Q or L or S or V; Residue 55 is N or D or Q or E or L or S; Residue 56 is L or S; Residue 58 is Q or E or H or I; Residue 60 is I or V; Residue 61 is E or T; Residue 62 is L or S or Y or V; Residue 63 is A or R or Q or H or S; Residue 66 is Q or E or L; Residue 72 is P or S; Residue 73 is H or Y; Residue 74 is L or Y; Residue 75 is H or W; Residue 79 is A or G; Residue 86 is G or K; Residue 87 is R or I or L or T or V; Residue 89 is R or Q or H or I or K; Residue 90 is R or Q or K; Residue 91 is A or L; Residue 92 is L or V; Residue 93 is R or Q or K; Residue 94 is L or M; Residue 95 is A or S; Residue 96 is A or R or Q; Residue 98 is N or E or T; Residue 99 is I or L or F or V; Residue 102 is Q or I or L or T or V; Residue 103 is I or L or V; Residue 104 is R or Q or E or K or S; Residue 105 is A or N or D or T; Residue 106 is I or L or W; Residue 107 is N or K; Residue 108 is A or P or S or V; Residue 109 is A or D or S or T; Residue 111 is I or L or V; Residue 112 is I or L or V; Residue 113 is L or F or Y or V; Residue 115 is I or L or M or F; Residue 117 is A or Q or I or L or V; Residue 118 is Q or P; Residue 120 is A or L; Residue 121 is E or S; Residue 123 is I or L or S or V; Residue 125 is N or L or K; Residue 126 is D or E or S; Residue 127 is R or Q or L; Residue 128 is N or G; Residue 129 is I or V; Residue 131 is A or G or S or V; Residue 132 is I or V; Residue 133 is R or K or P Residue 134 is L or F; Residue 135 is A or I or L or W; Residue 137 is F or S; Residue 139 is A or L or V; Residue 142 is L or F or W; Residue 143 is A or S; Residue 145 is I or L or F; Residue 146 is L or M or F or W or V; Residue 147 is N or Q or E or H; Residue 148 is H or F; Residue 149 is L or V; Residue 150 is R or D or K or T; Residue 151 is R or N or K; Residue 152 is L or P; Residue 154 is N or E or H or V; Residue 155 is E or P; Residue 157 is L or P; Residue 159 is E or P; Residue 160 is A or E; Residue 162 is R or D or E or H or K or M or S or Y; Residue 164 is R or S; Residue 165 is E or K; Residue 166 is R or I or L or W or Y; Residue 167 is A or E; Residue 168 is Q or L or K; Residue 169 is A or D or Q or V; Residue 170 is K or W; Residue 171 is H or L or M; Residue 172 is R or Y; Residue 174 is A or L or M; Residue 175 is M or F; Residue 176 is E or G; Residue 177 is R or K or T; Residue 178 is A or Q or E or G or V; Residue 179 is G or P; Residue 180 is D or K or P or T; Residue 181 is D or E; Residue 182 is R or D or E or L or K; Residue 183 is D or F; Residue 185 is L or F; Residue 186 is A or V; Residue 187 is D or E or K or P or V; Residue 188 is A or G or F; Residue 189 is R or N or Q or P or S; Residue 190 is A or N or C or K or M or S or T; Residue 191 is A or Q or G or K; Residue 192 is A or I or Y; Residue 193 is I or L or M or T or V; Residue 195 is I or M or V; Residue 196 is C or M; Residue 197 is S or T; Residue 200 is A or E or I or V; Residue 201 is I or L; Residue 206 is I or L or M; Residue 209 is C or L; Residue 210 is A or Q or M or S or T; Residue 211 is E or K or T; Residue 212 is L or W; Residue 213 is M or S or T; Residue 214 is N or G; Residue 215 is R or I or L or K or W or V; Residue 216 is Q or K; Residue 217 is I or V; Residue 222 is A or P; Residue 223 is P or S or T; Residue 224 is F or Y or V; Residue 225 is Q or L; Residue 226 is D or T or V; Residue 227 is A or L or P; Residue 228 is N or L or T or V; Residue 229 is F or P or T or Y; Residue 230 is N or D or E or L or S; Residue 232 is A or N or D or E or I or L; Residue 233 is R or D or G or S or Y; Residue 235 is I or K or M or P or S or V; Residue 238 is I or M; Residue 239 is R or D or K; Residue 242 is D or G; Residue 243 is K or T; Residue 245 is D or P; Residue 246 is E or L or P or T; Residue 247 is N or H; Residue 248 is A or S; Residue 249 is T or V; Residue 251 is F or Y; Residue 258 is A or Y; Residue 262 is R or K; Residue 264 is D or Q; Residue 265 is L or M; Residue 268 is I or L or V; Residue 270 is H or F or W; Residue 273 is E or V; Residue 276 is N or G; Residue 277 is A or I or S or V; Residue 279 is A or F; Residue 281 is I or W; Residue 283 is A or V; Residue 289 is A or R or E or V; Residue 290 is R or E or K; Residue 291 is A or R or Q or I or L or V; Residue 292 is R or N or H or L or T; Residue 293 is A or L; Residue 294 is E or I; Residue 295 is D or E; Residue 296 is A or V; Residue 299 is R or K or P or S; Residue 301 is F or T; Residue 302 is L or S; Residue 303 is D or E; Residue 305 is G or I or V; Residue 306 is R or G; Residue 307 is D or E; Residue 309 is A or G; Residue 310 is R or M or Y; Residue 311 is W or V; Residue 312 is R or L; Residue 313 is D or P or T; Residue 320 is C or H; Residue 323 is N or K or S; Residue 325 is K or P or S; Residue 326 is A or S; Residue 327 is I or T or V; Residue 331 is I or M or V; Residue 339 is I or W or V; Residue 340 is L or M; Residue 342 is A or S; Residue 343 is I or L; Residue 344 is D or H or Y; Residue 365 is I or L or M or V; Residue 366 is R or N or V; Residue 368 is L or M or W; Residue 370 is I or V; Residue 374 is I or V; Residue 375 is R or P or S or V; Residue 377 is R or D or Q; Residue 378 is D or E; Residue 379 is N or D or E or L; Residue 381 is R or K or S; Residue 382 is I or V; Residue 383 is H or P; Residue 385 is A or N or D or E or G; Residue 386 is A or E; Residue 389 is R or Q or E or K; Residue 390 is C or T or V; Residue 391 is I or L or V; Residue 392 is R or K; Residue 393 is D or E or S; Residue 395 is I or M or V; Residue 396 is N or C or G or F or S or T or V; Residue 397 is E or G; Residue 398 is A or K or P; Residue 399 is I or L or T; Residue 401 is Q or E; Residue 402 is N or E or I or K; Residue 405 is A or R or H or K; Residue 406 is N or K; Residue 408 is A or R or K; Residue 409 is D or E; Residue 410 is I or L; Residue 411 is A or G or S; Residue 412 is A or R or E or L or K or T; Residue 413 is R or N or K; Residue 414 is L or W; Residue 415 is R or K; Residue 416 is A or R or E or L or S; Residue 417 is A or R or I or K or T; Residue 419 is N or D or E or G or K; Residue 420 is E or P or V; Residue 423 is N or D or Q or G or T; Residue 424 is A or I or K; Residue 425 is A or L; Residue 426 is A or M or V; Residue 427 is E or L; Residue 428 is A or E; Residue 429 is L or F or Y; Residue 430 is I or L or K or M; Residue 431 is A or R or Q or L or K or S; Residue 433 is C or G or H; Residue 434 is R or Q or H or K or F or P or T or Y or V; Residue 435 is N or H or K; Residue 436 is R or L; Residue 437 is A or R or N or L or S; Residue 439 is A or L or K or F or S or T or Y; The polypeptide wherein residue 440 is N or K or V (SEQ ID NO:1228).

9. 2. The modified β-1,2-glycosyltransferase polypeptide of claim 1, comprising an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs:830-882.

10. A modified sucrose synthase polypeptide comprising an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

885.

11. A modified sucrose synthase polypeptide comprising an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

888.

12. 1. A modified sucrose synthase polypeptide comprising an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs:890-1227 and 1231-1332.

13. 13. The modified sucrose synthase polypeptide of claim 12, comprising an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs:890-924.

14. 13. The modified sucrose synthase polypeptide of claim 12, comprising an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs:925-1180.

15. 13. The modified sucrose synthase polypeptide of claim 12, comprising an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1231-1267.

16. 13. The modified sucrose synthase polypeptide of claim 12, comprising an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1268-1332.

17. A modified sucrose synthase polypeptide having a score of greater than 556 when scored by the PSSM shown in Table 24.

18. MIEXLXXXLXXXXXXXXXLRXXXXXXRXXXXXXDLXXXXXXFXXXXXXXXXXXXXXQEAXXXXXPWXXXAXRXRXXXWXYXRXHXEXLXVEEX XXXEXLXXKEXLVXXXXEGXAVXXXDXXDXXXXXQXXKDESTIGXGXXHLNRHLXGRXWXDXXXGXXXXXXXLXXHXXXXXXXLXLXXXXXXFDXLRXXXQY LGXXPXXXPXXXXXXXXXXGFEPGXGXTXXRXRXTXRLLXDXLDSPSPXXLEXFLXRXPXIXXXXIXSXHGXFXQXXVLGXPDTGGQVVXILDQXRALEX EXRXRLXXQGXDXEPXIXXXTRLIPXXXGTTCDQRLEPXXGXXXXXXILRXPFRXEXGXXXPXWISRFXXWPYLERXXXDXEXEXEXAELGXRPDXIIGNYSDG XLXAXXXXKXGXTQXNXAHALEKXKYXXSDLXWXXXEXXXHFXCQFTADXIAMNAADXIXTSTYQEIAGXDXXVGQYESXXXXTXPGLYRXXXGXDVFDX XFNIXSPGADXXXYFXYXXXEXRXXXLXPEIEXXXXXXXXXXRGVLXDXXKPXXXXXXRXDRIKNXXGXXEXXGXXXRLRXLLANLXXXXGHXDXXXSXDX EEXXXXXRXHXXXDXXXLDGQXRXXGXXLXKXXVGEXYRXXADXRGXXXQPALXEAFGLTVIEXMXSGLPXXATXXGGPXEIIEXGVSGFHIDPNXXXXXXXEXXXADXXEXXXXPXYWEXISXALXRVXXRYTWXXXAERXXTXRXXXGFWXXVXXREXQVXXRYLQMXRHLQXRPLAHAVPXE, Residue 4 is R or E or S or V; Residue 6 is R or D or E; Residue 7 is Q or E; Residue 8 is Q or F; Residue 10 is A or R or Q or H or L or K or S; Residue 11 is D or Q or E or S; Residue 12 is N or H or S; Residue 13 is R or P; Residue 14 is R or N or D or E; Residue 15 is A or D or Q or E or S or T; Residue 16 is L or W or V; Residue 17 is R or H or Y; Residue 18 is A or R or L; Residue 19 is L or F; Residue 22 is R or H; Residue 23 is L or Y; Residue 24 is L or V; Residue 25 is A or G; Residue 26 is Q or L or S; Residue 27 is Q or G; Residue 29 is D or G or P or T; Residue 30 is I or L or M or F or S; Residue 31 is L or W; Residue 32 is Q or L; Residue 33 is R or H; Residue 34 is H or S or T; Residue 37 is Q or W; Residue 38 is R or D or H; Residue 39 is A or E; Residue 40 is C or L or F; Residue 41 is A or D or E or L or K; Residue 42 is A or R or N or D or E or T; Residue 44 is R or C or Y; Residue 45 is A or E; Residue 46 is Q or E or P; Residue 47 is D or Q or E or P; Residue 48 is D or G or P; Residue 49 is N or E; Residue 50 is E or G; Residue 51 is E or S or Y; Residue 52 is A or E or G or L or P; Residue 53 is E or L; Residue 54 is A or D or C or G or L or K or M or T; Residue 55 is D or G or I or S or T; Residue 56 is G or S; Residue 57 is A or P or W or V; Residue 58 is L or F; Residue 59 is A or E or G; Residue 60 is R or D or Q or E; Residue 61 is A or L or F or V; Residue 62 is I or V; Residue 63 is A or R or Q or E or K; Residue 64 is A or R or H or K; Residue 65 is A or C or T; Residue 69 is A or I or V; Residue 70 is I or L or F or V; Residue 71 is R or D or E or H or L; Residue 72 is A or D; Residue 75 is A or I or L or M or F or V; Residue 76 is C or Y or V; Residue 77 is L or F; Residue 79 is I or L or W or V; Residue 81 is E or L or P; Residue 83 is P or V; Residue 84 is A or G; Residue 85 is R or V; Residue 87 is R or E or Y; Residue 89 is L or Y or V; Residue 91 is I or F; Residue 93 is I or L or V; Residue 95 is D or Q or T; Residue 97 is A or D or E or T or V; Residue 101 is I or L or V; Residue 102 is D or S or T; Residue 103 is T or V; Residue 104 is D or E or S; Residue 106 is F or Y; Residue 108 is A or Q; Residue 109 is A or F; Residue 112 is R or Q or E; Residue 115 is N or D or G or K or T; Residue 116 is G or L; Residue 117 is A or N or D or E or G; Residue 118 is A or Q or G or H or P or S; Residue 121 is N or D or E or P; Residue 124 is L or T or W; Residue 125 is E or K or T; Residue 126 is I or W or V; Residue 128 is L or F or W; Residue 129 is E or G; Residue 131 is L or F; Residue 132 is R or N; Residue 133 is R or P; Residue 134 is N or E or G or H or S or T or V; Residue 135 is F or S or V; Residue 137 is R or K; Residue 138 is L or M; Residue 146 is R or N or D; Residue 148 is L or V; Residue 149 is Q or E or T; Residue 156 is A or S; Residue 159 is I or L or M; Residue 161 is Q or G or T; Residue 163 is L or T; Residue 164 is A or G or K; Residue 165 is A or R or D or Q or E or G or K; Residue 167 is R or D or L or M; Residue 168 is D or Q or E or S or Y; Residue 169 is A or R or Q or K; Residue 170 is I or L; Residue 171 is I or L; Residue 172 is D or E; Residue 173 is F or W; Residue 175 is R or G or S; Residue 176 is L or V; Residue 178 is R or Q; Residue 179 is H or I or L or Y; Residue 180 is R or D; Residue 181 is N or G; Residue 182 is R or Q; Residue 183 is N or Q or H; Residue 185 is G or M; Residue 187 is N or S; Residue 188 is N or D or E; Residue 189 is R or G; Residue 190 is N or I or M or F; Residue 191 is R or Q or K or T; Residue 192 is D or S; Residue 195 is A or E or G or S; Residue 198 is R or Q; Residue 199 is A or T or V; Residue 200 is E or I or L or M or V; Residue 205 is R or G or K or T; Residue 206 is Q or L; Residue 208 is A or R or D or E or P; Residue 209 is D or E; Residue 210 is A or T; Residue 212 is L or W; Residue 213 is A or E or S; Residue 214 is D or E; Residue 215 is L or F or V; Residue 216 is A or R or Q or E or G; Residue 217 is E or H or P; Residue 218 is A or R or D or E or K or T; Residue 219 is L or M; Residue 220 is R or Q; Residue 221 is R or E; Residue 222 is R or L or M or W; Residue 228 is L or W; Residue 230 is R or N or D; Residue 232 is A or V; Residue 233 is A or Q or E or G; Residue 235 is I or M or V; Residue 237 is D or E; Residue 239 is L or M; Residue 243 is M or V; Residue 245 is I or L; Residue 252 is A or R or E or G or S; Residue 253 is A or N or T; Residue 256 is R or E or K or S or T; Residue 259 is A or D or G; Residue 261 is I or V; Residue 263 is L or M; Residue 265 is F or S; Residue 266 is N or S; Residue 267 is I or L or V; Residue 268 is A or I or L or T or V; Residue 270 is I or L or V; Residue 272 is I or P or V; Residue 275 is W or Y; Residue 277 is A or G; Residue 279 is A or D or S; Residue 280 is N or K; Residue 284 is R or L or Y; Residue 293 is F or W or Y; Residue 298 is A or V; Residue 303 is R or K; Residue 305 is I or L or M; Residue 307 is R or N or D or Q or E or K; Residue 310 is A or R or H or Y; Residue 311 is Q or E; Residue 314 is L or V; Residue 316 is I or V; Residue 319 is R or Q; Residue 321 is I or L; Residue 322 is I or V; Residue 323 is A or I or L or V; Residue 329 is D or E; Residue 330 is A or S; Residue 331 is R or D or E or G or K; Residue 342 is I or V; Residue 343 is H or S or V; Residue 345 is A or T; Residue 346 is R or E; Residue 347 is N or H or Y; Residue 348 is A or V; Residue 349 is R or Q or H or W; Residue 353 is I or V; Residue 357 is N or Y; Residue 359 is D or S; Residue 361 is R or N or E or T; Residue 362 is I or V; Residue 363 is H or I or L or V; Residue 365 is Q or H; Residue 371 is R or E or K; Residue 372 is I or V; Residue 379 is F or W or Y; Residue 380 is A or V; Residue 381 is R or Q or E or L or S; Residue 383 is A or L or V; Residue 385 is R or K; Residue 387 is I or L or V; Residue 388 is L or K; Residue 393 is G or S; Residue 397 is A or L or V; Residue 406 is N or G; Residue 408 is I or V; Residue 410 is S or T; Residue 411 is I or L; Residue 412 is I or L or M; Residue 413 is A or S; Residue 414 is Q or E; Residue 416 is L or W; Residue 418 is I or V; Residue 421 is C or I or M or T or V; Residue 423 is I or F; Residue 430 is S or T; Residue 433 is L or P; Residue 434 is D or G or Y; Residue 438 is H or Y; Residue 440 is R or K or P Residue 441 is R or D or L or K; Residue 442 is N or H or F or Y; Residue 444 is A or D or Q or E or P; Residue 445 is D or Q or K; Residue 446 is H or Y; Residue 449 is A or S; Residue 456 is L or W; Residue 464 is A or I or F or V; Residue 466 is I or V; Residue 476 is N or T; Residue 478 is R or N or D or H; Residue 479 is E or S; Residue 486 is H or Y; Residue 487 is A or Q or G or S or T; Residue 488 is A or D or H or S; Residue 489 is F or Y; Residue 491 is L or M; Residue 497 is I or V; Residue 498 is E or I or V; Residue 499 is N or H; Residue 501 is I or V; Residue 506 is P or S; Residue 507 is R or K; Residue 511 is I or V; Residue 517 is A or P; Residue 518 is R or D or E or S; Residue 519 is I or T or V; Residue 522 is P or S; Residue 524 is A or S or T; Residue 525 is R or D or E; Residue 526 is H or K or T; Residue 528 is R or E or K; Residue 530 is L or F; Residue 531 is S or T; Residue 532 is G or S; Residue 534 is H or W; Residue 539 is R or E or K or S; Residue 540 is I or L or M; Residue 541 is I or L or W or V; Residue 542 is F or Y; Residue 543 is G or S; Residue 544 is R or D or G or P; Residue 545 is D or E or P; Residue 546 is D or Q or E or P or T; Residue 547 is R or G; Residue 548 is A or G or P; Residue 549 is D or E or P; Residue 550 is A or H or I; Residue 555 is A or E or K or S; Residue 557 is R or P; Residue 558 is D or Q or S; Residue 561 is I or L or V; Residue 562 is I or L; Residue 563 is L or F; Residue 564 is S or T; Residue 565 is I or M or V; Residue 566 is A or M or S; Residue 568 is L or M; Residue 574 is Q or I or L or M; Residue 575 is S or T; Residue 577 is L or W; Residue 578 is A or L or M or V; Residue 580 is I or L or W; Residue 581 is F or Y or V; Residue 583 is A or R; Residue 584 is N or S; Residue 585 is A or N or E or P or S; Residue 589 is E or S; Residue 594 is I or L or V; Residue 595 is I or L or V; Residue 596 is I or V; Residue 597 is A or G; Residue 600 is I or V; Residue 602 is A or P or V; Residue 603 is A or N or Q or E or G or S; Residue 604 is A or R or N or Q or E or K; Residue 606 is A or R or N or D or G or M or S or T; Residue 608 is A or R or E or G or H or S; Residue 611 is R or Q; Residue 612 is A or E; Residue 613 is Q or E; Residue 614 is I or M or V; Residue 615 is A or R or Q or E or G or K; Residue 617 is I or L or M; Residue 619 is Q or E; Residue 620 is I or L; Residue 621 is I or L or M; Residue 623 is R or E or H or K; Residue 624 is H or Y; Residue 625 is N or Q or G; Residue 630 is A or M or F or V; Residue 632 is L or W; Residue 633 is I or L or V; Residue 635 is A or L or S or V; Residue 636 is Q or H; Residue 638 is N or D or E; Residue 640 is R or N or T or V; Residue 641 is R or L or W or V; Residue 645 is I or L; Residue 648 is W or Y or V; Residue 649 is I or L or V; Residue 652 is R or Q or G or H or K or T; Residue 655 is A or I or V; Residue 656 is F or W or V; Residue 657 is I or V; Residue 662 is F or Y; Residue 672 is A or V; Residue 674 is A or S or T; Residue 679 is T or V; Residue 680 is F or W; Residue 683 is R or C; Residue 684 is H or Y; Residue 688 is A or L; Residue 693 is D or H; Residue 704 is D or Q or H; Residue 705 is G or P; Residue 706 is D or E; Residue 707 is A or Q or E; Residue 708 is A or T or V; Residue 709 is A or L; Residue 711 is R or I or L or K; Residue 712 is I or L or M; Residue 715 is F or W; Residue 716 is L or F; Residue 718 is A or R or H or K; Residue 719 is A or C; Residue 720 is A or R or K; Residue 721 is A or N or E; Residue 722 is R or N or D or E; Residue 724 is D or G or K or S or T; Residue 728 is R or E or K; Residue 731 is R or D or Q; Residue 732 is A or G; Residue 735 is A or Q or E or K; Residue 738 is R or E or S or Y; Residue 739 is A or E or S; Residue 744 is E or K; Residue 745 is R or L; Residue 746 is W or Y; Residue 750 is L or M or W; Residue 751 is A or L or M; Residue 753 is I or L; Residue 754 is A or I or L or M or S or V; Residue 756 is A or C or I or V; Residue 757 is A or I or M or F or W or Y; Residue 761 is R or K; Residue 762 is F or Y; Residue 764 is L or S or T; Residue 765 is N or K or S; Residue 768 is R or H or S; Residue 771 is M or T; Residue 772 is R or E; Residue 778 is I or F; Residue 783 is F or W or Y; The polypeptide wherein residue 792 is L or M (SEQ ID NO:1229).

19. 13. The modified sucrose synthase polypeptide of claim 12, comprising an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1181-1227.

20. A modified sucrose synthase polypeptide having a score of greater than 569.5 when scored by the PSSM shown in Table 28.

21. MTXXLLXXXXXXSXXXXXLXQFXRXLXXXXKXYXLRNXILXAFXXYCXXXXXPXXXXXXSXLXKLXXYTQEIIXDXEXLXWIXRPXIAXQEVXRLXVXDXTXXP XTIXELLD XLXXLSDXXXXXXXXEXRFELQXLGXEPGXGXTXARVRXTLEXXXQXXDSPDHQVXEAXXSRIPMXFRXXXXSXHGWFGQEXVLGRPDTGGQVVXILDQXXXL EXQXX EDXX SDGNLXAFLLXRRXKXTQXNXAHALEKXKYLFSXLYWQDLEDKYHFSXQFTADLIXMNAAXXIXSSTYQEIVGTPDSIGQYESYQSFTMP PKFNVX PPG EEXXEIEKXXQXXXYXLXGKXRXLGIRLPKXDSGEXYRXXADXXGXFXQPALFEAFGLTILEXMIXGLPTFXTXFGGPLEIIQXXXNGFXINPTXLEEXAXXXXFXXXCXXDPXXWXXXSXXXIXRVXXXYTWKIXXXXXXLXXIXGXWNXXSQENREDXXRYXEAXXHLLXKPRAQXLLAEHLQR, Residue 3 is A or C or S; Residue 4 is D or E or V; Residue 7 is E or K; Residue 8 is A or S; Residue 9 is M or V; Residue 10 is I or L or W or V; Residue 11 is N or D or E; Residue 13 is D or E; Residue 14 is E or H; Residue 15 is R or K; Residue 16 is A or N or E or T; Residue 17 is A or D or E; Residue 19 is R or H; Residue 22 is I or L or F or S; Residue 24 is Q or I or L or Y; Residue 26 is R or D or Q; Residue 27 is A or R or D or L or T; Residue 28 is N or Q or G or K or S; Residue 29 is E or G; Residue 31 is R or G; Residue 33 is L or F; Residue 37 is D or E; Residue 40 is N or D or Q or G; Residue 43 is A or D or E or H; Residue 44 is D or E; Residue 47 is R or H; Residue 48 is A or N or D; Residue 49 is Q or L or K; Residue 50 is D or Q or E or G; Residue 51 is R or K; Residue 53 is A or E or V; Residue 54 is P or Y; Residue 55 is F or P or T; Residue 56 is P or Y; Residue 57 is D or H or L or S; Residue 58 is N or E or S; Residue 60 is R or G or S; Residue 62 is A or S or W; Residue 65 is I or V; Residue 66 is R or H or F or Y; Residue 73 is I or F or V; Residue 75 is N or D or E; Residue 77 is S or W; Residue 79 is C or W; Residue 82 is I or V; Residue 85 is R or Q or K; Residue 88 is R or Q; Residue 92 is C or W or Y or V; Residue 95 is H or L; Residue 97 is D or E; Residue 99 is L or M; Residue 101 is I or F or V; Residue 102 is E or V; Residue 104 is I or M; Residue 107 is Q or P; Residue 112 is A or L or F; Residue 119 is R or H; Residue 124 is D or E; Residue 127 is L or V; Residue 128 is L or F or W; Residue 130 is I or L; Residue 132 is M or W or V; Residue 133 is R or Q or E; Residue 135 is L or F; Residue 139 is F or S; Residue 141 is H or I or K or V; Residue 145 is A or P; Residue 150 is R or K; Residue 158 is F or Y; Residue 159 is I or L or M; Residue 163 is A or L; Residue 170 is G or W; Residue 172 is Q or E; Residue 173 is A or R or N or L or K or T; Residue 174 is L or F; Residue 175 is I or L or F; Residue 176 is N or D or Q; Residue 177 is F or W; Residue 178 is L or M; Residue 179 is R or Q; Residue 180 is I or V; Residue 182 is R or Q; Residue 186 is Q or I or Y; Residue 189 is G or L; Residue 190 is I or W or V; Residue 192 is D or E; Residue 196 is N or S; Residue 198 is Q or P; Residue 199 is Q or H; Residue 201 is A or L or M or S; Residue 204 is I or V; Residue 207 is A or L; Residue 209 is K or V; Residue 210 is A or I or F or T or W or Y or V; Residue 214 is R or Q; Residue 215 is A or P; Residue 216 is P or S; Residue 217 is A or D or T; Residue 218 is A or E; Residue 219 is A or P; Residue 220 is F or W or Y; Residue 221 is Q or E or S; Residue 223 is I or F; Residue 229 is N or E; Residue 232 is F or W; Residue 236 is L or W; Residue 238 is R or N or K; Residue 240 is A or V; Residue 245 is D or E; Residue 249 is I or L; Residue 250 is I or L or M or W; Residue 251 is A or D or L; Residue 253 is A or L or V; Residue 254 is A or I or L or M; Residue 262 is L or W; Residue 265 is L or F or W; Residue 266 is L or F or V; Residue 272 is I or L; Residue 275 is I or V; Residue 276 is A or I or L or V; Residue 277 is I or L; Residue 278 is I or V; Residue 280 is A or I or M or P or V; Residue 288 is N or G; Residue 301 is I or L or W or Y; Residue 306 is A or V; Residue 307 is R or Q or K; Residue 308 is A or N or S; Residue 311 is R or K; Residue 313 is I or L or M; Residue 314 is R or Q; Residue 317 is I or L; Residue 318 is Q or E or K; Residue 319 is E or L; Residue 323 is D or E or G; Residue 324 is W or V; Residue 327 is A or I; Residue 328 is R or Q; Residue 332 is I or L or V; Residue 333 is I or V; Residue 334 is A or I or L; Residue 341 is A or C or S; Residue 345 is R or L or T; Residue 348 is Q or E; Residue 353 is I or V; Residue 357 is N or D or E; Residue 358 is N or D or H; Residue 364 is I or V; Residue 379 is S or T; Residue 390 is A or F or W; Residue 391 is A or T or V; Residue 392 is I or L; Residue 394 is A or I or L or V; Residue 396 is R or K or T; Residue 398 is A or I or L or V; Residue 399 is R or L; Residue 402 is L or M or F; Residue 403 is Q or G; Residue 404 is G or H; Residue 405 is R or H or V; Residue 408 is L or V; Residue 419 is I or V; Residue 424 is A or S; Residue 427 is L or M or W; Residue 429 is I or V; Residue 432 is C or I or L or V; Residue 434 is I or M; Residue 441 is S or T; Residue 447 is N or D; Residue 461 is L or M; Residue 469 is A or T; Residue 474 is N or D; Residue 475 is A or F; Residue 477 is I or V; Residue 504 is D or E; Residue 507 is R or H; Residue 508 is I or V; Residue 512 is I or L; Residue 516 is H or S; Residue 522 is I or V; Residue 526 is A or V; Residue 529 is N or Q or E; Residue 535 is T or Y; Residue 536 is R or E or H; Residue 537 is R or N or Q or K or T or Y; Residue 538 is Q or E or T; Residue 539 is R or N or D or E or K; Residue 541 is L or V; Residue 543 is N or G or S; Residue 544 is D or E; Residue 545 is A or R; Residue 546 is Q or E; Residue 559 is Q or E or S; Residue 560 is Q or E; Residue 562 is F or Y or V; Residue 564 is N or H or K or Y; Residue 565 is I or L; Residue 566 is D or E or S; Residue 567 is A or N or D or H; Residue 568 is Q or L or P; Residue 569 is N or Q or E or H or K or S; Residue 572 is M or P; Residue 573 is I or L; Residue 577 is A or S; Residue 579 is A or L; Residue 585 is Q or I or L; Residue 589 is A or L or M; Residue 591 is A or C or L; Residue 592 is F or Y or V; Residue 594 is R or K; Residue 595 is N or S; Residue 596 is Q or K or P; Residue 597 is A or E or K; Residue 601 is R or Q or H or K; Residue 605 is I or V; Residue 606 is I or L or V; Residue 610 is K or Y; Residue 611 is A or L or V; Residue 612 is R or D; Residue 613 is P or T or V; Residue 614 is A or E; Residue 615 is D or G; Residue 617 is S or T; Residue 619 is R or S or Y; Residue 622 is R or I or K; Residue 623 is A or D; Residue 628 is I or L or M; Residue 629 is H or Y; Residue 631 is I or L; Residue 632 is I or M or V; Residue 633 is D or E or H or K; Residue 634 is Q or E; Residue 636 is N or Q; Residue 638 is N or Q or H or K or S; Residue 641 is A or I or V; Residue 643 is L or F or W; Residue 651 is A or N or G or I; Residue 656 is I or V; Residue 659 is I or V; Residue 660 is I or V; Residue 663 is R or H; Residue 664 is Q or G; Residue 666 is A or I or V; Residue 668 is A or V; Residue 683 is A or S; Residue 686 is S or T; Residue 692 is A or G; Residue 694 is R or Q; Residue 704 is N or D or H; Residue 705 is Q or G; Residue 706 is K or V; Residue 710 is H or Y; Residue 715 is D or H; Residue 719 is M or T; Residue 721 is E or K; Residue 722 is A or K or T; Residue 723 is I or L; Residue 724 is L or M or F or V; Residue 725 is R or K; Residue 727 is I or L or F; Residue 728 is A or E; Residue 729 is A or R or Q or H or K; Residue 731 is N or D; Residue 732 is R or Q or H or K; Residue 735 is N or Q or E; Residue 736 is Q or E or H or Y; Residue 738 is Q or E or Y; Residue 739 is R or E; Residue 740 is I or L; Residue 742 is Q or E or K; Residue 743 is A or R or K; Residue 744 is A or G or S; Residue 746 is D or Q or E; Residue 749 is R or Y; Residue 750 is E or S; Residue 751 is N or K or T; Residue 757 is H or F or W or Y; Residue 758 is A or C or T; Residue 759 is E or K or S or T; Residue 760 is R or K; Residue 761 is I or L or M or W; Residue 762 is L or M; Residue 763 is S or T; Residue 765 is A or I or S or V; Residue 766 is R or K; Residue 768 is M or Y; Residue 770 is L or F; Residue 773 is F or Y; Residue 774 is I or M or S or T or V; Residue 782 is L or M; Residue 783 is L or M or W; Residue 786 is I or L or M; Residue 789 is I or L or M; Residue 790 is F or Y; Residue 794 is F or Y; The polypeptide, wherein residue 800 is A or R or Q or K (SEQ ID NO:1230).

22. A method for transferring a sugar moiety to a substrate steviol glycoside, the method comprising contacting B12GT and sucrose synthase with one or more steviol glycosides, a non-UDP nucleotide diphosphate, and sucrose.

23. 23. The method of claim 22, wherein the B12GT polypeptide is a modified B12GT comprising an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-882 and 1333-1466.

24. 23. The method of claim 22, wherein the SuSy polypeptide is a modified sucrose synthase comprising an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs:883-1227 and 1231-1332.

25. (a) the B12GT polypeptide is a modified B12GT comprising an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-882 and 1333-1466; and 23. The method of claim 22, wherein (b) the SuSy polypeptide is a modified sucrose synthase comprising an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs:883-1227 and 1231-1332.

26. 23. The method of claim 22, wherein the B12GT polypeptide is a modified B12GT having a score of greater than 266.7 when scored by the PSSM shown in Table 17.

27. 23. The method of claim 22, wherein the SuSy polypeptide is an engineered sucrose synthase having a score of greater than 556 when scored by the PSSM set forth in Table 24.

28. 23. The method of claim 22, wherein the SuSy polypeptide is an engineered sucrose synthase having a score of greater than 194.5 when scored by the PSSM set forth in Table 28.

29. (a) the B12GT polypeptide is a modified B12GT having a score of greater than 266.7 when scored by the PSSM shown in Table 17; and 23. The method of claim 22, wherein (b) the SuSy polypeptide is an engineered sucrose synthase having a score of greater than 194.5 when scored by the PSSM set forth in Table 28.

30. (a) the B12GT polypeptide is a modified B12GT having a score of greater than 266.7 when scored by the PSSM shown in Table 17; and 23. The method of claim 22, wherein (b) the SuSy polypeptide is an engineered sucrose synthase having a score of greater than 556 when scored by the PSSM set forth in Table 24.

31. The substrate steviol glycoside is steviol, steviol-13-O-glucoside, steviol-19-O-glucoside, rubusoside, steviol-1,2-bioside, steviol-1,3-bioside, rubusoside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F, rebaudioside G, rebaudioside H, rebaudioside I, rebaudioside J, rebaudioside K, rebaudioside L, rebaudioside N, rebaudioside N, rebaudioside N, rebaudioside N, rebaudioside N, rebaudioside N, rebaudioside N, rebaudioside N, rebaudioside N, rebaudioside N, rebaudioside N, rebaudioside B, rebaudioside G, rebaudioside B, rebaudioside C, rebaudioside F, rebaudioside N ...

31. The method of any of claims 22-30, wherein the steviol glycoside is udioside A, rebaudioside I, rebaudioside E, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M, rebaudioside D, rebaudioside N, rebaudioside O, rebaudioside Q, an isomer thereof, a synthetic steviol glycoside, or a combination thereof.

32. The method according to any one of claims 22 to 30, wherein the substrate steviol glycoside is a mixture of stevioside and rebaudioside A.

33. The target steviol glycosides are steviol, steviol-13-O-glucoside, steviol-19-O-glucoside, rubusoside, steviol-1,2-bioside, steviol-1,3-bioside, rubusoside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F, rebaudioside G, rebaudioside H, rebaudioside I, rebaudioside J, rebaudioside K, rebaudioside L, rebaudioside N ...

33. The method of any of claims 22-32, wherein the steviol glycoside is rebaudioside A, rebaudioside I, rebaudioside E, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M, rebaudioside D, rebaudioside N, rebaudioside O, rebaudioside Q, isomers thereof, synthetic steviol glycosides, or combinations thereof.

34. 33. The method of any of claims 22-32, wherein the target steviol glycoside is a mixture of rebaudioside E and rebaudioside D.

35. 33. The method of any of claims 22-32, wherein the target steviol glycoside is rebaudioside D.

36. 33. The method of any of claims 22-32, wherein the target steviol glycoside is rebaudioside E.

37. The method of any one of claims 22 to 36, wherein the non-UDP nucleotide diphosphate is ADP, GDP, CDP, or TDP.

38. The method of any one of claims 22 to 37, wherein the non-UDP nucleotide diphosphate is ADP.

39. A polynucleotide encoding the polypeptide according to any one of claims 1 to 21.

40. A host microorganism heterologously expressing the polynucleotide of claim 39.

41. The modified β-1,2-glycosyltransferase polypeptide of claim 1, wherein the amino acid sequence includes H at position 15, D at position 114, D at position 357, and Q at position 358, numbered according to SEQ ID NO:

5.

42. The modified sucrose synthase polypeptide of claim 12, wherein the amino acid sequence comprises H at position 425, R at position 567, K at position 572, and E at position 663, numbered according to SEQ ID NO:

885.

43. The modified sucrose synthase polypeptide of claim 12, wherein the amino acid sequence comprises H at position 436, R at position 578, K at position 583, and E at position 674, numbered according to SEQ ID NO:

888.

44. The modified sucrose synthase polypeptide of claim 12, wherein the amino acid sequence selected from the group is sequence number 1132.

45. The modified sucrose synthase polypeptide of claim 12, wherein the amino acid sequence selected from the group is sequence number 1197.

46. ​​The method of claim 24, wherein the amino acid sequence selected from the group is sequence number 1132.

47. The method of claim 24, wherein the amino acid sequence selected from the group is sequence number 1197.

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