Compositions and methods for producing rebaudioside M

Abstract

The present disclosure provides enzymes and methods for using those enzymes to transfer sugar moieties to substrate steviol glycosides. The engineered beta-1,3-glycosyltransferases are capable of adding beta-1,3-linked glucose monomers to steviol glycosides. Specifically, the engineered beta-1,3-glycosyltransferases are used in a one-pot reaction with a beta-1,2-glycosyltransferase and sucrose synthase to convert stevioside and / or Reb A to Reb M.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 374,461, filed September 2, 2022, the entire contents of which are incorporated herein by reference.

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (ARZE_038_01WO_SeqList_ST26.xml, size: 681,823 bytes; and creation date: September 1, 2023) are incorporated herein by reference in their entirety.

[0003] The present disclosure relates to enzymatic 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. Healthcare systems incur exorbitant costs associated with treating these diseases. Replacing added sugar in foods with low-calorie, high-intensity sweeteners would have significant health and economic impacts.

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

[0006] Furthermore, rebaudioside M (Reb M) has an improved sweetness and bitterness profile compared to Reb A. However, Reb M is present in very low amounts in stevia leaves. Therefore, improved methods for converting glucose sources to lower steviol glycosides are needed. Summary of the Invention

[0007] The present disclosure provides enzymes, particularly unnatural enzymes, and methods for transferring sugar moieties to substrate steviol glycosides using these enzymes. Specifically, beta-1,3-glycosyltransferases (also referred to herein as "B13GT") capable of glycosylating the C3' of 13-O-glucose and / or 19-O-glucose of steviol glycoside substrates are disclosed. The present disclosure provides methods for producing Reb M using the disclosed beta-1,3-glycosyltransferases in combination with sucrose synthase (SuSy) and, optionally, beta-1,2-glycosyltransferase (B12GT).

[0008] In contrast to naturally occurring glycosyltransferases, the present disclosure provides glycosyltransferase polypeptides that can utilize adenosine diphosphate (ADP)-glucose as a sugar donor to glycosylate the C3' of 13-O-glucose and / or 19-O-glucose of steviol glycoside substrates. The present disclosure provides glycosyltransferase polypeptides comprising an amino acid sequence 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: 2-517. The glycosyltransferase polypeptides can comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-517. 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] The disclosure further provides methods utilizing the disclosed B13GT to produce steviol glycosides, specifically Reb M. Starting with a feed containing Reb A and / or stevioside, the enzymes B13GT, B12GT, and SuSy can be combined with sucrose, ADP, and the steviol glycoside feed to convert Reb A and / or stevioside to Reb M. 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 beta-1,3-glycosyltransferase, a beta-1,2 glycosyltransferase, and a sucrose synthase to produce Reb M. In another embodiment, a method comprises contacting a Stevia leaf extract purified to contain greater than 60% Reb A (RA60), ADP, and sucrose with beta-1,3-glycosyltransferase, beta-1,2 glycosyltransferase, and sucrose synthase to produce Reb M. In another embodiment, a method comprises contacting a Stevia leaf extract purified to contain greater than 90% stevioside (RA90), ADP, and sucrose with beta-1,3-glycosyltransferase, beta-1,2 glycosyltransferase, and sucrose synthase to produce Reb M. In another embodiment, a method comprises contacting a Stevia leaf extract purified to contain greater than 99% stevioside (RA99), ADP, and sucrose with beta-1,3-glycosyltransferase, beta-1,2 glycosyltransferase, and sucrose synthase to produce Reb M.

[0010] To enable a further understanding of the present disclosure, 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 explanation of the drawings]

[0011] [Figure 1] 1 shows the chemical structure of rebaudioside M. [Figure 2]1 shows a partial steviol glycoside network illustrating the conversion of stevioside and Reb A to Reb M by the addition of glucose monomers catalyzed by beta-1,2-glycosyltransferase (B12GT) and beta-1,3-glycosyltransferase (B13GT). For each molecule, the central hexagon represents the steviol glycoside core, the circles represent beta-1,2-linked glucose monomers, and the squares represent beta-1,3-linked glucose monomers. [Figure 3] 1 shows an SDS-PAGE gel of the engineered beta-1,3-glycosyltransferase expressed in Pichia pastoris and purified by immobilized metal affinity chromatography. DETAILED DESCRIPTION OF THE INVENTION

[0012] The disclosure provides enzymatic and biocatalytic processes for preparing compositions comprising a target steviol glycoside by contacting a feedstock composition comprising a substrate steviol glycoside, sucrose, and a nucleotide diphosphate (NDP) with one or more NDP-glycosyltransferase polypeptides and a sucrose synthase, thereby producing a composition comprising a target steviol glycoside that contains one or more additional glucose units than the substrate steviol glycoside.

[0013] definition As used herein, the term "a" or "one" refers to one or more of that entity, i.e., it can refer to a plural reference. Thus, the terms "a," "one," "one or more," and "at least one" are used interchangeably herein. In addition, reference to an "element" by the indefinite article "a" or "one" does not exclude the possibility that more than one element is present, unless the context clearly requires that there be only one element.

[0014] Throughout this application, ADP, GD, UDP, CDP, and TDP are used to refer to adenosine diphosphate, guanosine-5'-diphosphate, uridine diphosphate, cytidine diphosphate, and thymidine diphosphate, respectively.

[0015] Throughout this specification, the term "about" is used to indicate that a value includes the inherent variation of error for the device or method being used to determine the value, or the variation that exists between samples measured. Also, unless expressly stated otherwise or clear from context, the term "approximately" means within 10% of the reported numerical value (except when such number is greater than 100% or less than 0% of the possible values). When used in conjunction with a range or series of values, the term "about" applies to the endpoints of the range or each recited value in the series, unless otherwise indicated. As used in this application, the terms "about" and "approximately" are used as equivalent terms.

[0016] As used herein, the term "sequence identity" refers to the degree to which two optimally aligned polynucleotide or polypeptide sequences are invariant across a window of residue, e.g., nucleotide or amino acid, alignment. The "percent identity" of an aligned segment of a test sequence and a reference sequence is the number of identical residues shared by the two aligned sequences divided by the total number of residues in the reference sequence segment, i.e., the entire reference sequence or a defined small portion of the reference sequence. The "percent identity" is calculated by multiplying the percent identity by 100. Comparison of sequences to determine percent identity can be accomplished by several well-known methods, including, for example, using mathematical algorithms such as the BLAST suite of sequence analysis programs. Unless otherwise specified, the term "sequence identity" in the claims refers to sequence identity calculated by Clustal Omega® using default parameters.

[0017] As used herein, "biocatalyst" or "biocatalytic" refers to the use of natural catalysts, such as protein enzymes, to carry out chemical transformations on organic compounds. Biocatalysis is also known as biotransformation or biosynthesis. Both isolated biocatalytic methods and whole-cell biocatalytic methods are known in the art. Biocatalytic protein enzymes can be naturally occurring proteins or recombinant proteins.

[0018] As used herein, the term "steviol glycoside(s)" refers to glycosides of steviol, including, but not limited to, naturally occurring steviol glycosides, synthetic steviol glycosides, e.g., enzymatically glycosylated steviol glycosides, and combinations thereof. For example, the term steviol glycoside refers to the known steviol glycosides, 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 Rebaudioside C, Rebaudioside F, Rebaudioside A, Rebaudioside I, Rebaudioside E, Rebaudioside E2, Rebaudioside E3, Rebaudioside H, Rebaudioside L, Rebaudioside K, Rebaudioside J, Rebaudioside M, Rebaudioside D, Rebaudioside N, Rebaudioside O, Rebaudioside Q, and Rebaudioside AM.

[0019] As used herein, the term "feedstock composition" refers to any composition (generally an aqueous solution) containing one or more steviol glycosides, which serve as substrates for biotransformation.

[0020] As used herein, the terms "polynucleotide" or "nucleic acid" are used interchangeably, unless otherwise indicated by context, to refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, typically DNA.

[0021] 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, and the transcribed mRNA is subsequently translated into a polypeptide.

[0022] "Under transcriptional control" means that transcription of a polynucleotide, usually a DNA sequence, is dependent on being operably linked to elements that promote transcription.

[0023] "Operably linked" means that the polynucleotide element is positioned so that it can function within a cell, typically to produce a polypeptide within the cell; for example, the present disclosure provides a promoter operably linked to a downstream sequence that encodes a polypeptide.

[0024] 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 polynucleotide is said to encode the mRNA and the polypeptide. The antisense strand is the complementary strand of such a nucleic acid, from which the coding sequence can be deduced. Similarly, a "coding sequence" refers to the region of a nucleic acid that encodes an mRNA or a polypeptide.

[0025] As used herein, the term "promoter" refers to a control sequence that is a part of a polynucleotide sequence that controls the initiation and rate of transcription of a coding sequence. An "enhancer" is a regulatory element that increases expression of a target sequence. A "promoter / enhancer" is a polynucleotide having sequences that provide both promoter and enhancer function.

[0026] 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 naturally found 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. Often, 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 naturally found or one or more regulatory elements operably linked to the polynucleotide are not naturally found in the host cell.

[0027] 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 900 amino acids.

[0028] A "plasmid" is a DNA molecule that is typically separated from chromosomal DNA and can replicate independently of it. 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 for random or targeted stable integration into host chromosomes. Many plasmids are commercially available for various uses. The gene to be replicated is inserted into a copy of the plasmid, which also contains a gene that confers resistance to a specific antibiotic and a multiple cloning site (MCS, or polylinker), a short region containing several commonly used restriction sites that allows for easy insertion of DNA fragments into this location. Typically, the polypeptides disclosed herein are expressed from a plasmid.

[0029] The chemical structure of Reb M, shown in Figure 1, can be generated by adding a beta-1,3-linked glucose monomer to the lower steviol glycosides rebaudioside I (Reb I) or Reb AM, or a beta-1,2-linked glucose monomer to rebaudioside D (Reb D), as shown in Figure 2. The naturally occurring glycosyltransferases that carry out these conversions use uridine diphosphate (UDP)-glucose as the glucose source for transfer to the lower steviol glycosides.

[0030] The present disclosure provides non-naturally occurring, engineered beta-1,3-adenosine diphosphate (ADP) glycosyltransferases (B13GTs) that can add C3' beta-linked glucose monomers of 13-O-glucose and / or 19-O-glucose of steviol glycoside substrates using an ADP-glucose sugar donor. For example, the disclosed B13GTs can perform the following conversions: stevioside to Reb A, stevioside to rebaudioside E2 (Reb E2), Reb A to Reb I, rebaudioside E (Reb E) to Reb D, Reb E to rebaudioside AM (Reb AM), Reb E2 to Reb I, Reb D to Reb M, and Reb AM to Reb M. In certain embodiments, the glycosyltransferase polypeptide is one of SEQ ID NOs: 2-517. In another embodiment, the glycosyltransferase 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:2-517.

[0031] In bioinformatics, several methods have been developed to find and determine related polypeptide sequences. For example, percent sequence identity, position-specific scoring matrices (PSSMs), and hidden Markov models (HMMs) are all widely used to find sequences similar to a specific query sequence. Percent sequence identity calculates the number of amino acids shared between two sequences. Percent sequence identity is calculated based on a specific alignment between two sequences. Percent identity can be calculated using the alignment program Clustal Omega (available at / www.ebi.ac.uk / Tools / msa / clustalo / ) with default settings. The default transition matrix is ​​Gonnet, the gap opening penalty is 6 bits, and the 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.

[0032] 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 and 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 the different amino acids (or gaps) 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

[0033]

number

[0034] In some embodiments, the beta-1,3-glycosyltransferase polypeptide is prepared by expression in a host microorganism. Suitable host microorganisms include, but are not limited to, E. coli, Saccharomyces spp., Aspergillus spp., Pichia spp., and Bacillus spp. In certain embodiments, the glycosyltransferase is expressed in E. coli. In certain embodiments, the glycosyltransferase is expressed in Pichia pastoris.

[0035] The B13GT polypeptide can be provided in any suitable form, including free, immobilized, or whole cell systems. The glycosyltransferase polypeptide can 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 a membrane. In some embodiments, the glycosyltransferase polypeptide is immobilized on the solid support by covalent bonding, adsorption, crosslinking, entrapment, or encapsulation.

[0036] Furthermore, in another embodiment, the B13GT polypeptide is 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.

[0037] The present disclosure provides a biocatalytic process for preparing a composition comprising one or more target steviol glycosides from a feedstock composition comprising one or more substrate steviol glycosides, wherein the target steviol glycosides comprise steviol glycosides having one or more additional glucose units than the substrate steviol glycosides. The biocatalytic process comprises contacting an engineered B13GT, a sucrose synthase (SuSy) (unless ADP glucose is directly provided), and optionally B12GT, with a feedstock composition comprising one or more steviol glycosides, a non-UDP nucleotide diphosphate, and sucrose. In another embodiment, the biocatalytic process comprises contacting an engineered B13GT, SuSy, and optionally B12GT with a feedstock 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 an engineered B1,3 glycosyltransferase, B12GT, and SuSy to produce Reb M. In another embodiment, the method comprises contacting RA60, ADP, and sucrose with an engineered B1,3 glycosyltransferase, B12GT, and SuSy to produce Reb M. In another embodiment, the method comprises contacting stevioside, ADP, and sucrose with an engineered B1,3 glycosyltransferase, B12GT, and SuSy to produce Reb M. In another embodiment, the method comprises contacting Reb A, ADP, and sucrose with an engineered B1,3 glycosyltransferase, B12GT, and SuSy to produce Reb M. In another embodiment, the method comprises contacting RS90, ADP, and sucrose with engineered B13GT, B12GT, and SuSy to produce Reb M. In another embodiment, the method comprises contacting RS99, ADP, and sucrose with engineered B13GT, B12GT, and SuSy to produce Reb M.

[0038] In one embodiment, the B13GT polypeptide is one of SEQ ID NOs: 2-517. In another embodiment, the glycosyltransferase 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: 2-517.

[0039] In one embodiment, the sucrose synthase is any polypeptide having sucrose synthase activity. In another embodiment, the sucrose synthase is derived from an organism in the Bacteria domain. In another embodiment, the sucrose synthase is derived from an organism in the Plantae kingdom. In another embodiment, the sucrose synthase is derived from an organism belonging to the Proteobacteria, Deferribacteria, or Cyanobacteria phylum. In another embodiment, the sucrose synthase is derived from Acidithiobacillus caldus, Nitrosomonas europaea, Denitrovibrio acetiphilus, Thermosynechococcus elongatus, Oryza sativa, Arabidopsis thaliana, or Coffea arabica. 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 identical to a sucrose synthase derived from Acidithiobacillus caldus, Nitrosomonas europaea, Denitrovibrio acetiphilus, Thermosynechococcus elongatus, Oryza sativa, Arabidopsis thaliana, or Coffea arabica.

[0040] In one embodiment, B12GT is any polypeptide capable of glycosylating the C2' of 13-O-glucose and / or 19-O-glucose of steviol glycoside substrates. In another embodiment, B12GT is derived from an organism in the kingdom Plantae. In another embodiment, B12GT is derived from an organism from the angiosperm clade. In another embodiment, B12GT is derived from the species Hordeum vulgare, Oryza sativa, Solanum lycopersicum, Lycium barbarum, Solanum tuberosum, or Stevia rebaudiana. In another embodiment, the B12GT is an engineered B12GT 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 identical to B12GT derived from the species Hordeum vulgare, Oryza sativa, Solanum lycopersicum, Lycium barbarum, Solanum tuberosum, or Stevia rebaudiana.

[0041] In some embodiments, the B13GT, B12GT, 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 spp., Aspergillus spp., Pichia spp., and Bacillus spp. In certain embodiments, the glycosyltransferases and sucrose synthase are expressed in E. coli. In other embodiments, the glycosyltransferases and sucrose synthase are expressed in Pichia pastoris. In other embodiments, the B13GT, B12GT, and / or sucrose synthase polypeptides are prepared by cell-free expression.

[0042] The B13GT, B12GT, and sucrose synthase polypeptides can be provided in any suitable form, including free, immobilized, or whole cell systems. The purity of the polypeptides can vary, and they can be provided, for example, as crude enzyme preparations, semi-purified enzyme preparations, or purified enzyme preparations. In one embodiment, the B13GT, B12GT, and / or SuSy polypeptides are free. In another embodiment, the B13GT, B12GT, and / or SuSy polypeptides are immobilized on a solid support, e.g., 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 B13GT, B12GT, and / or SuSy polypeptides are immobilized on the solid support by covalent bonding, adsorption, crosslinking, entrapment, or encapsulation.

[0043] Furthermore, in another embodiment, the B13GT, 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.

[0044] The steviol glycoside component(s) of the feedstock composition, as described herein, serve as substrate(s) for the production of target steviol glycoside(s), which differ chemically from the corresponding substrate steviol glycoside by one or more additional glucose units.

[0045] The raw steviol glycoside composition can 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 F, rebaudioside G, or rebaudioside B. The starting steviol glycoside composition may be selected from the group consisting of rebaudioside A, rebaudioside I, rebaudioside E, rebaudioside E2, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M, rebaudioside AM, rebaudioside D, rebaudioside N, rebaudioside O, rebaudioside Q, isomers thereof, synthetic steviol glycosides, or combinations thereof. In another embodiment, the starting steviol glycoside composition comprises stevioside and Reb A. In another embodiment, the starting steviol glycoside composition comprises stevioside. In yet another embodiment, the starting steviol glycoside composition comprises Reb A.

[0046] The raw steviol glycoside composition can be synthetic or purified (partially or completely), commercially available, or prepared. One example of a raw material 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 material composition is a commercially available stevia extract in a solvent solution. Yet another example of a raw material composition is a commercially available mixture of steviol glycosides in a solvent solution. Other suitable raw material compositions include by-products of processes for isolating and purifying steviol glycosides.

[0047] In one embodiment, the feedstock composition comprises purified substrate steviol glycosides. For example, the feedstock composition may comprise, on a moisture-free basis, greater than 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99.6% by weight of one or more substrate steviol glycosides.

[0048] In another embodiment, the feedstock composition comprises a partially purified substrate steviol glycoside composition, e.g., the feedstock composition comprises greater than 0.5%, greater than 1%, greater than 2%, greater than 3%, greater than 4%, greater than 5%, greater than 10%, greater than 20%, greater than 30%, greater than 40%, or greater than 50% by weight of one or more substrate steviol glycosides on an anhydrous basis.

[0049] 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% rebaudioside A or an isomer by weight on an anhydrous basis. In another embodiment, the substrate steviol glycoside comprises partially purified rebaudioside A. In certain embodiments, the substrate steviol glycoside comprises greater than 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% rebaudioside A by weight on an anhydrous basis.

[0050] In yet 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 other embodiments, the substrate steviol glycoside comprises partially purified stevioside. In certain embodiments, the substrate steviol glycoside comprises greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% by weight stevioside on an anhydrous basis.

[0051] In yet another embodiment, the substrate steviol glycoside is a combination of stevioside and rebaudioside A. In certain embodiments, the substrate steviol glycoside is, on an anhydrous basis, greater than 5% by weight stevioside and greater than 5% by weight Reb A, greater than 10% by weight stevioside and greater than 10% by weight Reb A, greater than 20% by weight stevioside and greater than 20% by weight Reb A, greater than 30% by weight stevioside and greater than 30% by weight Reb A, greater than 40% by weight stevioside and greater than 40% by weight Reb A, greater than 45% by weight stevioside and greater than 45% by weight Reb A, greater than 40% by weight stevioside and greater than 50% by weight Reb A, greater than 30% by weight stevioside and greater than 60% by weight Reb A, greater than 20% by weight stevioside and greater than 70% by weight Reb A, greater than 10% by weight stevioside and greater than 80% by weight Reb A. A, more than 5% by weight of stevioside and more than 90% by weight of Reb A, more than 50% by weight of stevioside and more than 40% by weight of Reb A, more than 60% by weight of stevioside and more than 30% by weight of Reb A, more than 70% by weight of stevioside and more than 20% by weight of Reb A, more than 80% by weight of stevioside and more than 10% by weight of Reb A, or more than 90% by weight of stevioside and more than 5% by weight of Reb A.

[0052] In another embodiment, the substrate steviol glycoside is derived from a stevia leaf extract. In one embodiment, RA50 (a 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 100 mg / mL. In another embodiment, RA60, a stevia leaf extract purified to contain 60% or more Reb A, is used as the steviol glycoside substrate. In another embodiment, RA60 is used at a concentration of about 1 to 800 mg / mL. In another embodiment, RA60 is used at a concentration of 100 mg / mL. In another embodiment, RA90, a stevia leaf extract purified to contain 90% or more stevioside, is used as the steviol glycoside substrate. In another embodiment, RA99, a stevia leaf extract purified to contain 99% or more stevioside, is used as the steviol glycoside substrate. In another embodiment, RA90 is used at a concentration of about 1 to 800 mg / mL, hi another embodiment, RA90 is used at a concentration of about 100 mg / mL.

[0053] The 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 ADP, GDP, UDP, CDP, or TDP. In some embodiments, the nucleotide can be a non-UDP nucleotide (i.e., ADP, GDP, CDP, or TDP). In other embodiments, the nucleotide is ADP. In certain embodiments, the reaction can be carried out with ADP at a concentration of 0.01 mM 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.

[0054] The reaction can be carried out at a concentration of sucrose between 10 mM and 2 M, e.g., 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 450 mM.

[0055] In one embodiment, the reaction is carried out at any temperature. In another embodiment, the 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.

[0056] The reaction medium for the conversion is typically aqueous, such as purified water, a buffer solution, or a combination thereof. In certain embodiments, the reaction medium is a buffer solution. Suitable buffer solutions include, but are not limited to, acetate buffer, citrate buffer, HEPES, and phosphate buffer. In certain embodiments, the reaction medium is a phosphate buffer solution. The reaction medium can have a pH of 4 to 10. In certain embodiments, the pH of the reaction medium is 6. Alternatively, the reaction medium can be an organic solvent.

[0057] The step of contacting the feedstock composition with the glycosyltransferase and sucrose synthase polypeptide can be carried out for a period of time ranging from 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, or 6 days to 7 days. In certain embodiments, the reaction is carried out for 6 hours.

[0058] Reactions can be monitored by any appropriate method, including, but not limited to, high performance liquid chromatography (HPLC), liquid chromatography mass spectrometry (LCMS), thin layer chromatography (TLC), infrared spectroscopy (IR), or nuclear magnetic resonance (NMR).

[0059] The target steviol glycoside can be any steviol glycoside. In one embodiment, the target steviol glycoside is 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 I, rebaudioside II, rebaudioside III, rebaudioside I, rebaudioside II, rebaudioside III, rebaudioside I, rebaudioside I, rebaudioside II ... Rebaudioside E2, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M, rebaudioside AM, 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 I or an isomer thereof. In another embodiment, the target steviol glycoside is rebaudioside M or an isomer thereof. In another embodiment, the target steviol glycoside is rebaudioside AM or an isomer thereof. In another embodiment, the target steviol glycoside is rebaudioside E2 or an isomer thereof. In yet another embodiment, the target steviol glycosides are Reb I, Reb AM, Reb D, and Reb M.

[0060] In one embodiment, conversion of the raw steviol glycoside composition to Reb I is at least 2% complete, as determined by any of the methods described above. In certain embodiments, conversion of the raw steviol glycoside composition to Reb I 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, conversion of the raw steviol glycoside composition to Reb I is at least 95% complete. In some embodiments, at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the raw steviol glycoside composition is converted to Reb I.

[0061] In one embodiment, conversion of the raw steviol glycoside composition to Reb M is at least 2% complete, as determined by any of the methods described above. In certain embodiments, conversion of the raw steviol glycoside composition to Reb M 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, conversion of the raw steviol glycoside composition to Reb M is at least 95% complete. In some embodiments, at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the raw steviol glycoside composition is converted to Reb M.

[0062] In one embodiment, conversion of the raw steviol glycoside composition to Reb AM is at least 2% complete, as determined by any of the methods described above. In certain embodiments, conversion of the raw steviol glycoside composition to Reb AM 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, conversion of the raw steviol glycoside composition to Reb AM is at least 95% complete. In some embodiments, at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the raw steviol glycoside composition is converted to Reb AM.

[0063] In one embodiment, conversion of the raw steviol glycoside composition to Reb E2 is at least 2% complete, as determined by any of the above methods. In certain embodiments, conversion of the raw steviol glycoside composition to Reb E2 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, conversion of the raw steviol glycoside composition to Reb E2 is at least 95% complete. In some embodiments, at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the raw steviol glycoside composition is converted to Reb E2.

[0064] In one embodiment, conversion of the raw steviol glycoside composition to Reb I, Reb AM, Reb D, and Reb M is at least 2% complete, as determined by any of the methods described above. In certain embodiments, conversion of the raw steviol glycoside composition to Reb I, Reb AM, Reb D, and Reb M 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, conversion of the raw steviol glycoside composition to Reb E2 is at least 95% complete. In some embodiments, at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the raw steviol glycoside composition is converted to Reb I, Reb AM, Reb D, and Reb M.

[0065] The target steviol glycoside(s) may be in any polymorphic or amorphous form, including hydrates, solvates, anhydrates, or combinations thereof.

[0066] Optionally, the methods of the present disclosure further include separating the target steviol glycoside(s) from the target composition. The target steviol glycoside(s) can be separated by any suitable method, such as, for example, crystallization, membrane separation, centrifugation, extraction, chromatographic separation, or a combination of such methods.

[0067] In one embodiment, separation of the target steviol glycoside(s) produces a composition comprising greater than 80% by weight of the target steviol glycoside on an anhydrous basis, i.e., a highly purified steviol glycoside composition. In another embodiment, separation produces a composition comprising greater than 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.6% by weight of the target steviol glycoside. In certain embodiments, the composition comprises greater than 95% by weight of the target steviol glycoside(s) on an anhydrous basis.

[0068] The purified target steviol glycosides can be used as sweeteners in consumer products, including, but not limited to, foods, beverages, pharmaceutical compositions, tobacco products, dietary supplement compositions, oral hygiene compositions, and cosmetic compositions.

[0069] Plasmids containing nucleic acids encoding enzymes having SEQ ID NOs: 2-517 are listed in Table 1 below.

[0070] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Example]

[0071] Example 1: Computational design of ADP glucose-dependent B13GT Computational design was performed to improve native B13GT from Stevia rebaudiana (SEQ ID NO: 1) and convert it into an ADP-glycosyltransferase. A structural model of the B13GT variant of SEQ ID NO: 1 was generated and used as a starting point for computational design. Visual inspection of the structural model identified residues H24 as the catalytic histidine, D123 as an activating histidine, and two sugar-binding residues D379 and Q380 (numbering relative to SEQ ID NO: 2). Computational design incorporating coevolutionary information was performed to improve B13GT. Fifty-three of these computational designs were selected for experimental validation (SEQ ID NOs: 2-54).

[0072] Polynucleotides encoding the designed B13GT amino acid sequence were synthesized (Twist Bioscience) and inserted into the pARZ4 expression vector. The polynucleotides encoded the B13GT enzyme with an N-terminal His tag. The polynucleotides were sequenced as full-length genes or gene fragments, which were then assembled using Gibson assembly. The recombinant vectors were transformed into E. coli HMS174(DE3) (Novagen) or T7 Express lysY / Iq (New England BioLabs) using the heat shock method to generate recombinant microorganisms.

[0073] The transformed recombinant microorganisms were inoculated into 1 ml of LB-kanamycin medium and grown overnight at 37°C with shaking. The culture was then inoculated into 5 ml of TB-kanamycin medium and grown for 2 hours at 37°C, followed by 1 hour at 25°C. The culture was induced with 50 μL of 50 mM IPTG and grown overnight. Finally, the culture was centrifuged at maximum speed for 5 minutes and stored at -80°C.

[0074] Microorganisms were lysed in lysis buffer (lysozyme, DNAse I, 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 220 rpm at 25°C for 30 minutes. The disrupted solution was centrifuged at 2200 × 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 × g for 4 minutes, then washed twice with 500 μL of binding buffer (300 ml of 20 mM HEPES pH 7.5, 500 mM NaCl, 20 mM imidazole) and centrifuged at 500 × g for 2 minutes. The protein was eluted with 150 μL of elution buffer (15 ml of 20 mM HEPES pH 7.5, 500 mM NaCl, 500 mM imidazole) and shaken for 1 minute at a maximum shaking speed of 0.25, followed by centrifugation at 500 × g for 2 minutes. The recovered protein was desalted in enzyme activity assay buffer (50 mM HEPES pH 7.5, 50 mM NaCl).

[0075] Each B13GT variant was assayed in a one-pot reaction using the B12GT and SuSy enzymes. B12GT and SuSy were used in excess, resulting in complete conversion of the raw RA50 feed to RebD / E in the absence of the B13GT variant. Purified B13GT, B12GT, and SuSy were reacted with 100 mg / ml RA50, 450 mM sucrose, and 0.5 mM ADP in 50 mM pH 6 phosphate buffer and 50 mM NaCl at 60°C for 24 hours.

[0076] The steviol glycosides, stevioside, Reb A, Reb D, Reb E, Reb I, Reb M, and other trace steviol glycosides produced and consumed during the reaction were monitored by LCMS using an Agilent 6470 QQQ mass spectrometer (column: Waters ACQUITY UPLC HSS T3 column, 100 mm x 2.1 mm). QQQ was performed by multiple reaction monitoring (MS / MS) to accurately quantify the steviol glycosides of the present invention. Several engineered enzymes were successfully expressed and could efficiently produce Reb M (Table 2). Table 2. Purified protein concentration and Reb M conversion of successful B13GT design variants expressed in E. coli.

[0077] [Table 2-1] [Table 2-2]

[0078] Example 2: Pichia pastoris expression of engineered B13GT and SuSy Polynucleotides optimized for expression in Pichia pastoris from the top 10 B13GTs designed in Example 1 were synthesized (Twist Bioscience) and inserted into a Pichia shuttle vector. These polynucleotides encoded B13GT enzymes with a C-terminal His tag instead of the N-terminal His tag used in Example 1. Two unique polynucleotides were sequenced for each B13GT amino acid sequence. The vectors were transformed into a commercially available Pichia pastoris strain (ATCC). 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 in desalting buffer (20 mM KPO4 (pH 6), 50 mM NaCl). The designed B13GT was solublely expressed (FIG. 3), and the enzyme activity was measured in the same manner as in Example 1 (Table 3).

[0079] The polynucleotide encoding the top-performing B13GT was amplified from a Pichia shuttle vector and integrated into the Pichia genome. Four integrated Pichia strains expressing the engineered B13GT were grown in 1 L fermenters. The Pichia microorganisms were grown for approximately 24 hours using glycerol as the primary carbon source, followed by feeding with methanol for approximately 72 hours to allow expression of the desired B13GT. Cells were harvested and lysed using a French press. The expressed protein was purified by immobilized metal affinity chromatography (IMAC) and dialyzed into desalting buffer (20 mM KPO4 (pH 6), 50 mM NaCl). All four strains successfully expressed active B13GT in fermentation.

[0080] [Table 3]

[0081] Example 3: Computer-aided design of an improved B13GT A previously developed protein sequence-based deep learning model was fine-tuned with the results from Example 1. This model was used to design an improved B13GT enzyme. Eighty computational designs were selected for experimental validation (SEQ ID NOS: 55-134). A polynucleotide encoding the B13GT enzyme with a C-terminal His tag was synthesized (Twist Bioscience). An E. coli strain expressing the designed B13GT was generated as described in Example 1. The designed B13GT enzyme was expressed and purified as described in Example 1.

[0082] Each B13GT variant was assayed in a one-pot reaction using B12GT and SuSy enzymes. B12GT and SuSy were used in excess, resulting in complete conversion of the raw RA50 feed to RebD / E in the absence of the B13GT variant. Purified B13GT, B12GT, and SuSy were reacted with 71.5 mg / ml RA50, 250 mM sucrose, and 0.5 mM ADP in 50 mM pH 6 phosphate buffer and 50 mM NaCl at 60°C for 24 hours. Steviol glycosides produced and consumed during the reaction were measured as described in Example 1. The designed enzymes were solubly expressed and could efficiently produce Reb M (Table 4).

[0083] [Table 4-1] [Table 4-2]

[0084] Structural models of the top B13GT designs were generated and analyzed. To this end, further improved B13GT enzymes were designed using a structure-based deep learning method trained to predict protein sequences for a given protein scaffold. 121 of these computational designs were selected for experimental validation (SEQ ID NOS: 135-255). The machine learning model was retrained with crystal structures and structural models of proteins from thermophilic organisms. An additional 69 B13GT enzymes were selected for experimental validation (SEQ ID NOS: 256-324). Polynucleotides encoding B13GT enzymes (SEQ ID NOS: 135-324) with C-terminal His tags were synthesized (Twist Bioscience). E. coli strains expressing the designed B13GT were generated as described in Example 1. The designed B13GT enzymes were expressed and purified as described in Example 1.

[0085] Each B13GT variant was assayed in a one-pot reaction using B12GT and SuSy enzymes. B12GT and SuSy were used in excess, resulting in complete conversion of the raw RA50 feed to RebD / E in the absence of the B13GT variant. Purified B13GT, B12GT, and SuSy were reacted with 71.5 mg / ml RA50, 450 mM sucrose, and 0.5 mM ADP in 50 mM pH 6 phosphate buffer and 50 mM NaCl at 60°C for 24 hours. Steviol glycosides produced and consumed during the reaction were measured as described in Example 1. The designed enzymes were solubly expressed and could efficiently produce Reb M (Table 5).

[0086] [Table 5-1] [Table 5-2]

[0087] Example 4: Further improved computational design of B13GT The protein-based deep learning model from Example 3 was fine-tuned with the results from Examples 1 and 3. This sequence-based model was used to design an improved B13GT enzyme. 100 computational designs were selected for experimental validation (SEQ ID NOS: 325-424). Polynucleotides encoding B13GT enzymes (SEQ ID NOS: 325-424) with C-terminal His tags were synthesized (Twist Bioscience). E. coli strains expressing the designed B13GT were generated as described in Example 1. The designed B13GT enzymes were expressed and purified as described in Example 1.

[0088] Each B13GT variant was assayed in a one-pot reaction using B12GT and SuSy enzymes. B12GT and SuSy were used in excess, resulting in complete conversion of the raw RA50 feed to RebD / E in the absence of the B13GT variant. Purified B13GT, B12GT, and SuSy were reacted with 71.5 mg / ml RA60, 450 mM sucrose, and 0.5 mM ADP in 50 mM pH 6 phosphate buffer and 250 mM NaAcetate at 60°C for 24 hours. Steviol glycosides produced and consumed during the reaction were measured as described in Example 1. The designed enzymes were solubly expressed and could efficiently produce Reb M (Table 6).

[0089] [Table 6]

[0090] Structural models of the top B13GT designs of Example 3 were generated and analyzed. Further improved B13GT enzymes were generated using a structure-based deep learning method trained to predict protein sequences for a given protein scaffold. This method was used to design improved B13GT enzymes. Ninety-three of these calculated designs were selected for experimental validation (SEQ ID NOS: 425-517). Polynucleotides encoding B13GT enzymes with C-terminal His tags were synthesized (Twist Bioscience). E. coli strains expressing the designed B13GT were generated as described in Example 1. The designed B13GT enzymes were expressed and purified as described in Example 1.

[0091] Each B13GT variant was assayed in a one-pot reaction using B12GT and SuSy enzymes. B12GT and SuSy were used in excess, resulting in complete conversion of the raw RA50 feed to RebD / E in the absence of the B13GT variant. Purified B13GT, B12GT, and SuSy were reacted with 71.5 mg / ml RA60, 450 mM sucrose, and 0.5 mM ADP in 50 mM pH 6 phosphate buffer and 250 mM NaAcetate at 60°C for 24 hours. Steviol glycosides produced and consumed during the reaction were measured as described in Example 1. The designed enzymes were solubly expressed and could efficiently produce Reb M (Table 7).

[0092] [Table 7]

[0093] Example 5: Representation of successful B13GT designs with PSSM The successful B13GT designs from Example 1 were used to generate PSSMs (Table 8). PSSMs are a concise way of representing successful designs and related sequences. Sequences with a PSSM score of greater than 332 are considered related to the active B13GT computational designs described herein. To score a sequence with a PSSM, it must first be aligned to the representative sequence, SEQ ID NO: 2. For example, the following successful designs, AA29162, AA29163, AA29161, and AA29165, have PSSM scores of 332.2, 335.5, 332.5, and 337.3, while the wild-type B13GT, SEQ ID NO: 1, has a PSSM score of only 326.7.

[0094] The successful B13GT designs from Example 3 were used to generate PSSMs (Table 9). PSSMs are a concise way of representing successful designs and related sequences. Sequences with a PSSM score of greater than 54.5 are considered related to the active B13GT computational designs described herein. To score a sequence with a PSSM, it must first be aligned to the representative sequence, SEQ ID NO: 2. For example, the following successful designs, AA40232, AA40239, AA40205, and AA40243, have PSSM scores of 70.74, 80.53, 78.75, and 76.08, while the wild-type B13GT SEQ ID NO: 1 has a PSSM score of only 51.16.

[0095] The successful B13GT designs from Example 4 were used to generate PSSMs (Table 10). PSSMs are a concise way of representing successful designs and related sequences. Sequences with a PSSM score of greater than 58.3 are considered related to the active B13GT computational designs described herein. To score a sequence with a PSSM, it must first be aligned to the representative sequence, SEQ ID NO: 2. For example, the following successful designs, AA41333, AA41342, AA41345, and AA41346, have PSSM scores of 90.04, 92.68, 89.16, and 97.08, while the wild-type B13GT SEQ ID NO: 1 has a PSSM score of only 51.16.

[0096]

Table 8-1

Table 8-2

Table 8-3

Table 8-4

Table 8-5

Table 8-6

Table 8-7

Table 8-8

Table 8-9

Table 8-10

Table 8-11

[0097]

Table 9-1

Table 9-2

Table 9-3

Table 9-4

Table 9-5

Table 9-6

Table 9-7

Table 9-8

Table 9-9

Table 9-10

Table 9-11

Table 9-12

[0098]

Table 10-1

Table 10-2

Table 10-3

Table 10-4

[0099] Example 6: E. coli fermentation of engineered B13GT E. coli strains with a constitutive promoter controlling B13GT expression were generated for the upper B13GT gene in Example 3. The E. coli strains were grown in 1-liter fermentors for 48 hours using a glycerol feed. Fermentations of strains expressing AA37959, AA40205, AA40232, and AA40243 yielded wet cell masses of 171, 60, 182, and 190 grams per liter of fermentation. 20-30 grams of cells from each fermentation were resuspended and lysed using a homogenizer. All lysates were tested and confirmed to contain B13GT activity. The clarified lysates were purified using IMAC. The protein yields of the resulting purified protein samples A37959, AA40205, AA40232, and AA40243 were 0.47, 0.15, 1.20, and 1.42 mg / ml, respectively.

[0100] Incorporation by Reference All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entirety for all purposes. However, no mention is made of the references, articles, patent publications, patent publications, and patent applications cited herein as an acknowledgement or suggestion that they form part of the general knowledge in any country in the world.

Claims

1. An engineered beta-1,3-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: 4-517, 2 and 3.

2. 2. The engineered beta-1,3-glycosyltransferase polypeptide of claim 1, comprising an amino acid sequence identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-517, 2, and 3.

3. An engineered beta-1,3-glycosyltransferase polypeptide having a score of greater than 332 when scored by the PSSM set forth in Table 8, wherein the engineered beta-1,3-glycosyltransferase polypeptide is aligned to SEQ ID NO:2 prior to scoring.

4. An engineered beta-1,3-glycosyltransferase polypeptide having a score of greater than 54.5 when scored by the PSSM set forth in Table 9, wherein the engineered beta-1,3-glycosyltransferase polypeptide is aligned to SEQ ID NO:2 prior to scoring.

5. An engineered beta-1,3-glycosyltransferase polypeptide having a score of greater than 58.3 when scored by the PSSM set forth in Table 10, wherein the engineered beta-1,3-glycosyltransferase polypeptide is aligned to SEQ ID NO:2 prior to scoring.

6. A polypeptide having the sequence: MPNTXXTXXRXXXXXXFPVPFPGHXNPMLQLANXLYXXGFXIXIXXXXFNXPKTSXYPHFXFXXXXDXXPQXXXLRRNLPTXGPGXGARIPXINXHGXXXXXXLXXXXXXXXXDXXXXXCXIXDALWYFXXXVXXXLXXXLVLXTXSLFNFXXXLXXXLPXFXXXGXLXXXXXXXXXXXXXXXGXPXLXXXDIXXAYSHXXEXKPILXKMXXXTXX XSGXIWNSXKELEESELETXXREIPAPSFLXPLPKHXXASSXSLLDXDXXXXXWLXXQXXXSVXYVXFGSQSXXXXXXXXXXGLXXSXXXLWLVRPGFXKGXXWXXXXPXG XXGXXXRXVKSXPQQEVLAHXAIGAFWTHGGWNGTXEXVCEGVPMIXSXFGLDQPLNAXYMXXXXXXGXYLXXGXXXXXXXXAXXXXXXXX SSYESLEXLXSYISSL wherein residue 5 is N or S; Residue 6 is P or T; Residue 8 is T or V; Residue 9 is R or V; Residue 11 is R or D or E or H or K or S or T; Residue 12 is R or D or L or K; Residue 13 is R or H or T or W; Residue 14 is I or V; residue 15 is A or I or V; Residue 16 is I or L or M; residue 25 is I or L or W or V; Residue 34 is L or V; Residue 37 is R or S; Residue 38 is R or K; residue 41 is A or R or N or D or Q or E or K or S; residue 43 is I or T or V; residue 45 is I or L or F; Residue 46 is H or Y; Residue 47 is M or T; residue 48 is A or R or N or Q or E or K or S or T; residue 51 is A or R or L or K; Residue 56 is N or L; Residue 61 is Q or E or T; residue 63 is R or E or I or L or K or V; Residue 64 is R or N or D or Q or E or L or F or P or S or T or Y; residue 65 is I or L or F or T or V; Residue 66 is I or L; Residue 68 is R or N or E; Residue 69 is D or E; Residue 72 is D or P or T; Residue 73 is D or E; Residue 74 is W or Y; Residue 81 is H or T; residue 85 is A or V; Residue 91 is Q or I or L or V; Residue 94 is Q or E or K; Residue 97 is A or K; residue 98 is A or D or E or K or P; residue 99 is A or R or Q or E or K or P or T or V; Residue 100 is L or F; Residue 101 is R or E or L or K or Y; residue 102 is A or R or D or Q or E or K; residue 103 is A or E or I or L or K or M or T or V; residue 105 is R or Q or E or L or K or S; residue 106 is A or R or N or D or Q or E or I or L or K or S; residue 107 is A or C or E or I or L or M or F or Y or V; residue 108 is I or L or K or M; residue 109 is A or R or N or D or Q or E or L or K or S; Residue 110 is A or N or D or E or S; Residue 111 is S or T; residue 112 is A or E or G or K or P; residue 113 is D or E or K or S; residue 115 is E or V; residue 116 is R or Q or E or K or P or T or V; residue 117 is I or L or V; Residue 118 is A or R or S; Residue 120 is L or F or V; Residue 122 is A or T, residue 129 is A or T; residue 130 is A or N or Q or E or G or L or K or M or T or Y; Residue 131 is D or E or L or P or S; Residue 133 is A or T, residue 134 is R or D or Q or E or K; residue 135 is R or E or K or S; Residue 137 is N or G; Residue 138 is I or L; Residue 139 is R or K or P; Residue 140 is R or L or P; Residue 144 is Q or M; Residue 146 is G or S; Residue 152 is H or L; residue 153 is A or C; residue 155 is A or V; Residue 156 is C or S; residue 159 is A or R or D or Q or E or H or L or K; Residue 161 is R or D or E or I or L; residue 162 is A or R or D or Q or E or K; residue 163 is A or R or D or Q or L or K; residue 165 is I or L or W or Y; Residue 167 is N or D; Residue 168 is L or P; residue 169 is A or N or D or E or S or T; Residue 170 is N or D or S or T; Residue 171 is N or Q or K or P or S or T; Residue 172 is R or E or H or L or K or S or T or W; residue 173 is A or R or N or D or G or K or P; Residue 174 is D or E or L or P; residue 175 is D or E; residue 176 is A or D or Q or E or K or T; Residue 177 is Q or P; Residue 178 is A or V; Residue 179 is E or K or P or S or V; Residue 181 is A or L or F or Y; Residue 183 is R or Q or E or H or I or L or M or F or W or Y; Residue 185 is R or L or K or T; Residue 186 is A or R or N or W or V; Residue 187 is A or R or G or K; Residue 190 is R or L or K; Residue 191 is R or K or S; Residue 196 is D or I or K or S or W; Residue 197 is D or Q; Residue 199 is A or S; Residue 204 is D or E or G or M or S; residue 207 is I or T or V; Residue 208 is E or K or T; residue 209 is A or Q or E or M or T; Residue 211 is R or K; residue 212 is A or R or N or Q or E or L or K; Residue 213 is A or S; residue 216 is I or V; residue 221 is A or F; residue 232 is I or V; Residue 233 is R or Q or I or W; residue 243 is I or L; residue 249 is L or F or Y; Residue 250 is R or T; Residue 254 is S or T; residue 259 is E or T; Residue 261 is R or P; Residue 262 is S or T; residue 263 is A or F or T or V; Residue 264 is A or F; residue 265 is A or D or Q or E or P; Residue 268 is A or D; residue 269 is R or Q or G or K or T; Residue 271 is A or P; residue 272 is A or D or E or K or P; Residue 273 is R or N or E or G or K or S; Residue 276 is L or V; Residue 279 is G or S; residue 285 is Q or E or S; residue 286 is I or L or M or F or V; residue 287 is D or E or S or T; residue 288 is A or R or E or L or K or P or V; residue 289 is A or D or E or K; residue 290 is D or Q or E or T; Residue 291 is A or F; Residue 292 is L or K; residue 293 is A or R or N or E or K or T; Residue 294 is I or L; residue 295 is A or L or M or V; Residue 296 is R or D or Q or E or H or K; residue 299 is R or I or K or V; residue 300 is A or R or D or E or L or K; Residue 302 is E or G or K; residue 303 is Q or E or H or I or L or K or V; Residue 304 is R or N or K or P or S; residue 305 is A or F or T or V; residue 314 is I or V; residue 317 is A or R or Q or E or H or L or K or S or Y; residue 318 is A or R or D or E or K or S or T; residue 320 is I or L or T or V; Residue 321 is D or E or S; residue 322 is A or D or I or L or F or P or S or V; residue 323 is I or L; residue 325 is D or E or P; residue 327 is R or L or K or F or Y; residue 328 is G or L or M or F; residue 330 is D or E or P; residue 331 is A or R or N or Q or L or K; residue 332 is G or I; residue 334 is I or V; residue 338 is A or C; residue 347 is R or E or G or K or P; Residue 362 is L or M or V; Residue 364 is A or S; residue 373 is C or F; Residue 375 is D or P; Residue 385 is A or R or T or V; Residue 388 is A or S or T or V; Residue 389 is R or N or D or E or K; residue 390 is E or I or K or V; Residue 391 is R or L or K; Residue 392 is R or N or E or G or H or K; residue 393 is I or V; residue 395 is I or V; Residue 398 is R or D or E; Residue 399 is R or N or D or E or K; Residue 401 is F or W; Residue 402 is R or N or D or Q or E or I or K or V; residue 403 is A or R or E or K or P or T or V; Residue 404 is A or R or D or Q or E or G or K; Residue 405 is A or R or N or D or E or K; Residue 406 is I or V; residue 407 is A or I or V; Residue 408 is A or R or N or D or E or K; residue 410 is A or I or L or V; Residue 411 is R or N or Q or E or H or K; Residue 412 is R or E or H or L or K or T or Y; residue 413 is I or V; Residue 414 is L or M; residue 415 is R or Q or E or L or K or T or V; residue 416 is A or D or E or S; residue 417 is A or E or K or P; residue 418 is A or R or E or K or F or S; residue 420 is A or R or Q or E or L or K; residue 421 is A or E or G or H or K or P or Y or V; Residue 422 is I or M or Y; Residue 423 is A or R or K; Residue 424 is A or R or N or D or Q or E or K; Residue 425 is R or N; residue 427 is A or R or Q or E or L or K; residue 428 is A or R or E or L or K or V; Residue 430 is A or K; Residue 431 is A or R or D or Q or E; Residue 433 is A or L; residue 435 is R or G or V; residue 437 is I or L; Residue 438 is A or M; Residue 439 is K or P; Residue 449 is R or S; Residue 451 is Q or V (SEQ ID NO:518).

7. The polypeptide according to any one of claims 1 to 6, wherein the polypeptide is expressed in a host microorganism.

8. 8. The polypeptide of claim 7, wherein the host microorganism is E. coli, Saccharomyces species, Aspergillus species, Pichia species, or Bacillus species.

9. The polypeptide of any one of claims 1 to 8, wherein the polypeptide is immobilized on a solid support.

10. 10. The polypeptide of claim 9, wherein the solid support is derivatized from cellulose, glass, ceramic, methacrylate, styrene, acrylic, metal oxide, or a membrane.

11. The polypeptide of claim 9 or 10, wherein the polypeptide is immobilized on the solid support by covalent bonding, adsorption, cross-linking, entrapment, or encapsulation.

12. 1. A method for transferring a sugar moiety to a substrate steviol glycoside, comprising contacting a beta-1,3-glycosyltransferase polypeptide, a sucrose synthase, and optionally a beta-1,2-glycosyltransferase polypeptide with a feedstock composition comprising one or more steviol glycosides, a nucleotide diphosphate, and sucrose.

13. 13. The method of claim 12, wherein the beta-1,3-glycosyltransferase polypeptide is an engineered beta-1,3-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: 4-517, 2 and 3.

14. The sucrose synthase is selected from the group consisting of Acidithiobacillus caldus, Nitrosomonas europaea, Denitrovibrio acetiphilus, Thermosynechococcus elongatus, Oryza sativa, Arabidopsis thaliana, and Coffea 13. The method of claim 12, wherein the engineered sucrose synthase has 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 identical to a sucrose synthase derived from A. arabica.

15. 13. The method of claim 12, wherein the beta-1,2-glycosyltransferase polypeptide is an engineered beta-1,2-glycosyltransferase polypeptide 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 identical to a beta-1,2-glycosyltransferase polypeptide derived from the species Hordeum vulgare, Oryza sativa, Solanum lycopersicum, Lycium barbarum, Solanum tuberosum, or Stevia rebaudiana.

16. 13. The method of claim 12, (a) the beta-1,3-glycosyltransferase polypeptide is an engineered beta-1,3-glycosyltransferase polypeptide comprising an amino acid sequence 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:4-517, 2 and 3; (b) the sucrose synthase is selected from the group consisting of Acidithiobacillus caldus, Nitrosomonas europaea, Denitrovibrio acetiphilus, Thermosynechococcus elongatus, Oryza sativa, Arabidopsis thaliana, and Coffea 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 identical to a sucrose synthase derived from A. arabica; (c) the beta-1,2-glycosyltransferase polypeptide is an engineered beta-1,2-glycosyltransferase polypeptide 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 identical to a beta-1,2-glycosyltransferase polypeptide derived from the species Hordeum vulgare, Oryza sativa, Solanum lycopersicum, Lycium barbarum, Solanum tuberosum, or Stevia rebaudiana.

17. 13. The method of claim 12, wherein the beta-1,3-glycosyltransferase polypeptide is an engineered beta-1,3-glycosyltransferase polypeptide having a score of greater than 332 when scored by the PSSM set forth in Table 8, and wherein the engineered beta-1,3-glycosyltransferase polypeptide is aligned to SEQ ID NO: 2 prior to scoring.

18. 13. The method of claim 12, wherein the beta-1,3-glycosyltransferase polypeptide is an engineered beta-1,3-glycosyltransferase polypeptide having a score of greater than 54.5 when scored by the PSSM set forth in Table 9, and wherein the engineered beta-1,3-glycosyltransferase polypeptide is aligned to SEQ ID NO: 2 prior to scoring.

19. 13. The method of claim 12, wherein the beta-1,3-glycosyltransferase polypeptide is an engineered beta-1,3-glycosyltransferase polypeptide having a score of greater than 58.3 when scored by the PSSM set forth in Table 10, and wherein the engineered beta-1,3-glycosyltransferase polypeptide is aligned to SEQ ID NO: 2 prior to scoring.

20. The substrate steviol glycoside may be 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 B, rebaudioside C, rebaudioside F, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside I, rebaudioside G, rebaudioside I ...

20. The method of any one of claims 12-19, comprising at least one steviol glycoside selected from the group consisting of rebaudioside E, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M, rebaudioside D, rebaudioside E2, rebaudioside AM, rebaudioside N, rebaudioside O, rebaudioside Q, isomers thereof, and synthetic steviol glycosides.

21. 21. The method of claim 20, wherein the substrate steviol glycoside comprises a mixture of stevioside and rebaudioside A.

22. 21. The method of claim 20, wherein the substrate steviol glycoside comprises stevioside.

23. 21. The method of claim 20, wherein the substrate steviol glycoside comprises one or more selected from the group consisting of Reb A, RS50, RS60, RS90, and RS99.

24. 33. The method of any one of claims 12 to 32, further comprising producing a target steviol glycoside, The target steviol glycosides include 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 B, rebaudioside C, rebaudioside D, rebaudioside F, rebaudioside G, rebaudioside B, rebaudioside G, rebaudioside C, rebaudioside F, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside G, rebaudioside B, rebaudioside C, rebaudioside F, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside G, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside B, rebaudioside G, rebaudioside C, rebaudioside F, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D ... C, rebaudioside D, rebaudioside B, rebaudioside C, rebaudioside D, rebaudio at least one steviol glycoside selected from udioside I, rebaudioside E, rebaudioside E2, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside AM, rebaudioside M, rebaudioside D, rebaudioside N, rebaudioside O, rebaudioside Q, isomers thereof, and synthetic steviol glycosides.

25. 25. The method of claim 24, wherein the target steviol glycoside comprises at least one steviol glycoside selected from the group consisting of stevioside rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside I, rebaudioside D, rebaudioside AM, and rebaudioside M.

26. 25. The method of claim 24, wherein the target steviol glycoside comprises at least one steviol glycoside selected from the group consisting of rebaudioside I, rebaudioside D, rebaudioside AM, and rebaudioside M.

27. 25. The method of claim 24, wherein the target steviol glycoside comprises rebaudioside M.

28. 25. The method of claim 24, wherein the target steviol glycoside comprises rebaudioside AM.

29. 29. The method of any one of claims 12 to 28, wherein the nucleotide diphosphate comprises one selected from the group consisting of adenosine diphosphate, uridine diphosphate, cytidine diphosphate, thymidine diphosphate, and guanosine diphosphate.

30. 29. The method of any one of claims 12 to 28, wherein the nucleotide diphosphate comprises one selected from the group consisting of adenosine diphosphate, guanosine diphosphate, cytidine diphosphate, or thymidine diphosphate.

31. The method of any one of claims 12 to 28, wherein the nucleotide diphosphate comprises adenosine diphosphate.

32. 32. The method of any one of claims 12 to 31, wherein the raw material composition comprises, on an anhydrous basis, greater than about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.6% by weight of steviol glycosides.

33. 33. The method of any of claims 12-32, further comprising producing a target composition, wherein the target composition comprises greater than about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.6% by weight of the target steviol glycoside on an anhydrous basis.

34. A polynucleotide encoding the polypeptide according to any one of claims 1 to 6.

35. A host microorganism that heterologously expresses the polynucleotide of claim 34.

36. 36. The microorganism of claim 35, wherein the host microorganism is E. coli, a Saccharomyces species, an Aspergillus species, a Pichia species, or a Bacillus species.

37. 1. A method for transferring a sugar moiety to a substrate steviol glycoside, comprising contacting a beta-1,3-glycosyltransferase polypeptide and a sucrose synthase with one or more steviol glycosides, a nucleotide diphosphate, and sucrose.

38. 2. The polypeptide of claim 1, wherein the polypeptide comprises H at position 24, D at position 123, D at position 379, and Q at position 380, the position numbers being based on SEQ ID NO:

2.

39. 1. An engineered beta-1,3-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 SEQ ID NO:4, wherein the polypeptide comprises an H at position 24, a D at position 123, a D at position 379, and a Q at position 380.

40. 1. An engineered beta-1,3-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 SEQ ID NO:3, wherein the polypeptide comprises an H at position 24, a D at position 123, a D at position 379, and a Q at position 380.

41. 1. An engineered beta-1,3-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 SEQ ID NO:219, wherein the polypeptide comprises an H at position 24, a D at position 123, a D at position 379, and a Q at position 380.

42. 1. An engineered beta-1,3-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 SEQ ID NO:208, wherein the polypeptide comprises an H at position 24, a D at position 123, a D at position 379, and a Q at position 380.

43. 1. An engineered beta-1,3-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 SEQ ID NO: 181, wherein the polypeptide comprises an H at position 24, a D at position 123, a D at position 379, and a Q at position 380.

Images