Compositions and methods for producing rebaudioside m

Abstract

The present disclosure provides enzymes and a method to use those enzymes to transfer a sugar moiety to a substrate steviol glycoside. The designed beta-1,3-glycosyltransferases can add beta-1,3 linked glucose monomers to steviol glycosides. Specifically, the designed beta-1,3-glycosyltransfersases are used in a one-pot reaction with a beta-1,2-glycosyltransferase and sucrose synthase to convert stevioside and / or Reb A into Reb M.

Description

COMPOSITIONS AND METHODS FOR PRODUCING REBAUDIOSIDE MCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 374,461, filed on September 2, 2022, the content of which is herein incorporated by reference in its entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (ARZE_038_01WO_Se- qList_ST26.xml; Size: 681,823 bytes; and Date of Creation: September 1, 2023) are herein incorporated by reference in its entirety.FIELD OF THE DISCLOSURE

[0003] The present disclosure relates to enzymes and biocatalytic processes for producing ste- viol glycosides. The present disclosure particularly relates to the use of glycosyltransferases that can transfer a glucose moiety from an ADP-glucose sugar donor to steviol glycosides.BACKGROUND

[0004] Excess sugar consumption has been linked to worldwide health epidemics including diabetes and heart disease. Healthcare systems incur exorbitant costs associated with treating these diseases. Replacing added sugar in food with a low calorie, high-intensity sweetener would have significant health and economic impact.

[0005] The species Stevia rebaudlana is commonly grown for its sweet leaves, which have traditionally been used as a sweetener. Stevia extract is 200-300 times sweeter than sugar and is used commercially as a high intensity sweetener. The main glycoside components of stevia leaf are steviosides and rebaudiosides. Over ten different steviol glycosides are present in appreciable quantities in the leaf. The principal sweetening compounds are stevioside and rebau- dioside A. Rebaudioside A (Reb A) is considered higher value compared to stevioside, because of its increased sweetness and decreased bitterness.

[0006] Further, when compared to Reb A, the sweetness and bitterness profile of rebaudioside M (Reb M) is improved. Reb M, however, is present at very low quantities in the stevia leaf. Accordingly, improved methods for converting glucose sources into low-order steviol glycosides are required.SUMMARY OF THE INVENTION

[0007] The present disclosure provides enzymes, particularly non-natural enzymes, and methods to use those enzymes to transfer a sugar moiety to a substrate steviol glycoside. Specifically, beta-l,3-glycosyltransferases (also referred to herein as “B13GTs”) are disclosed that can glycosylate the C3’ of the 13-O-glucose and / or the 19-O-glucose of a Steviol glycoside substrate. The present disclosure provides methods to use the disclosed beta- 1,3 -glycosyltransferases in combination with a sucrose synthase (SuSy) and optionally a beta-l,2-glycosyltrans- ferase (B12GT) to make Reb M.

[0008] In contrast to native glycosyltransferases, the disclosure provides glycosyltransferase polypeptides that can utilize adenosine diphosphate (ADP)-glucose as the sugar donor to glycosylate the C3’ of the 13-O-glucose and / or 19-O-glucose of a steviol glycoside substrate. 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: 2-517. The glycosyltransferase polypeptide may comprise, or consist of, an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-517. The polypeptides may comprise one or more peptide tags used for solubility, expression and / or purification; for example, a polyhistidine tag of between 4 and 10 histidine residues, preferably 6 histidine residues. Other suitable tags include, but are not limited to, glutathione 5-transferase (GST), FLAG, maltose binding protein (MBP), calmodulin binding peptide (CBP), and Myc tag. Suitable linkers include, but are not limited to, polypeptides composed of glycine and serine, such as GSGS, polyglycine linkers, EAAAK repeats, and sequences containing cleavage sites for enzymes such as factor Xa, enterokinase, and thrombin.

[0009] The disclosure additionally provides a method to utilize the disclosed B13GTs to make 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 the Reb A and / or stevioside to Reb M. In some embodiments, the method comprises contacting a stevia leaf extract purified to contain greater than 50% Reb A (RA50), ADP, and sucrose with a beta-l,3-glycosyltransferase, beta-1,2 glycosyltransferase and sucrose synthase to make Reb M. In another embodiment, the method comprises contacting a stevia leaf extract purified to contain greater than 60% Reb A (RA60), ADP, and sucrose with a beta- 1,3 -glycosyltransferase, beta- 1,2 glycosyltransferase, and sucrose synthase to make Reb M. In another embodiment, the method comprises contacting a stevia leaf extract purified- to contain greater than 90% stevioside (RS90), ADP, and sucrose with a beta- 1,3 -glucosyl - transferase, beta-l,2-glucosyltransferase, and sucrose synthase to make Reb M. In another embodiment, the method comprises contact a stevia leaf extract purified to contain greater than 99% stevioside (RS99), ADP, and sucrose with a beta-l,3-glucosyltransferase, beta-l,2-gluco- syltransferase, and sucrose synthase to make Reb M.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The accompanying drawings are included to provide a further understanding of the dis- closure. The drawings illustrate embodiments of the disclosure and together with the descrip- tion, serve to explain the principles of the embodiments of the disclosure.

[0011] FIG. 1 Chemical structure of rebaudioside M.

[0012] FIG. 2 Partial steviol glycoside network showing conversion of stevioside and Reb A to Reb M through additions of glucose monomers catalyzed by a beta-l,2-glycosyltransferase (B12GT) and beta- 1,3 -glycosyltransferase (B13GT). For each molecule, the central hexagon represents the steviol glycoside core, circles represent beta-l,2-linked glucose monomers and squares represent beta- 1,3 -linked glucose monomers.

[0013] FIG. 3 SDS-PAGE gel of designed beta- 1,3 -glycosyltransferases expressed in Pichia pastoris and purified by immobilized metal affinity chromatography.DETAILED DESCRIPTION

[0014] The present disclosure provides enzymes and biocatalytic processes for preparing a composition comprising a target steviol glycoside by contacting a starting composition com- prising a substrate steviol glycoside, sucrose, and nucleotide diphosphate (NDP) with one or more NDP-glycosyltransferase polypeptides and a sucrose synthase, thereby producing a com- position comprising a target steviol glycoside comprising one or more additional glucose units than the substrate steviol glycoside.Definitions

[0015] The term “a” or “an” refers to one or more of that entity, i.e. can refer to plural referents. As such, the terms “a,” “an,” “one or more,” and “at least one” are used interchangeably herein. In addition, reference to “an element” by the indefinite article “a” or “an” does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there is one and only one of the elements.

[0016] Throughout this application, ADP, GDP, UDP, CDP, and TDP are used to refer, respec- tively, to adenosine diphosphate, guanosine-5 ’-diphosphate, uridine diphosphate, cytidine di- phosphate, and thymidine diphosphate.

[0017] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device or the method being employed to determine the value, or the variation that exists among the samples being measured. Unless otherwise stated or otherwise evident from the context, the term “about” means within 10% above or below the reported numerical value (except where such number would exceed 100% of a possible value or go below 0% ). When used in conjunction with a range or series of values, the term “about” applies to the endpoints of the range or each of the values enumerated in the series, unless otherwise indicated. As used in this application, the terms “about” and “approximately” are used as equivalents.

[0018] As used herein the term “sequence identity” refers to the extent to which two optimally aligned polynucleotides or polypeptide sequences are invariant throughout a window of alignment of residues, e.g. nucleotides or amino acids. An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical residues which are 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 smaller defined part of the reference sequence. “Percent identity” is the identity fraction times 100. Comparison of sequences to determine percent identity can be accomplished by a number of well-known methods, including for example by using mathematical algorithms, such as, for example, those in the BLAST suite of sequence analysis programs. Unless noted otherwise, the term “sequence identity” in the claims refers to sequence identity as calculated by Clustal Omega® using default parameters.

[0019] As used herein, “biocatalysis” or “biocatalytic” refers to the use of natural catalysts, such as protein enzymes, to perform chemical transformations on organic compounds. Bio- catalysis is alternatively known as biotransformation or biosynthesis. Both isolated and whole- cell biocatalysis methods are known in the art. Biocatalyst protein enzymes can be naturally occurring or recombinant proteins.

[0020] As used herein, the term “steviol glycoside(s)” refers to a glycoside of steviol, includ- ing, 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 glycosides includes the known steviol glycosides steviol- 13 -O-glucoside, steviol- 19-0- glucoside, rubusoside, steviol- 1,2-bioside, steviol- 1,3 -bioside, rubusoside, dulcoside B,-ulcoside A, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F, re- baudioside A, rebaudioside I, rebaudioside E, rebaudioside E2, rebaudioside E3, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M, rebaudioside D, rebaudi- oside N, rebaudioside O, rebaudioside Q, and rebaudioside AM.

[0021] As used herein, “starting composition” refers to any composition (generally an aqueous solution) containing one or more steviol glycosides, where the one or more steviol glycosides serve as the substrate for the biotransformation.

[0022] As used herein, the terms “polynucleotide" or “nucleic acid” are used interchangeably, unless indicated by context, and is used to refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, typically DNA.

[0023] As used herein, "expression" refers to either or both steps, depending on context, of the two-step process by which polynucleotides are transcribed into mRNA and the transcribed mRNA is subsequently translated into a polypeptides.

[0024] "Under transcriptional control" means that transcription of a polynucleotide, usually a DNA sequence, depends on its being operatively linked to an element that promotes transcrip- ------0025] "Operatively linked" means that the polynucleotide elements are arranged in a manner that allows them to function in a cell; typically to produce polypeptides in the cell; for example the disclosure provides promoters operatively linked to the downstream sequences encoding polypeptides.

[0026] The term "encode" refers to the ability of a polynucleotide to produce an mRNA or a polypeptide if it can be transcribed to produce the mRNA and then translated to produce the polypeptide or a fragment thereof. In each case, the polynucleotide is referred to as encoding the mRNA and encoding the polypeptide. The antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom. Similarly, a “coding se- quence” refers to a region of a nucleic acid that encodes an mRNA or a polypeptide.

[0027] 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 se- quence. An “enhancer” is a regulatory element that increases the expression of a target se- quence. A "promoter / enhancer" is a polynucleotide with sequences that provide both promoter and enhancer functions.

[0028] The regulatory elements, e.g. enhancers and promoters, may be "homologous" or "het- erologous." A "homologous" regulatory element is one which is naturally linked with a givenpolynucleotide in the genome; for example, it may be the promoter found natively in the or- ganism upstream of the encoded polypeptide. A "heterologous" regulatory element is one which is placed in juxtaposition to a polynucleotide by means of recombinant molecular bio- logical techniques but is not a combination found in nature. Often, promoters, enhancers and other regulatory elements are heterologous so as to facilitate expression of a polypeptide in a host cell other than one in which a polypeptide naturally occurs. Thus, “heterologous expression”, as used herein, refers to producing an mRNA and / or a polypeptide in a host cell, such as a microorganism, where the polynucleotide is not found naturally or one or more regulatory elements are not naturally found operably linked to the polynucleotide in the host cell.

[0029] The term "polypeptide” is used here to refer to a molecule of two or more subunits of amino acids linked by peptide bonds. Typically, though not always, the polypeptides contain several hundred amino acids; for example, about 400 to about 900 amino acids.

[0030] A "plasmid" is a DNA molecule that is typically separate from and capable of replicating independently of the chromosomal DNA. In many cases, it is circular and double-stranded. It is known in the art that while plasmid vectors often exist as extrachromosomal circular DNA molecules, plasmid vectors may also be designed to be stably integrated into a host chromo- some either randomly or in a targeted manner. Many plasmids are commercially available for varied uses. The gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics, and a multiple cloning site (MCS, or polylinker), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this location. Typically, the polypeptides disclosed herein are expressed from plasmids.

[0031] Reb M, the chemical structure of which is shown in FIG. 1, can be made by adding a beta- 1,3 -linked glucose monomer to the lower-order steviol glycosides Rebaudioside I (Reb I), or Reb AM or adding a beta- 1 ,2-linked glucose monomer to Rebaudioside D (Reb D), as shown in FIG. 2. Native glycosyltransferases that perform these conversions use uridine diphosphate (UDP)-glucose as the glucose source for transferring to the lower-order steviol glycosides.

[0032] The present disclosure provides non-natural, engineered beta-l,3-adenosine diphos- phate (ADP) glycosyltransferases (B13GTs) that can use an ADP-glucose sugar donor to add a beta-linked glucose monomer to the C3’ of the 13-O-glucose and / or 19-O-glucose of a steviol glycoside substrate. For example, the disclosed B13GTs can perform the following conver- sions: stevioside to Reb A, stevioside to Rebaudioside E2 (Reb E2), Reb A to Reb I, Rebaudi- oside E (Reb E) to Reb D, Reb E to Rebaudioside AM (Reb AM), Reb E2 to Reb I, Reb D toReb M and Reb AM to Reb M . In a particular embodiment, 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.

[0033] In bioinformatics, several methods have been developed to find and determine related polypeptide sequences. For example, percent sequence identity, position-specific scoring ma- trices (PSSMs), and hidden Markov models (HMMs) are all commonly employed to find se- quences that are similar to a given query sequence. Percent sequence identity calculates the number of amino acids that are shared between two sequences. Percent sequence identity is calculated in the context of a given alignment between two sequences. Percentage identity may 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, gap opening penalty is 6 bits, and gap extension is 1 bit. Clustal Omega uses the HHalign algorithm and its default settings as its core alignment engine. The algorithm is de- scribed in Sbding, J. (2005) 'Protein homology detection by HMM-HMM comparison’. Bioin- formatics 21, 951-960.

[0034] Position-specific scoring matrices (PSSMs) are a concise way to represent many related sequences. PSSMs are often generated using multiple sequence alignments. The sequence search tool PSI-BLAST generates PSSMs and uses them to search for related polypeptide se- quences. A PSSM used to score polypeptide sequences is a matrix (i.e. table) composed of 21 columns by N rows, where N is the length of the related sequences. Each row corresponds to a position within the polypeptide sequence and each column represents a different amino acid (or gap) that the residue position can take on. Each entry in the PSSM represents a score for the specific amino acid at the specific position within the polypeptide sequence. A sequence can be scored with a PSSM by first aligning the sequence to a reference sequence, and then calculating the following sum: SPSSM= X PSSM(i, aai'), where z is the sequence position and act, is the amino acid at position z. Related polypeptide sequences will all have high PSSM scores, while unrelated sequences will yield low scores.

[0035] In some embodiments, the beta- 1,3 -glycosyltransferase polypeptide is prepared by ex- -ression in a host microorganism. Suitable host microorganisms include, but are not limitedto, E. coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp. In a particular embodi- -ent, the glycosyltransferase is expressed in E. coll. In a particular embodiment, the glycosyl- transferase is expressed in Pichia pastoris.

[0036] The B13GT polypeptide can be provided in any suitable form, including free, immobi- lized or as a whole cell system. The degree of purity of the glycosyltransferase polypeptide may vary, e.g., it may be provided as a crude, semi -purified or purified enzyme preparation(s). In one embodiment, the glycosyltransferase polypeptide is free. In another embodiment, the glycosyltransferase polypeptide is immobilized to a solid support, for example on an inorganic or organic support. In some embodiments, the solid support is derivatized cellulose, glass, ce- ramic, methacrylate, styrene, acrylic, a metal oxide, or a membrane. In some embodiments, the glycosyltransferase polypeptide is immobilized to the solid support by covalent attachment, adsorption, cross-linking, entrapment, or encapsulation.

[0037] In yet another embodiment, the B13GT polypeptide is provided in the form of a whole cell system, for example as a living fermentative microbial cell, or as dead and stabilized mi- crobial cell, or in the form of a cell lysate.

[0038] The present disclosure provides a biocatalytic process for the preparation of a compo- sition comprising one or more target steviol glycosides from a starting composition comprising one or more substrate steviol glycosides, wherein the target steviol glycoside comprises steviol glycosides with one or more additional glucose units than the substrate steviol glycoside. The biocatalytic process comprises contacting an engineered B13GT, a sucrose synthase (SuSy) (unless ADP-glucose is directly supplied), and optionally a B12GT with a starting composition containing one or more steviol glycosides, a non-UDP nucleotide diphosphate, and sucrose. In another embodiment, the biocatalytic process comprises contacting an engineered B13GT, a SuSy, and optionally a B12GT with a starting composition containing one or more steviol gly- cosides, a non-UDP nucleotide diphosphate, and sucrose. In some embodiments, the method comprises contacting RA50, ADP, and sucrose with an engineered Bl,3 glycosyltransferase, a B12GT, and a SuSy to make Reb M. In another embodiment, the method comprises contacting RA60, ADP, and sucrose with an engineered B13GT, a B12GT, and a SuSy to make Reb M. In another embodiment, the method comprises contacting stevioside, ADP, and sucrose with an engineered Bl,3 glycosyltransferase, a B12GT, and a SuSy to make Reb M. In another embodiment, the method comprises contacting Reb A, ADP, and sucrose with an engineered B13GT, a B 12GT, and a SuSy to make Reb M. In another embodiment, the method comprises contacting RS90, ADP, and sucrose with an engineered B l 3GT, a B12GT, and a SuSy to makeReb M. In another embodiment, the method comprises contacting RS99, ADP, and sucrose with an engineered B13GT, B12GT, and a SuSy to make Reb M.

[0039] 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.

[0040] In one embodiment the sucrose synthase is any polypeptide with sucrose synthase ac- tivity. In another embodiment, the sucrose synthase is derived from an organism from the Bac- teria domain. In another embodiment, the sucrose synthase is derived from an organism from the Plantae kingdom. In another embodiment, the sucrose synthase is derived from an organism from the proteobacteria, deferribacteres, or cyanobacteria phylum. In another embodiment, the sucrose synthase is derived from the species Acidithiobacillus caldus, Nitrosomonas europaea, Denitrovibrio acetiphilus, Thermosynechococcus elongatus, Oryza saliva, Arabidopsis thali- ana, or Coffea arabica. In another embodiment, the sucrose synthase is an engineered sucrose synthase with 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 a sucrose synthase derived from the species Acidithiobacillus caldus, Nitrosomonas europaea, Denitrovibrio acetiphilus, Thermosynechococcus elongatus, Oryza sativa, Ara- bidopsis thaliana, or Coffea arabica.

[0041] In one embodiment the B12GT is any polypeptide that can glycosylate the C2’ of the 13-O-glucose and / or 19-O-glucose of a steviol glycoside substrate. In another embodiment, the B12GT is derived from an organism from the Plantae kingdom. In another embodiment, the B12GT is derived from an organism from the Angiosperm clade. In another embodiment, the 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 with 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 a B 12GT derived from the species Hordeum vulgare, Oryza sativa, Solanum lycopersicum, Lycium barbarum, Solanum tuberosum, or Stevia rebaudiana.

[0042] 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 sp., Aspergillus sp., Pichia sp., Bacillus sp. In a partic- ular embodiment, the glycosyltransferase and sucrose synthase are expressed in E. coli. hi an-other embodiment, the glycosyltransferase and sucrose synthase are expressed in Pichia pas- toris. In another embodiment, the B13GT, B12GT and / or sucrose synthase polypeptides are prepared by cell-free expression.

[0043] The B 13GT, B12GT and sucrose synthase polypeptides can be provided in any suitable form, including free, immobilized or as a whole cell system. The degree of purity of the poly- peptides may vary, e.g., they may be provided as a crude, semi-purified or purified enzyme preparation(s). In one embodiment, the B13GT, B12GT and / or SuSy polypeptide is free. In another embodiment, the B13GT, B12GT and / or SuSy polypeptide is immobilized to a solid support, for example on an inorganic or organic support. In some embodiments, the solid sup- port is derivatized cellulose, glass, ceramic, methacrylate, styrene, acrylic, a metal oxide, or a membrane. In some embodiments, the B13GT, B12GT and / or SuSy polypeptide is immobi- lized to the solid support by covalent attachment, adsorption, cross-linking, entrapment, or en- capsulation.

[0044] In yet another embodiment, the B13GT, B12GT and / or SuSy polypeptide is provided in the form of a whole cell system, for example as a living fermentative microbial cell, or as dead and stabilized microbial cell, or in the form of a cell lysate.

[0045] The steviol glycoside component(s) of the starting composition serve as a substrate(s) for the production of the target steviol glycoside(s), as described herein. The target steviol gly- cosides differ chemically from their corresponding substrate steviol glycoside(s) by the addi- tion of one or more glucose units.

[0046] The starting steviol glycoside composition can contain at least one substrate steviol glycoside. In one embodiment, the substrate steviol glycoside is selected from the group con- sisting of steviol, steviol- 13-O-glucoside, steviol- 19-O-glucoside, rubusoside, steviol- 1,2-bio- side, steviol-l,3-bioside, rubusoside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F, 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, an iso- mer thereof, a synthetic steviol glycoside 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.

[0047] The starting steviol glycoside composition may be synthetic or purified (partially or entirely), commercially available or prepared. One example of a starting composition useful inthe method of the present disc losure is an extract obtained from purification of Stevia rebaudi- ana plant material (e.g. leaves). Another example of a starting composition is a commercially available stevia extract brought into solution with a solvent. Yet another example of a starting composition is a commercially available mixture of steviol glycosides brought into solution with a solvent. Other suitable starting compositions include by-products of processes to isolate and purify steviol glycosides.

[0048] In one embodiment, the starting composition comprises a purified substrate steviol gly- coside. For example, the starting composition may comprise greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 85%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, or greater than 99.6% of one or more substrate steviol glycosides by weight on an anhydrous basis.

[0049] In another embodiment, the starting composition comprises a partially purified sub- strate steviol glycoside composition. For example, the starting composition contains 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%, of one or more substrate steviol glycosides by weight on an anhydrous basis.

[0050] In another embodiment, the substrate steviol glycoside is purified rebaudioside A, or isomers thereof. In a particular embodiment, the substrate steviol glycoside contains greater than 99% rebaudioside A, or isomers thereof, by weight on an anhydrous basis. In another embodiment, the substrate steviol glycoside comprises partially purified rebaudioside A. In a particular embodiment, the substrate steviol glycoside contains greater than 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% rebaudioside A by weight on an anhydrous basis.

[0051] In yet another embodiment, the substrate steviol glycoside comprises purified stevio- side, or isomers thereof. In a particular embodiment, the substrate steviol glycoside contains greater than 99% stevioside, or isomers thereof, by weight on an anhydrous basis. In another embodiment, the substrate steviol glycoside comprises partially purified stevioside. In a par- ---ular embodiment, the substrate steviol glycoside contains greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% stevioside by weight on an anhydrous basis.

[0052] In yet another embodiment, the substrate steviol glycoside is a combination of stevio- side and rebaudioside A. In a particular embodiment, the substrate steviol glycoside contains greater than 5% stevioside and greater than 5% Reb A, greater than 10% stevioside and greater than 10% Reb A, greater than 20% stevioside and greater than 20% Reb A, greater than 30%stevioside and greater than 30% Reb A, greater than 40% stevioside and greater than 40% Reb A, greater than 45% stevioside and greater than 45% Reb A, greater than 40% stevioside and greater than 50% Reb A, greater than 30% stevioside and greater than 60% Reb A, greater than 20% stevioside and greater than 70% Reb A, greater than 10% stevioside and greater than 80% Reb A, greater than 5% stevioside and greater than 90% Reb A, greater than 50% stevioside and greater than 40% Reb A, greater than 60% stevioside and greater than 30% Reb A, greater than 70% stevioside and greater than 20% Reb A, greater than 80% stevioside and greater than 10% Reb A, or greater than 90% stevioside and greater than 5% Reb A by weight on an anhydrous basis.

[0053] In still another embodiment, the substrate steviol glycoside is derived from stevia leaf extract. In one embodiment, RA50, stevia leaf extract purified to contain greater than 50% Reb A, is used as the steviol glycoside substrate. In one embodiment, RA50 is used at a concentration between 1 mg / ml and 800 mg / ml. hi another embodiment, RA50 is used at a concentration of 100 mg / ml. In another embodiment, RA60, stevia leaf extract purified to contain greater than 60% Reb A, is used as the steviol glycoside substrate, hi another embodiment, RA60 is used at a concentration between 1 mg / ml and 800 mg / ml. In another embodiment, RA60 is used at a concentration of 100 mg / ml. In another embodiment, RS90, stevia leaf extract purified to contain greater than 90% stevioside, is used as the steviol glycoside substrate. In another embodiment, RS99, stevia leaf extract purified to contain greater than 99% stevioside, is used as the steviol glycoside substrate. In another embodiment, RS90 is used at a concentration between 1 mg / ml and 800 mg / ml. In another embodiment, RS90 is used at a concentration of 100 mg / ml.

[0054] The reaction can be carried out with a nucleotide cofactor that can be converted to an 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 another embodiment, the nucleotide is ADP. In a particular embodiment, the reaction can be carried out with ADP at a concentration between 0.01 mM and 10 mM, such as, for example, between 0.01 mM and 0.05 mM, between 0.05 mM and 0.1 mM, between 0.1 mM and 0.5 mM, between 0.5 mM and 1 mM, between 1 mM and 5 mM, or between 5 mM and 10 mM. In a particular embodiment, ADP is used at a concentration of 0.5 mM.

[0055] The reaction can be carried out with a sucrose concentration between 10 mM and 2M, such as, for example, greater than 10 mM, greater than 50 mM, greater than 100 mM, greaterthan 250 mM, greater than 500 mM, greater than 1 M, greater than 1.5 M and greater than 2 M. In a particular embodiment, sucrose is used at a concentration of 450 mM.

[0056] In one embodiment, the reaction is run at any temperature. In another embodiment, the reaction is run at a temperature between 10 °C and 80 °C. Such as, for example, between 10° C to 20° C, between 20° C to 30° C, between 30° C to 40° C, between 40° C to 50° C, between 50° C to 60° C, between 60° C to 70° C, between 70° C to 80° C or 80° C. In a particular embodiment, the one-pot reaction is carried out at 60 °C.

[0057] The reaction medium for conversion is generally aqueous, e.g., purified water, buffer, or a combination thereof. In a particular embodiment, the reaction medium is a buffer. Suitable buffers include, but are not limited to, acetate buffer, citrate buffer, HEPES, and phosphate buffer, hi a particular embodiment, the reaction medium is phosphate buffer. The reaction me- dium can have a pH between 4 and 10. In a particular embodiment, the reaction medium has a pH of 6. The reaction medium can also be, alternatively, an organic solvent.

[0058] The step of contacting the starting composition with the glycosyltransferase and sucrose synthase polypeptides can be carried out in a duration of time between 1 hour and 1 week, such as, for example, between 30 minutes and 1 hours, between 1 hour and 4 hours, between 4 hours and 6 hours, between 6 hours and 12 hours, between 12 hours and 24 hours, between 1 day and 2 days, between 2 days and 3 days, 3 days and 4 days, between 4 days and 5 days, between 6 days and 7 days. In a particular embodiment, the reaction is carried out for 6 hours.

[0059] The reaction can be monitored by suitable methods including, but not limited to, high- performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LCMS), thin layer chromatography (TEC), infrared spectrometry (IR), or nuclear magnetic resonance (NMR).

[0060] 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 -bi- oside, steviol-l,3-bioside, rubusoside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F, 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, a re- baudioside with 7 covalently attached glucose units (e.g. rebaudioside M plus 1 glucose unit), a synthetic steviol glycoside, an isomer thereof, and / or a steviol g-ycoside composition. In an- other embodiment, the target steviol glycoside is rebaudioside I, or isomers thereof. In another embodiment, the target steviol glycoside is rebaudioside M, or isomers thereof. In another em-bodiment, the target steviol glycoside is rebaudioside AM, or isomers thereof. In another em- bodiment, the target steviol glycoside is rebaudioside E2, or isomers thereof. In still another embodiment, the target steviol glycosides are Reb I, Reb AM, Reb D and Reb M.

[0061] In one embodiment, the conversion of the starting steviol glycoside composition to Reb I is at least 2% complete, as determined by any of the methods mentioned above. In a particular embodiment, the conversion of the starting steviol glycoside composition to Reb I is at least 10% complete, at least 20% complete, at least 30% complete, at least 40% complete, at least 50% complete, at least 60% complete, at least 70% complete, at least 80% complete or at least 90% complete. In a particular embodiment, the conversion of starting steviol glycoside composition to Reb I is at least 95% complete. In some embodiments, wherein at least 5%, 10%, 20%, 30? / o, 40%, 50%, 60%, 70%, 80%, or 90% of the starting steviol glycoside composition is converted to Reb I.

[0062] In one embodiment, the conversion of the starting steviol glycoside composition to Reb M is at least 2% complete, as determined by any of the methods mentioned above. In a partic- ular embodiment, the conversion of the starting steviol glycoside composition to Reb M is at least 10% complete, at least 20% complete, at least 30% complete, at least 40% complete, at least 50% complete, at least 60% complete, at least 70% complete, at least 80% complete or at least 90% complete. In a particular embodiment, the conversion of the starting steviol glycoside composition to Reb M is at least 95% complete. In some embodiments, wherein at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80? / o, or 90% of the starting steviol glycoside composition is converted to Reb M.

[0063] In one embodiment, the conversion of the starting steviol glycoside composition to Reb AM is at least 2% complete, as determined by any of the methods mentioned above. In a par- ticular embodiment, the conversion of the starting steviol glycoside composition to Reb AM is at least 10% complete, at least 20% complete, at least 30% complete, at least 40% complete, at least 50% complete, at least 60% complete, at least 70% complete, at least 80% complete or at least 90% complete. In a particular embodiment, the conversion of the starting steviol glycoside composition to Reb AM is at least 95% complete. In some embodiments, wherein at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80? / o, or 90% of the starting steviol glycoside compo- sition is converted to Reb AM.

[0064] In one embodiment, the conversion of the starting steviol glycoside composition to Reb E2 is at least 2% complete, as determined by any of the methods mentioned above. In a partic- ular embodiment, the conversion of the starting steviol glycoside composition to Reb E2 is at least 10% complete, at least 20% complete, at least 30% complete, at least 40% complete, atleast 50% complete, at least 60% complete, at least 70% complete, at least 80% complete or at least 90% complete. In a particular embodiment, the conversion of the starting steviol glycoside composition to reb E2 is at least 95% complete. In some embodiments, wherein at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the starting steviol glycoside compo- sition is converted to Reb E2.

[0065] In one embodiment, the conversion of the starting 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 mentioned above. In a particular embodiment, the conversion of the starting steviol glycoside composition to Reb I, Reb AM, Reb D and Reb M is at least 10% complete, at least 20% complete, at least 30% complete, at least 40% complete, at least 50% complete, at least 60% complete, at least 70% complete, at least 80% complete or at least 90% complete. In a particular embodiment, the conversion of the starting steviol glycoside composition to Reb E2 is at least 95% complete. In some embodiments, wherein at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the starting steviol glycoside composition is converted to Reb I, Reb AM, Reb D and Reb M.

[0066] The target steviol glycoside(s) can be in any polymorphic or amorphous form, including hydrates, solvates, anhydrous or combinations thereof.

[0067] Optionally, the method of the present disclosure further comprises separating the target steviol glycoside from the target composition. The target steviol glycoside(s) can be separated by any suitable method, such as, for example, crystallization, separation by membranes, cen- trifugation, extraction, chromatographic separation or a combination of such methods.

[0068] In one embodiment, the separation of target steviol glycosides produces a composition comprising greater than 80% by weight of the target steviol glycoside(s) on an anhydrous basis, i.e., a highly purified steviol glycoside composition, hi another embodiment, separation produces a composition comprising 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%, greater than 50%, greater than 60%, greater than 70%, greater than80%, greater than 85%, greater than 90%, greater than 91%, greater than 92%, greater than93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than98%, greater than 99%, or greater than 99.6% by weight of the target steviol glycosides. In particular embodiments, the composition comprises greater than 95% by weight of the target steviol glycoside(s) on an anhydrous basis.

[0069] Purified target steviol glycosides can be used in consumable products as a sweetener. Suitable consumer products include, but are not limited to, food, beverages, pharmaceuticalcompositions, tobacco products, nutraceutical compositions, oral hygiene compositions, and cosmetic compositions.

[0070] Plasmids containing nucleic acids encoding enzymes having SEQ ID NOs: 2-517 are described in Table 1 below. Table 1. Designed B13GTsEXAMPLESExample 1: Computational Design of ADP-GIucose Dependent B13GTs

[0071] Computational designs were conducted to improve and convert the native Bl 3GT from Stevia rebaudiana (Seq ID NO: 1) to an ADP-glycosyltransferase. Structural models of a B13GT variant of SEQ ID NO: 1 were generated and used as the starting point for computational designs. Visual inspection of the structural models identified residues H24 as the catalytic histidine, DI 23 which activates the histidine, and two sugar binding residues D379 and Q38O (numbering relative to SEQ ID NO: 2). Computational designs incorporating coevolu- tionary information were conducted to improve the B13GT. 53 of these computational designs were chosen for experimental validation (SEQ ID NOs: 2-54).

[0072] Polynucleotides encoding the amino acid sequences for the designed B 13GTs were synthesized (Twist Bioscience) and inserted into the pARZ4 expression vector. The polynucleotides encoded the B13GT enzymes with an N-terminal His tag. Polynucleotides were either ordered as full-length genes or ordered as gene fragments and then assembled using Gibson assembly. The recombinant vectors were used in a heat shock method to transform E. coli HMS174(DE3) (Novagen) or T7 Express lysY / Iq (New England BioLabs), thereby preparing a recombinant microorganism.

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

[0074] The microorganisms were dissolved in a lysis buffer (lysozyme, DNAsel, Bugbuster, 300ml 20 mM HEPES pH 7.5, 500mM NaCl, and 20mM Imidazole). Two to three glass beads were added to each well and were disrupted by shaking at 25° C and 220 rpm for 30 minutes. The disrupted liquid was centrifuged at 2200 x g for 6-10 minutes. The obtained supernatant was loaded onto a Ni-NTA plate and shaken for 10 minutes at room temperature. The plate was centrifuged for 4 minutes at 100 x g followed by two washes of 500 pl binding buffer (300 ml 20mM HEPES pH 7.5, 500mM NaCl, 20mM Imidazole) and two-minute centrifugation (500 x g). The proteins were eluted with 150 pl elution buffer (15 ml 20mM HEPES pH 7.5, 500mM NaCl, 500mM Imidazole) and shaken for 1 minute at 0.25 maximum shaking speed followed by centrifugation for 2 minutes at 500 x g. The recovered protein was desalted into a buffer solution for enzyme activity evaluation (50mM HEPES pH 7.5, 50mM NaCl).

[0075] Each B13GT variant was assayed in a one-pot reaction with a B12GT and SuSy enzyme. B12GT and SuSy were used in excess such that in the absence of the B13GT variant, the starting RA50 feed was fully converted to RebD / E. The purified B 13GT, B 12GT 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 for 24 hours at 60 °C.

[0076] Steviol glycosides produced and consumed during the reaction, stevioside, Reb A, Reb D, Reb E, Reb I, Reb M and other minor steviol glycosides, were monitored by LCMS using an Agilent 6470 QQQ mass spectrometer (column: Waters ACQUITY UPLC HSS T3 Column, 100 mm x 2.1 mm). The QQQ was run with multi reaction monitoring (MS / MS) to accurately quantitate steviol glycosides of interest. Several designed enzymes expressed well and were able to efficiently produce Reb M (Table 2).Table 2. Purified Protein Concentration and Reb M Conversion of Successful B13GT De- sign Variants Expressed in E.coliExample 2: Pichia pastoris expression of designed B13GTs and SuSys

[0077] Polynucleotides of the top ten designed Bl 3GTs from Example 1 optimized for Pichia pastoris expression were synthesized (Twist Bioscience) and inserted into a Pichia shuttle vec- tor. The polynucleotides encoded the B13GT enzymes with a C-terminal His tag instead of the N-terminal His tag used in Example 1. Two unique polynucleotides for each B13GT amino acid sequence were ordered. The vectors were transformed into a commercially available Pichia pastoris strain (ATCC). The transformed microorganisms were grown in BMGY (buffered glycerol complex) media and protein expression was induced by feeding of methanol. The 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 into desalt buffer (20mM KPO4 pH6, 50mM NaCl). The designed B13GTs were solubly expressed (FIG. 3) and enzyme activity was measured, similar to Example 1 (Table 3).

[0078] Polynucleotides encoding the top performing B13GTs were amplified from the Pichia shuttle vectors and integrated into the Pichia genome. Four integrated Pichia strains expressing designed B13GTs were grown in IL fermentors. The Pichia microorganisms were grown with glycerol as the main carbon source for ~24 hours, and then were fed methanol for -72 hours to express the desired Bl 3GT. The cells were collected and lysed by French press. The expressed protein was purified by immobilized metal affinity chromatography (IMAC) and dialyzed into desalt buffer (20mM KPO4 pH6, 50mM NaCl). All four strains successfully expressed the active B13GT in the fermentations.Table 3. Purified Protein Concentration and Reb M Conversion of Top B13GT Designs Expressed in Pichia -Example 3: Computational Designs of Improved B13GTs

[0079] A previously-developed protein sequence-based deep learning model was fine-tuned with the results from Example 1. The model was used to design improved B13GT enzymes. Eighty of these computational designs were chosen for experimental validation (SEQ ID NOs : 55-134). Polynucleotides encoding the B13GT enzymes with a C-terminal His tag were syn- thesized (Twist Bioscience). E. coli strains expressing the designed B13GTs were created as described in Example 1. The designed B13GT enzymes were expressed and purified as de- scribed in Example 1.

[0080] Each B13GT variant was assayed in a one-pot reaction with a B12GT and SuSy en- zyme. B12GT and SuSy were used in excess such that in the absence of the B13GT variant, the starting RA50 feed was fully converted to RebD / E. The purified B 13GT, 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 for 24 hours at 60 °C. Steviol glycosides produced and consumed during the reaction were measured as described in Example 1. The designed en- zymes solubly expressed and were able to efficiently produce Reb M (Table 4).Table 4. Purified Protein Concentration and Reb M Conversion o f Top B13GT Design Variants Expressed in E. coli

[0081] Structural models of the top B13GT designs were generated and analyzed. To this end, a structure-based deep learning method trained to predict protein sequences for a given protein backbone was used to design further improved B13GT enzymes. 121 of these computational designs were chosen for experimental validation (SEQ ID NOs: 135-255). The machine learn-ing model was retrained on crystal structures and structural models of proteins from thermo- philic organisms. An additional 69 B13GT enzymes were selected for experimental validation (SEQ ID NOs: 256-324). Polynucleotides encoding the B13GT enzymes (SEQ ID NOs: 135- 324) with a C-terminal His tag were synthesized (Twist Bioscience). E. coli strains expressing the designed B13GTs were created as described in Example 1. The designed Bl 3GT enzymes were expressed and purified as described in Example 1.

[0082] Each B13GT variant was assayed in a one-pot reaction with a B12GT and SuSy en- zyme. B12GT and SuSy were used in excess such that in the absence of the B13GT variant, the starting RA50 feed was fully converted to RebD / E. The 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 for 24 hours at 60 °C. Steviol glycosides produced and consumed during the reaction were measured as described in Example 1. The designed en- zymes solubly expressed and were able to efficiently produce Reb M (Table 5). Table 5. Purified Protein Concentration and Reb M Conversion of Top B13GT Design Variants Expressed in E. coli - -Example 4: Computational Designs of Further Improved B13GTs

[0083] 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 improved B13GT en- zymes. One hundred of these computational designs were chosen for experimental validation (SEQ ID NOs: 325-424). Polynucleotides encoding the B13GT enzymes (SEQ ID NOs: 325- 424) with a C-terminal His tag were synthesized (Twist Bioscience). E. coli strains expressing the designed B13GTs were created as described in Example 1. The designed B13GT enzymes were expressed and purified as described in Example 1.

[0084] Each B13GT variant was assayed in a one-pot reaction with a B12GT and SuSy en- zyme. B12GT and SuSy were used in excess such that in the absence of the B13GT variant, the starting RA50 feed was fully converted to RebD / E. The purified Bl 3GT, 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 for 24 hours at 60 °C. Steviol glycosides produced and consumed during the reaction were measured as described in Example 1. The designed enzymes solubly expressed and were able to efficiently produce Reb M (Table 6).Table 6. Purified Protein Concentration and Reb M Conversion of Top B13GT Design Varian ts Expressed in E. coli

[0085] Structural models of the top B 13GT designs from Example 3 were generated and ana- lyzed. The structure-based deep learning method trained to predict protein sequences for a given protein backbone was used to generate further improved B13GT enzymes. The method was used to design improved Bl 3GT enzymes. 93 of these computational designs were chosen for experimental validation (SEQ ID NOs: 425-517). Polynucleotides encoding the B13GT enzymes with a C-terminal His tag were synthesized (Twist Bioscience). E. coll strains ex- -ressing the designed B13GTs were created as described in Example 1. The designed Bl 3GT enzymes were expressed and purified as described in Example 1.

[0086] Each B13GT variant was assayed in a one-pot reaction with a B12GT and SuSy en- zyme. B12GT and SuSy were used in excess such that in the absence of the B13GT variant, the starting RA50 feed was fully converted to RebD / E. The 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 for 24 hours at 60 °C. Steviol glycosides produced and consumed during tire reaction were measured as described in Example 1. The designed enzymes solubly expressed and were able to efficiently produce Reb M (Table 7).Table 7. Purified Protein Concentration and Reb M Conversion of Top B13GT Design Variants Expressed in E. coli -Example 5: Representing Successful B13GTs Designs with a PSSM

[0087] The successful B13GT designs from Example 1 were used to generate a PSSM (Table 8). The PSSM is a concise way to represent the successful designs and related sequences. Se- quences that have a PSSM score greater than 332 are considered related to the active B13GT computational designs described herein. To score a sequence with the PSSM, it must first be aligned with the representative sequence, SEQ ID NO: 2. For example, the following successful designs, AA29162, AA29163, AA29161, AA29165, have the following PSSM scores: 332.2, 335.5, 332.5, 337.3, while the wild-type B13GT SEQ ID NO: 1, has a PSSM score of only 326.7.

[0088] The successfol B13GT designs from Example 3 were used to generate a PSSM (Table 9). The PSSM is a concise way to represent successfol designs and related sequences. Se- quences that have a PSSM score greater than 54.5 are considered related to the active B13GT computational designs described in herein. To score a sequence with the PSSM, it must first be aligned with the representative sequence, SEQ ID NO: 2. For example, the following successful designs, AA40232, AA40239, AA40205, AA40243, have the following PSSM scores: 70.74, 80.53, 78.75, 76.08, while the wild-type B13GT SEQ ID NO: 1, has a PSSM score of only 51.16.

[0089] The successful B13GT designs from Example 4 were used to generate a PSSM (Table 10). The PSSM is a concise way to represent successful designs and related sequences. Se- quences that have a PSSM score greater than 58.3 are considered related to the active B13GT computational designs described herein. To score a sequence with the PSSM, it must first be aligned with the representative sequence, SEQ ID NO: 2. For example, the following successful designs, AA41333, AA41342, AA41345, AA41346, have the following PSSM scores: 90.04, 92.68, 89.16, 97.08, while the wild-type B13GT SEQ ID NO: 1, has a PSSM score of only51.16.Table 8. Position Specific Scoring Matrix (PSSM) of Successful B13GT DesignsTable 9. Position Specific Scoring Matrix (PSSM) of Successful B13GT DesignsTable 10. Position Specific Scoring Matrix (PSSM) of Successful B13GT DesignsExample 6: E. coli Fermentation of Designed B13GTs

[0090] E. coli strains that control B13GT expression with a constitutive promoter were made for the top B13GTs from Example 3. The E. coli strains were grown in 1 liter fermenters with a glycerol feed for 48 hours. The fermentations of strains expressing AA37959, AA40205, AA40232, and AA40243 yielded 171-, 60-, 182-, and 190-grams wet cell mass per liter fermentation. Twenty to thirty grams of cells from each fermentation were resuspended and lysed with a homogenizer. All lysates were tested and confirmed to have B13GT activity. The clarified lysate was purified using IMAC. The resulting purified protein samples of AA37959, AA40205, AA40232, and AA40243 had protein yields of 0.47, 0.15, 1.20 and 1.42 mg / ml, respectively.INCORPORATION BY REFERENCE

[0091] All references, articles, publications, patents, patent publications, and patent applica- tions cited herein are incorporated by reference in their entireties for all purposes. However, mention of any reference, article, publication, patent, patent publication, and patent applicationcited herein is not, and should not be taken as an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any coun- try in the world.

Claims

CLAIMS:

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

3. An engineered beta-l,3-glycosyltransferase polypeptide that has a score greater than 332 when scored by the PSSM shown in Table 8, wherein the engineered beta- 1,3- glycosyltransferase polypeptide has been aligned to SEQ ID NO: 2 prior to scoring.

4. An engineered beta- 1 ,3-glycosyltransferase polypeptide that has a score greater than 54.5 when scored by the PSSM shown in Table 9, wherein the engineered beta-1,3- glycosyltransferase polypeptide has been aligned to SEQ ID NO: 2 prior to scoring.

5. An engineered beta-l,3-glycosyltransferase polypeptide that has a score greater than 58.3 when scored by the PSSM shown in Table 10, wherein the engineered beta-1, 3- glycosyltransferase polypeptide has been aligned to SEQ ID NO: 2 prior to scoring.

6. A polypeptide having the sequence of:wherein residue 5 is N or S wherein residue 6 is P or Twherein residue 8 is T or V wherein residue 9 is R or V wherein residue 1 1 is R or D or E or H or K or S or T wherein residue 12 is R or D or L or K wherein residue 13 is R or H or T or W wherein residue 14 is I or V wherein residue 15 is A or I or V wherein residue 16 is I or L or M wherein residue 25 is I or L or W or V wherein residue 34 is L or V wherein residue 37 is R or S wherein residue 38 is R or K wherein residue 41 is A or R or N or D or Q or E or K or S wherein residue 43 is I or T or V wherein residue 45 is I or L or F wherein residue 46 is H or Y wherein residue 47 is M or T wherein residue 48 is A or R or N or Q or E or K or S or T wherein residue 51 is A or R or L or K wherein residue 56 is N or L wherein residue 61 is Q or E or T wherein residue 63 is R or E or I or L or K or V wherein residue 64 is R or N or D or Q or E or L or F or P or S or T or Y wherein residue 65 is I or L or F or T or V wherein residue 66 is I or L wherein residue 68 is R or N or E wherein residue 69 is D or E wherein residue 72 is D or P or T wherein residue 73 is D or E wherein residue 74 is W or Y wherein residue 81 is H or T wherein residue 85 is A or V wherein residue 91 is Q or I or L or V wherein residue 94 is Q or E or Kwherein residue 97 is A or K wherein residue 98 is A or D or E or K or P wherein residue 99 is A or R or Q or E or K or P or T or V wherein residue 100 is L or F wherein residue 101 is R or E or L or K or Y wherein residue 102 is A or R or D or Q or E or K wherein residue 103 is A or E or I or L or K or M or T or V wherein residue 105 is R or Q or E or L or K or S wherein residue 106 is A or R or N or D or Q or E or I or L or K or S wherein residue 107 is A or C or E or 1 or L or M or F or Y or V wherein residue 108 is I or L or K or M wherein residue 109 is A or R or N or D or Q or E or L or K or S wherein residue 110 is A or N or D or E or S wherein residue 1 1 1 is S or T wherein residue 112 is A or E or G or K or P wherein residue 113 is D or E or K or S wherein residue 115 is E or V wherein residue 1 16 is R or Q or E or K or P or T or V wherein residue 117 is I or L or V wherein residue 118 is A or R or S wherein residue 120 is L or F or V wherein residue 122 is A or T wherein residue 129 is A or T wherein residue 130 is A or N or Q or E or G or L or K or M or T or Y wherein residue 131 is D or E or L or P or S wherein residue 133 is A or T wherein residue 134 is R or D or Q or E or K wherein residue 135 is R or E or K or S wherein residue 137 is N or G wherein residue 138 is I or L wherein residue 139 is R or K or P wherein residue 140 is R or L or P wherein residue 144 is Q or M wherein residue 146 is G or Swherein residue 152 is H or L wherein residue 153 is A or C wherein residue 155 is A or V wherein residue 156 is C or S wherein residue 159 is A or R or D or Q or E or H or L or K wherein residue 161 is R or D or E or I or L wherein residue 162 is A or R or D or Q or E or K wherein residue 163 is A or R or D or Q or L or K wherein residue 165 is I or L or W or Y wherein residue 167 is N or D wherein residue 168 is L or P wherein residue 169 is A or N or D or E or S or T wherein residue 170 is N or D or S or T wherein residue 171 is N or Q or K or P or S or T wherein residue 172 is R or E or El or L or K or S or T or W wherein residue 173 is A or R or N or D or G or K or P wherein residue 174 is D or E or L or P wherein residue 175 is D or E wherein residue 176 is A or D or Q or E or K or T wherein residue 177 is Q or P wherein residue 178 is A or V wherein residue 179 is E or K. or P or S or V wherein residue 181 is A or L or F or Y wherein residue 183 is R or Q or E or H or I or L or M or F or W or Y wherein residue 185 is R or L or K or T wherein residue 186 is A or R or N or W or V wherein residue 187 is A or R or G or K wherein residue 190 is R or L or K wherein residue 191 is R or K or S wherein residue 196 is D or I or K or S or W wherein residue 197 is D or Q wherein residue 199 is A or S wherein residue 204 is D or E or G or M or S wherein residue 207 is I or T or Vwherein residue 208 is E or K or T wherein residue 209 is A or Q or E or M or T wherein residue 21 1 is R or K wherein residue 212 is A or R or N or Q or E or L or K wherein residue 213 is A or S wherein residue 216 is I or V wherein residue 221 is A or F wherein residue 232 is I or V wherein residue 233 is R or Q or I or W wherein residue 243 is I or L wherein residue 249 is L or F or Y wherein residue 250 is R or T wherein residue 254 is S or T wherein residue 259 is E or T wherein residue 261 is R or P wherein residue 262 is S or T wherein residue 263 is A or F or T or V wherein residue 264 is A or F wherein residue 265 is A or D or Q or E or P wherein residue 268 is A or D wherein residue 269 is R or Q or G or K or T wherein residue 271 is A or P wherein residue 272 is A or D or E or K or P wherein residue 273 is R or N or E or G or K or S wherein residue 276 is L or V wherein residue 279 is G or S wherein residue 285 is Q or E or S wherein residue 286 is I or L or M or F or V wherein residue 287 is D or E or S or T wherein residue 288 is A or R or E or L or K or P or V wherein residue 289 is A or D or E or K wherein residue 290 is D or Q or E or T wherein residue 291 is A or F wherein residue 292 is L or Kwherein residue 293 is A or R or N or E or K or T wherein residue 294 is I or L wherein residue 295 is A or L or M or V wherein residue 296 is R or D or Q or E or H or K wherein residue 299 is R or I or K or V wherein residue 300 is A or R or D or E or L or K wherein residue 302 is E or G or K wherein residue 303 is Q or E or H or I or L or K or V wherein residue 304 is R or N or K or P or S wherein residue 305 is A or F or T or V wherein residue 314 is I or V wherein residue 317 is A or R or Q or E or FI or L or K or S or Y wherein residue 318 is A or R or D or E or K or S or T wherein residue 320 is I or L or T or V wherein residue 321 is D or E or S wherein residue 322 is A or D or I or L or F or P or S or V wherein residue 323 is I or L wherein residue 325 is D or E or P wherein residue 327 is R or L or K or F or Y wherein residue 328 is G or L or M or F wherein residue 330 is D or E or P wherein residue 331 is A or R or N or Q or L or K. wherein residue 332 is G or I wherein residue 334 is I or V wherein residue 338 is A or C wherein residue 347 is R or E or G or K or P wherein residue 362 is L or M or V wherein residue 364 is A or S wherein residue 373 is C or F wherein residue 375 is D or P wherein residue 385 is A or R or T or V wherein residue 388 is A or S or T or V wherein residue 389 is R or N or D or E or K wherein residue 390 is E or I or K or Vwherein residue 391 is R or L or K wherein residue 392 is R or N or E or G or H or K wherein residue 393 is I or V wherein residue 395 is I or V wherein residue 398 is R or D or E wherein residue 399 is R or N or D or E or K wherein residue 401 is F or W wherein residue 402 is R or N or D or Q or E or I or K or V wherein residue 403 is A or R or E or K or P or T or V wherein residue 404 is A or R or D or Q or E or G or K. wherein residue 405 is A or R or N or D or E or K wherein residue 406 is I or V wherein residue 407 is A or I or V wherein residue 408 is A or R or N or D or E or K wherein residue 410 is A or I or L or V wherein residue 411 is R or N or Q or E or H or K wherein residue 412 is R or E or H or L or K or T or Y wherein residue 413 is I or V wherein residue 414 is L or M wherein residue 415 is R or Q or E or L or K or T or V wherein residue 416 is A or D or E or S wherein residue 417 is A or E or K or P wherein residue 418 is A or R or E or K or F or S wherein residue 420 is A or R or Q or E or L or K wherein residue 421 is A or E or G or El or K or P or Y or V wherein residue 422 is I or M or Y wherein residue 423 is A or R or K wherein residue 424 is A or R or N or D or Q or E or K wherein residue 425 is R or N wherein residue 427 is A or R or Q or E or L or K wherein residue 428 is A or R or E or L or K or V wherein residue 430 is A or K wherein residue 431 is A or R or D or Q or E wherein residue 433 is A or Lwherein residue 435 is R or G or V wherein residue 437 is I or L wherein residue 438 is A or M wherein residue 439 is K or P wherein residue 449 is R or S wherein residue 451 is Q or V (SEQ ID NO: 518). The polypeptide of any one of claims 1-6, wherein the polypeptide is expressed in a host microorganism. The polypeptide of claim 7, wherein the host microorganism is E. coli, Saccharomyces sp., Aspergillus sp., Pichia sp., or Bacillus sp. The polypeptide of any one of claims 1 -8, wherein the polypeptide is immobilized to a solid support. . The polypeptide of claim 9, wherein the solid support is derivatized from cellulose, glass, ceramic, methacrylate, styrene, acrylic, a metal oxide, or a membrane. . The polypeptide of claim 9 or 10, wherein the polypeptide is immobilized to the solid support by covalent attachment, adsorption, cross-linking, entrapment, or encapsulation. . A method for transferring a sugar moiety to a substrate steviol glycoside, the method comprising contacting a beta-l,3-glycosyltransferase polypeptide, a sucrose synthase and optionally a beta-l,2-glycosyltransferase polypeptide with a starting composition comprising one or more steviol glycosides, a nucleotide diphosphate, and sucrose. . The method of claim 12, wherein the beta- 1,3 -glycosyl transferase polypeptide is an engi- neered beta- 1,3 -glycosyltransferase polypeptide that comprises 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 se- quence selected from the group consisting of SEQ ID NOs: 4-517, 2, and 3.The method of claim 12, wherein the sucrose synthase is an engineered sucrose synthase with 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 a sucrose synthase derived from the species Acidithiobacillus caldus, Nitrosomonas europaea, Denitrovibrio acetiphilus, Thermosynechococcus elongatus, Oryza saliva, Arabidopsis thaliana, or Coffea arabica. The method of claim 12, wherein the beta-l,2-glycosyltransferase polypeptide is an engineered beta- 1,2-glycosyl transferase polypeptide with 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 a beta-1, 2- glycosyltransferase polypeptide derived from the species Hordeum vulgare, Oryza sativa, Solanum lycopersicum, Lycium barbarum, Solanum tuberosum, or Stevia rebaudiana. The method of claim 12, wherein(a) the beta- 1,3 -glycosyltransferase polypeptide is an engineered beta-l,3-glycosyl- transferase polypeptide that comprises 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.(b) the sucrose synthase is an engineered sucrose synthase with 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 a sucrose synthase derived from the species Acidithiobacillus caldus, Nitrosomonas europaea, Denitrovibrio acetiphilus, Thermosynechococcus elongatus, Oryza sativa, Arabidopsis thaliana, or Coffea arabica.(c) the beta-l,2-glycosyltransferase polypeptide is an engineered beta- 1,2- glycosyltransferase polypeptide with 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 a beta- 1,2- glycosyltransferase polypeptide derived from the species Hordeum vulgare, Oryza sativa, Solanum lycopersicum, Lycium barbarum, Solanum tuberosum, or Stevia rebaudiana.The method of claim 12, wherein the beta-l,3-glycosyltransferase polypeptide is an engineered beta- 1 ,3-glycosyltransferase polypeptide that has a score greater than 332 when scored by the PSSM shown in Table 8, wherein the engineered beta- 1 ,3- glycosyltransferase polypeptide has been aligned to SEQ ID NO: 2 prior to scoring. The method of claim 12, wherein the beta-l ,3-glycosyltransferase polypeptide is an engineered beta- 1 ,3-glycosyltransferase polypeptide that has a score greater than 54.5 when scored by the PSSM shown in Table 9, wherein the engineered beta- 1,3- glycosyltransferase polypeptide has been aligned to SEQ ID NO: 2 prior to scoring. The method of claim 12, wherein the beta- 1,3 -glycosyltransferase polypeptide is an engineered beta-l,3-glycosyltransferase polypeptide that has a score greater than 58.3 when scored by the PSSM shown in Table 10, wherein the engineered beta-1, 3- glycosyltransferase polypeptide has been aligned to SEQ ID NO: 2 prior to scoring. The method of any one of claims 12 to 19, wherein the substrate steviol glycoside comprises at least one steviol glycoside selected from the group consisting of 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 E2, rebaudioside AM, rebaudioside N, rebaudioside O, rebaudioside Q, an isomer thereof, and a synthetic steviol glycoside. The method of claim 20, wherein the substrate steviol glycoside comprises a mixture of stevioside and rebaudioside A. The method of claim 20, wherein the substrate steviol glycoside comprises stevioside. 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. The method of any one of claims 12 to 23, further comprising producing a target steviol glycoside, wherein the target steviol glycoside comprises at least one steviol glycosideselected from the group consisting of steviol, steviol-13-O-glucoside, steviol- 19-0- 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 E2, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside AM, rebaudioside M, rebaudioside D, rebaudioside N, rebaudioside O, rebaudioside Q, an isomer thereof, and a synthetic steviol glycoside. 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. 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. The method of claim 24, wherein the target steviol glycoside comprises rebaudioside M. The method of claim 24, wherein the target steviol glycoside comprises rebaudioside AM. The method of any 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. The method of any 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. The method of any of claims 12 to 28, wherein the nucleotide diphosphate comprises adenosine diphosphate. The method of any of claims 12 to 31 , wherein the starting composition comprises greater than about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about20%, 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%, about 99.6% of steviol glycoside by weight on an anhydrous basis. The method of any of claims 12 to 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%, about 99.6% of the target steviol glycoside by weight on an anhydrous basis. A polynucleotide encoding a polypeptide of any of claims 1 to 6. A host microorganism heterologously expressing a polynucleotide of claim 34. A microorganism of claim 35 where the host microorganism is E. coli, Saccharomyces sp., Aspergillus sp., Pichia sp., or Bacillus sp.. A method for transferring a sugar moiety to a substrate steviol glycoside, the method comprising contacting a beta-l,3-glycosyltransferase polypeptide and a sucrose synthase with one or more steviol glycosides, a nucleotide diphosphate, and sucrose. 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, and wherein the position numbers are based on SEQ ID NO:

2. An engineered beta- 1,3 -glycosyltransferase polypeptide that comprises 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, and wherein the polypeptide comprises H at position 24, D at position 123, D at position 379, and Q at position 380.An engineered beta-l,3-glycosyltransferase polypeptide that comprises 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, and wherein the polypeptide comprises H at position 24, D at position 123, D at position 379, and Q at position 380. An engineered beta-l ,3-glycosyltransferase polypeptide that comprises 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, and wherein the polypeptide comprises H at position 24, D at position 123, D at position 379, and Q at position 380. An engineered beta-l,3-glycosyltransferase polypeptide that comprises 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, and wherein the polypeptide comprises H at position 24, D at position 123, D at position 379, and Q at position 380. An engineered beta-l,3-glycosyltransferase polypeptide that comprises 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, and wherein the polypeptide comprises H at position 24, D at position 123, D at position 379, and Q at position 380.

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