Compositions and methods for producing rebaudioside M

Abstract

The present disclosure provides enzymes and methods of using those enzymes to transfer sugar moieties to the substrate steviol glycosides. The designed beta-1, 3-glycosyltransferase can be used for adding a glucose monomer connected with beta-1, 3 to steviol glycosides. In particular, the designed beta-1, 3-glycosyltransferase is used in a one-pot reaction together with a beta-1, 2-glycosyltransferase and a sucrose synthase to convert stevioside and / or Reb A to Reb M.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 374,461, filed on September 2, 2022, the contents of which are incorporated herein by reference in their entirety.

[0003] Reference to an electronic sequence listing

[0004] 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. Technical Field

[0005] The present disclosure relates to enzymes and biocatalytic methods for producing steviol glycosides. The present disclosure specifically relates to the use of glycosyltransferases that can transfer glucose moieties from ADP-glucose sugar donors to steviol glycosides. Background Art

[0006] Excess sugar consumption has been linked to health epidemics worldwide, including diabetes and heart disease. Healthcare systems incur high costs from treating these illnesses. Replacing added sugar in food products with low-calorie, high-intensity sweeteners would have a significant impact on health and the economy.

[0007] Stevia (Stevia rebaudiana) species is widely planted for its sweet leaves and has been used as a sweetener traditionally. The sweetness of stevia extract is 200-300 times that of sugar and is used as a high-intensity sweetener commercially. The main glycoside components of stevia leaves are stevioside and rebaudioside. There are a large number of more than ten different steviol glycosides in the leaves. The main sweet compounds are stevioside and rebaudioside A. Compared with stevioside, rebaudioside A (Reb A) is considered to have a higher value because its sweetness increases and bitterness decreases.

[0008] In addition, the sweetness and bitterness characteristics of rebaudioside M (Reb M) are improved when compared to Reb A. However, Reb M is present in very low amounts in stevia leaves. Therefore, there is a need for improved methods for converting glucose sources into lower steviol glycosides. Summary of the invention

[0009] The present disclosure provides enzymes, particularly non-natural enzymes, and methods of using those enzymes to transfer sugar moieties to substrate steviol glycosides. Specifically, disclosed are β-1,3-glycosyltransferases (also referred to herein as "B13GTs") that can glycosylate the C3' of 13-O-glucose and / or 19-O-glucose of steviol glycoside substrates. The present disclosure provides methods of preparing Reb M using the disclosed β-1,3-glycosyltransferases in combination with sucrose synthase (SuSy) and optionally β-1,2-glycosyltransferases (B12GTs).

[0010] In contrast to natural glycosyltransferases, the present disclosure provides glycosyltransferase polypeptides that can utilize adenosine diphosphate (ADP)-glucose as a sugar donor to glycosylate 13-O-glucose and / or 19-O-glucose C3' of steviol glycoside substrates. The present disclosure provides glycosyltransferase polypeptides comprising an amino acid sequence having 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% identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 2-517. The glycosyltransferase polypeptide may comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NO: 2-517. The polypeptide may comprise one or more peptide tags for dissolution, expression and / or purification; for example, a polyhistidine tag having between 4 and 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 consisting 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.

[0011] The present disclosure further provides a method for preparing steviol glycosides, particularly Reb M, using the disclosed B13GT. Starting from a feed containing Reb A and / or stevioside, the enzymes B13GT, B12GT, and SuSy can be compounded with sucrose, ADP, and steviol glycoside feed to convert 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 β-1,3-glycosyltransferase, a β-1,2 glycosyltransferase, and a sucrose synthase to prepare 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 β-1,3-glycosyltransferase, a β-1,2 glycosyltransferase, and a sucrose synthase to prepare 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 β-1,3-glucosyltransferase, a β-1,2-glucosyltransferase, and a sucrose synthase to produce Reb M. In another embodiment, the method comprises contacting a stevia leaf extract purified to contain greater than 99% stevioside (RS99), ADP, and sucrose with a β-1,3-glucosyltransferase, a β-1,2-glucosyltransferase, and a sucrose synthase to produce Reb M. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The accompanying drawings are included to provide a further understanding of the present disclosure. The drawings illustrate embodiments of the present disclosure and together with the description serve to explain the principle of embodiments of the present disclosure.

[0013] Figure 1 Chemical structure of rebaudioside M.

[0014] Figure 2 A partial steviol glycoside network showing the conversion of stevioside and Reb A to Reb M through the addition of glucose monomers catalyzed by β-1,2-glycosyltransferase (B12GT) and β-1,3-glycosyltransferase (B13GT). For each molecule, the central hexagon represents the steviol glycoside core, the circles represent β-1,2-linked glucose monomers and the squares represent β-1,3-linked glucose monomers.

[0015] Figure 3 SDS-PAGE gel of the designed β-1,3-glycosyltransferase expressed in Pichia pastoris and purified by immobilized metal affinity chromatography. DETAILED DESCRIPTION

[0016] The present disclosure provides enzymes and biocatalytic methods for preparing a composition comprising a target steviol glycoside by contacting a starting 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 comprising one or more additional glucose units compared to the substrate steviol glycoside.

[0017] definition

[0018] The term "a" or "an" refers to one or more of the entity, i.e., can refer to multiple referents. Therefore, the terms "a", "an", "one or more", and "at least one" are used interchangeably in this article. In addition, an "element" referred to by the indefinite article "a" or "an" does not exclude the possibility that more than one element is present, unless the context clearly requires the presence of one and only one element.

[0019] Throughout this application, ADP, GDP, UDP, CDP and TDP are used to refer to adenosine diphosphate, guanosine-5'-diphosphate, uridine diphosphate, cytidine diphosphate and thymidine diphosphate, respectively.

[0020] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of the error of the device or method used to determine the value, or the variation that exists between samples being measured. Unless otherwise noted or otherwise apparent from the context, the term "about" means within 10% above or below the reported value (unless this number will exceed 100% of the possible value or be below 0%). When used in conjunction with a range or series of values, unless otherwise indicated, the term "about" applies to each value in the values ​​listed in the endpoints of the range or the series. As used in this application, the terms "about" and "approximately" are used as equivalents.

[0021] As used herein, the term "sequence identity" refers to the degree to which two optimally aligned polynucleotide or polypeptide sequences are unchanged in the alignment window of residues (e.g., nucleotides or amino acids). The "identity score" of the alignment segment of the test sequence and the 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 smaller defined portion of the reference sequence). "Identity percentage" is the identity score multiplied by 100. Comparing sequences to determine the identity percentage can be achieved by many well-known methods, including, for example, by using mathematical algorithms, such as those in the BLAST suite of sequence analysis programs. Unless otherwise indicated, the term "sequence identity" in the claims refers to the sequence identity calculated by Clustal Omega® using default parameters.

[0022] As used herein, "biocatalysis" or "biocatalytic" refers to the chemical transformation of organic compounds using natural catalysts, such as proteases. Biocatalysis is also known as biotransformation or biosynthesis. Both isolated and whole-cell biocatalytic methods are known in the art. The biocatalyst protease can be a naturally occurring protein or a recombinant protein.

[0023] As used herein, the term "steviol glycoside" 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 glycosides includes 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 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.

[0024] As used herein, "starting composition" refers to any composition (generally an aqueous solution) containing one or more steviol glycosides, wherein the one or more steviol glycosides are used as substrates for bioconversion.

[0025] Unless the context indicates otherwise, as used herein, the terms "polynucleotide" or "nucleic acid" are used interchangeably and are used to refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, typically DNA.

[0026] As used herein, "expression" refers to either or both steps, depending on the context, of a two-step process by which a polynucleotide is transcribed into mRNA and the transcribed mRNA is subsequently translated into a polypeptide.

[0027] "Under transcriptional control" means that transcription of a polynucleotide, typically a DNA sequence, is dependent upon it being operably linked to elements that facilitate transcription.

[0028] "Operably linked" means that polynucleotide elements are arranged in a manner that allows them to function in a cell; typically for production of a polypeptide in a cell; for example, the disclosure provides a promoter operably linked to a downstream sequence encoding a polypeptide.

[0029] The term "coding" refers to the ability of a polynucleotide to produce mRNA or a polypeptide, provided that the polynucleotide can be transcribed to produce mRNA, which is then translated to produce a polypeptide or a fragment thereof. In each case, the polynucleotide is referred to as encoding mRNA and encoding polypeptide. The antisense strand is the complementary strand of this nucleic acid, and the coding sequence can be derived therefrom. Similarly, a "coding sequence" refers to a nucleic acid region that encodes an mRNA or a polypeptide.

[0030] As used herein, the term "promoter" refers to a control sequence that is part of a polynucleotide sequence that controls the initiation of transcription and the rate of transcription of a coding sequence. An "enhancer" is a regulatory element that increases the expression of a target sequence. A "promoter / enhancer" is a polynucleotide having sequences that provide both promoter and enhancer functions.

[0031] Regulatory elements, such as enhancers and promoters, can be "homologous" or "heterologous". A "homologous" regulatory element is a regulatory element naturally associated with a given polynucleotide in a genome; for example, it can be a promoter that occurs naturally in an organism upstream of the encoded polypeptide. A "heterologous" regulatory element is a regulatory element that is juxtaposed with a polynucleotide by means of recombinant molecular biology techniques but is not in a combination found in nature. Typically, promoters, enhancers, and other regulatory elements are heterologous to promote expression of a polypeptide in a host cell other than the host cell in which the polypeptide occurs naturally. Thus, as used herein, "heterologous expression" refers to the production of mRNA and / or polypeptides in a host cell, such as a microorganism, in which the polynucleotide is not found naturally, or one or more regulatory elements are not found naturally operably linked to a polynucleotide in a host cell.

[0032] The term "polypeptide" is used herein to refer to a molecule of two or more amino acid subunits linked by peptide bonds. Typically, although not always, a polypeptide contains several hundred amino acids; for example, about 400 to about 900 amino acids.

[0033] "Plasmid" is a DNA molecule that is usually separated from chromosomal DNA and can be replicated independently of the chromosomal DNA. In many cases, it is circular and double-stranded. As known in the art, although plasmid vectors are usually present as extrachromosomal circular DNA molecules, plasmid vectors can also be designed to be stably integrated into the host chromosome randomly or in a targeted manner. Many plasmids are commercially available for a variety of purposes. The gene to be replicated is inserted into a plasmid copy containing a gene that makes the cell resistant to a specific antibiotic and a multiple cloning site (MCS, or polylinker), which is a short region containing several commonly used restriction sites, thereby allowing DNA fragments to be easily inserted at this position. Typically, polypeptides disclosed herein are expressed from plasmids.

[0034] Its chemical structure is Figure 1Reb M shown in FIG. 1 can be prepared by adding a β-1,3-linked glucose monomer to the lower steviol glycoside rebaudioside I (Reb I) or Reb AM or by adding a β-1,2-linked glucose monomer to rebaudioside D (Reb D), such as Figure 2 The natural glycosyltransferases that perform these conversions use uridine diphosphate (UDP)-glucose as a source of glucose to transfer to lower steviol glycosides.

[0035] The present disclosure provides non-natural, engineered β-1,3-adenosine diphosphate (ADP) glycosyltransferases (B13GTs) that can use an ADP-glucose sugar donor to add β-linked glucose monomers to the C3' of 13-O-glucose and / or 19-O-glucose of a steviol glycoside substrate. 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 a specific 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.

[0036] In bioinformatics, several methods have been developed to find and determine related polypeptide sequences. For example, sequence identity percentage, position-specific scoring matrix (PSSM) and hidden Markov model (HMM) are all commonly used to find sequences similar to a given query sequence. The sequence identity percentage calculates the number of amino acids shared between two sequences. The sequence identity percentage is calculated in the case of a given comparison between two sequences. The alignment program ClustalOmega (available at / www.ebi.ac.uk / Tools / msa / clustalo / ) can be used to calculate the identity percentage with default settings. The default transfer matrix is ​​Gonnet, and the gap open penalty is 6, and the gap extension is 1. Clustal Omega uses the HHalign algorithm and its default settings as its core alignment engine. This algorithm is described in Söding, J. (2005) 'Protein homology detection by HMM-HMM comparison'. Bioinformatics 21, 951-960.

[0037] A position-specific scoring matrix (PSSM) is a concise way to represent many related sequences. PSSMs are typically generated using multiple sequence alignments. The sequence search tool PSI-BLAST generates PSSMs and uses them to search for related polypeptide sequences. The PSSM used to score polypeptide sequences is a matrix (i.e., a table) consisting of 21 columns × N rows, where N is the length of the related sequence. Each row corresponds to a position within the polypeptide sequence, and each column represents a different amino acid (or gap) that can be present at the residue position. Each entry in the PSSM represents the score of a specific amino acid at a specific position within the polypeptide sequence. The PSSM can be used to score a sequence by first aligning the sequence with a reference sequence and then calculating the following sum: , where i is the sequence position and aa i is the amino acid at position i. Related polypeptide sequences will all have high PSSM scores, while unrelated sequences will receive low scores.

[0038] In some embodiments, the β-1,3-glycosyltransferase polypeptide is prepared by expression in a host microorganism. Suitable host microorganisms include, but are not limited to, Escherichia coli (E. coli), Saccharomyces species, Aspergillus species, Pichia species, Bacillus species. In a specific embodiment, the glycosyltransferase is expressed in Escherichia coli. In a specific embodiment, the glycosyltransferase is expressed in Pichia pastoris.

[0039] The B13GT polypeptide can be provided in any suitable form, including free, fixed or as a whole cell system. The purity of the glycosyltransferase polypeptide can vary, for example, it can be provided as a crude, semi-purified or purified enzyme preparation. In one embodiment, the glycosyltransferase polypeptide is free. In another embodiment, the glycosyltransferase polypeptide is fixed to a solid support, for example, on an inorganic or organic support. In some embodiments, the solid support is derived from cellulose, glass, ceramic, methacrylate, styrene, acrylic, metal oxide or film. In some embodiments, the glycosyltransferase polypeptide is fixed to a solid support by covalent attachment, adsorption, cross-linking, embedding or encapsulation.

[0040] In another embodiment, the B13GT polypeptide is provided in the form of a whole cell system, for example as live fermented microbial cells, or as dead and stabilized microbial cells, or in the form of a cell lysate.

[0041] The present disclosure provides a biocatalytic method for preparing a composition comprising one or more target steviol glycosides from a starting composition comprising one or more substrate steviol glycosides, wherein the target steviol glycoside comprises a steviol glycoside having one or more additional glucose units compared to the substrate steviol glycosides. The biocatalytic method includes contacting an engineered B13GT, sucrose synthase (SuSy) (unless ADP-glucose is directly supplied) and an optional B12GT with a starting composition containing one or more steviol glycosides, non-UDP nucleotide diphosphates and sucrose. In another embodiment, the biocatalytic method includes contacting an engineered B13GT, SuSy and an optional B12GT with a starting composition containing one or more steviol glycosides, non-UDP nucleotide diphosphates and sucrose. In some embodiments, the method includes contacting RA50, ADP and sucrose with an engineered B1,3 glycosyltransferase, B12GT and SuSy to prepare Reb M. In another embodiment, the method comprises contacting RA60, ADP and sucrose with engineered B13GT, B12GT and SuSy to prepare Reb M. In another embodiment, the method comprises contacting stevioside, ADP and sucrose with engineered B1,3 glycosyltransferase, B12GT and SuSy to prepare Reb M. In another embodiment, the method comprises contacting Reb A, ADP and sucrose with engineered B13GT, B12GT and SuSy to prepare Reb M. In another embodiment, the method comprises contacting RS90, ADP and sucrose with engineered B13GT, B12GT and SuSy to prepare Reb M. In another embodiment, the method comprises contacting RS99, ADP and sucrose with engineered B13GT, B12GT and SuSy to prepare Reb M.

[0042] 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.

[0043] In one embodiment, sucrose synthase is any polypeptide with sucrose synthase activity. In another embodiment, sucrose synthase is derived from an organism in the bacterial domain. In another embodiment, sucrose synthase is derived from an organism in the plant kingdom. In another embodiment, sucrose synthase is derived from an organism in the proteobacteria, deferribacteres or cyanobacteria phylum. In another embodiment, sucrose synthase is derived from the following species: 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 having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a sucrose synthase derived from the following species: Thermoacidithiobacillus, Nitrosomonas europeanus, Denitrificovibrio acetophilus, Thermosynechococcus elongatus, Oryza sativa, Arabidopsis thaliana, or Coffea arabica.

[0044] In one embodiment, B12GT is any polypeptide that can glycosylate 13-O-glucose and / or 19-O-glucose C2' of a steviol glycoside substrate. In another embodiment, B12GT is derived from an organism in the plant kingdom. In another embodiment, B12GT is derived from an organism in the angiosperm clade. In another embodiment, B12GT is derived from the following species: barley (Hordeum vulgare), rice (Oryza sativa), tomato (Solanum lycopersicum), wolfberry (Lycium barbarum), potato (Solanum tuberosum) or stevia (Stevia rebaudiana). In another embodiment, the B12GT is an engineered B12GT having a polypeptide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a B12GT derived from barley, rice, tomato, wolfberry, potato, or stevia.

[0045] In some embodiments, B13GT, B12GT and / or sucrose synthase polypeptides are prepared by expression in a host microorganism. Suitable host microorganisms include, but are not limited to, Escherichia coli, Saccharomyces species, Aspergillus species, Pichia species, Bacillus species. In a specific embodiment, glycosyltransferases and sucrose synthases are expressed in Escherichia coli. In another embodiment, glycosyltransferases and sucrose synthases are expressed in Pichia pastoris. In another embodiment, B13GT, B12GT and / or sucrose synthase polypeptides are prepared by cell-free expression.

[0046] B13GT, B12GT and sucrose synthase polypeptides can be provided in any suitable form, including free, fixed or as a whole cell system. The purity of the polypeptide can vary, for example, they can be provided as a crude, semi-purified or purified enzyme preparation. In one embodiment, B13GT, B12GT and / or SuSy polypeptides are free. In another embodiment, B13GT, B12GT and / or SuSy polypeptides are fixed to a solid support, such as an inorganic or organic support. In some embodiments, the solid support is derived from cellulose, glass, ceramics, methacrylate, styrene, acrylic acid, metal oxide or film. In some embodiments, B13GT, B12GT and / or SuSy polypeptides are fixed to a solid support by covalent attachment, adsorption, cross-linking, embedding or encapsulation.

[0047] In another embodiment, the B13GT, B12GT and / or SuSy polypeptide is provided in the form of a whole cell system, for example as live fermented microbial cells, or as dead and stabilized microbial cells, or in the form of a cell lysate.

[0048] As described herein, the steviol glycoside component of the starting composition is used as a substrate for producing target steviol glycosides. The target steviol glycosides are chemically different from their corresponding substrate steviol glycosides by the addition of one or more glucose units.

[0049] The starting steviol glycoside composition may contain at least one substrate steviol glycoside. In one embodiment, the substrate steviol glycoside is 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 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 another embodiment, the starting steviol glycoside composition comprises Reb A.

[0050] The starting steviol glycoside composition can be synthetic or purified (partially or completely), commercially available or prepared. An example of a starting composition that can be used for the method of the present disclosure is an extract obtained from the purification of stevia plant material (e.g., leaves). Another example of a starting composition is a commercially available stevia extract made into a solution with a solvent. Another example of a starting composition is a commercially available steviol glycoside mixture made into a solution with a solvent. Other suitable starting compositions include byproducts of the process of separating and purifying steviol glycosides.

[0051] In one embodiment, the starting composition comprises purified substrate steviol glycosides. 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.

[0052] In another embodiment, the starting composition comprises a partially purified substrate 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% by weight on an anhydrous basis. One or more substrate steviol glycosides.

[0053] In another embodiment, the substrate steviol glycoside is purified rebaudioside A or an isomer thereof. In a specific embodiment, the substrate steviol glycoside contains greater than 99% rebaudioside A or an isomer thereof by weight on an anhydrous basis. In another embodiment, the substrate steviol glycoside comprises partially purified rebaudioside A. In a specific 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.

[0054] In another embodiment, the substrate steviol glycoside comprises purified stevioside or its isomer. In a specific embodiment, by weight on an anhydrous basis, the substrate steviol glycoside contains greater than 99% stevioside or its isomer. In another embodiment, the substrate steviol glycoside comprises partially purified stevioside. In a specific embodiment, by weight on an anhydrous basis, the substrate steviol glycoside contains greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% stevioside.

[0055] In another embodiment, the substrate steviol glycoside is a combination of stevioside and rebaudioside A. In a specific embodiment, the substrate steviol glycosides contain 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. 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.

[0056] In another embodiment, the substrate steviol glycosides are derived from stevia leaf extract. In one embodiment, RA50, a stevia leaf extract purified to contain greater than 50% Reb A, is used as a steviol glycoside substrate. In one embodiment, RA50 is used at a concentration between 1 mg / ml and 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 greater than 60% Reb A, is used as a steviol glycoside substrate. In 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, a stevia leaf extract purified to contain greater than 90% stevioside, is used as a steviol glycoside substrate. In another embodiment, RS99, a stevia leaf extract purified to contain greater than 99% stevioside, is used as a steviol glycoside substrate. In another embodiment, RS90 is used at a concentration of between 1 mg / ml and 800 mg / ml. In another embodiment, RS90 is used at a concentration of 100 mg / ml.

[0057] The reaction can be carried out using a nucleotide cofactor that can be converted into 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 specific embodiment, the reaction can be carried out with a concentration between 0.01 mM and 10 mM, 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 ADP. In a specific embodiment, ADP is used at a concentration of 0.5 mM.

[0058] The reaction can be performed using sucrose concentrations between 10 mM and 2 M, such as 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 a particular embodiment, sucrose is used at a concentration of 450 mM.

[0059] 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. For example, between 10°C and 20°C, between 20°C and 30°C, between 30°C and 40°C, between 40°C and 50°C, between 50°C and 60°C, between 60°C and 70°C, between 70°C and 80°C, or 80°C. In a specific embodiment, a one-pot reaction is performed at 60°C.

[0060] The reaction medium used for conversion is generally aqueous, for example, purified water, buffer or a combination thereof. In a specific embodiment, the reaction medium is a buffer. Suitable buffer includes but is not limited to acetate buffer, citrate buffer, HEPES and phosphate buffer. In a specific embodiment, the reaction medium is a phosphate buffer. The reaction medium can have a pH between 4 and 10. In a specific embodiment, the pH of the reaction medium is 6. Alternatively, the reaction medium can also be an organic solvent.

[0061] The step of contacting the starting composition with the glycosyltransferase and sucrose synthase polypeptides can be carried out for a duration of between 1 hour and 1 week, such as between 30 minutes and 1 hour, 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, between 3 days and 4 days, between 4 days and 5 days, between 6 days and 7 days. In a specific embodiment, the reaction is carried out for 6 hours.

[0062] The reaction can be monitored by a suitable 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).

[0063] 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 E2, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M, rebaudioside AM, rebaudioside D, rebaudioside N, rebaudioside O, rebaudioside Q, rebaudioside having 7 covalently attached glucose units (e.g., rebaudioside M plus 1 glucose unit), a synthetic steviol glycoside, an isomer thereof, and / or a steviol glycoside composition. 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 another embodiment, the target steviol glycoside is Reb I, Reb AM, Reb D, and Reb M.

[0064] 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 specific 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 specific embodiment, the conversion of the starting steviol glycoside composition to Reb I 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.

[0065] 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 specific 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 specific 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%, or 90% of the starting steviol glycoside composition is converted to Reb M.

[0066] 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 specific embodiment, the conversion of the starting steviol glycoside composition to Reb AM is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. In a specific embodiment, the conversion of the starting steviol glycoside composition to Reb AM is at least 95%. In some embodiments, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the starting steviol glycoside composition is converted to Reb AM.

[0067] In one embodiment, the conversion of the starting steviol glycoside composition to Reb E2 is completed by at least 2%, as determined by any of the methods mentioned above. In a specific embodiment, the conversion of the starting steviol glycoside composition to Reb E2 is completed by at least 10%, completed by at least 20%, completed by at least 30%, completed by at least 40%, completed by at least 50%, completed by at least 60%, completed by at least 70%, completed by at least 80%, or completed by at least 90%. In a specific embodiment, the conversion of the starting steviol glycoside composition to reb E2 is completed by at least 95%. In some embodiments, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the starting steviol glycoside composition is converted to Reb E2.

[0068] 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 specific 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 specific 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.

[0069] The target steviol glycoside may be in any polymorphic form or amorphous form, including hydrates, solvates, anhydrates, or combinations thereof.

[0070] Optionally, the method of the present disclosure further comprises separating the target steviol glycoside from the target composition. The target steviol glycoside can be separated by any suitable method, such as crystallization, membrane separation, centrifugation, extraction, chromatography, or a combination of such methods.

[0071] In one embodiment, separation of target steviol glycosides produces a composition comprising target steviol glycosides greater than 80% by weight on an anhydrous basis, i.e., a highly purified steviol glycoside composition. In another embodiment, separation produces a composition comprising target steviol glycosides greater than 0.5% by weight, greater than 1% by weight, greater than 2% by weight, greater than 3% by weight, greater than 4% by weight, greater than 5% by weight, greater than 10% by weight, greater than 20% by weight, greater than 30% by weight, greater than 40% by weight, greater than 50% by weight, greater than 60% by weight, greater than 70% by weight, greater than 80% by weight, greater than 85% by weight, greater than 90% by weight, greater than 91% by weight, greater than 92% by weight, greater than 93% by weight, greater than 94% by weight, greater than 95% by weight, greater than 96% by weight, greater than 97% by weight, greater than 98% by weight, greater than 99% by weight, or greater than 99.6% by weight. In a specific embodiment, the composition comprises target steviol glycosides greater than 95% by weight on an anhydrous basis.

[0072] Purified target steviol glycosides can be used as sweeteners in consumer products. Suitable consumer products include, but are not limited to, foods, beverages, pharmaceutical compositions, tobacco products, nutritional compositions, oral hygiene compositions, and cosmetic compositions.

[0073] Plasmids containing nucleic acids encoding enzymes having SEQ ID NOs: 2-517 are described in Table 1 below.

[0074] Table 1. Designed B13GT

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0082] Example

[0083] Example 1: Computational design of ADP-glucose dependent B13GT

[0084] Computational design was performed to improve the native B13GT from Stevia (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 residue H24 as a catalytic histidine, D123 that activates the histidine, and two sugar binding residues D379 and Q380 (numbered relative to SEQ ID NO: 2). Computational design incorporating co-evolutionary information was performed to improve B13GT. 53 of these computational designs were selected for experimental validation (SEQ ID NO: 2-54).

[0085] The polynucleotides encoding the designed amino acid sequence of B13GT were synthesized (Twist Bioscience) and inserted into the pARZ4 expression vector. The polynucleotides encode the B13GT enzyme with an N-terminal His tag. The polynucleotides were ordered as full-length genes or as gene fragments and then assembled using Gibson assembly. The recombinant vectors were used to transform Escherichia coli HMS174 (DE3) (Novagen) or T7 Express lysY / Iq (New England BioLabs) in a heat shock method to prepare recombinant microorganisms.

[0086] The transformed recombinant microorganism was inoculated into 1 ml of LB-kanamycin medium and cultured overnight at 37°C by shaking. The culture was 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 high speed for 5 minutes and stored at -80°C.

[0087] The microorganisms were dissolved in lysis buffer (lysozyme, DNAseI, Bugbuster, 300 ml 20 mM HEPES pH7.5, 500 mM NaCl and 20 mM imidazole). 2 to 3 glass beads were added to each well and broken by shaking at 25 ° C and 220 rpm for 30 minutes. The broken liquid was centrifuged at 2200 xg for 6-10 minutes. The supernatant obtained was loaded onto the Ni-NTA plate and shaken at room temperature for 10 minutes. The plate was centrifuged at 100 xg for 4 minutes, then washed twice with 500 µl binding buffer (300 ml 20mM HEPES pH 7.5, 500mM NaCl, 20mM imidazole) and centrifuged for two minutes (500 xg). The protein was eluted with 150 µl elution buffer (15 ml 20 mM HEPES pH 7.5, 500 mM NaCl, 500 mM imidazole) and shaken at 0.25 maximum shaking speed for 1 minute, followed by centrifugation at 500 xg for 2 minutes. The recovered protein was desalted into a buffer solution for enzyme activity assessment (50 mM HEPES pH 7.5, 50 mM NaCl).

[0088] Each B13GT variant was assayed using the B12GT and SuSy enzymes in a one-pot reaction. B12GT and SuSy were used in excess to allow complete conversion of the starting 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.

[0089] The steviol glycosides, stevioside, Reb A, Reb D, Reb E, Reb I, Reb M and other minor 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). The QQQ was run by multiple reaction monitoring (MS / MS) to accurately quantify the steviol glycosides of interest. Several designed enzymes expressed well and were able to efficiently produce Reb M (Table 2).

[0090] Table 2. Purified protein concentrations and Reb M conversion rates of successful B13GT design variants expressed in E. coli

[0091]

[0092]

[0093] Example 2: Expression of designed B13GT and SuSy in Pichia pastoris

[0094] The polynucleotides of the first ten designed B13GTs optimized for Pichia pastoris expression from Example 1 were synthesized (Twist Bioscience) and inserted into a Pichia shuttle vector. The polynucleotides encode a B13GT enzyme with a C-terminal His tag instead of the N-terminal His tag used in Example 1. Two unique polynucleotides were ordered for each B13GT amino acid sequence. The vectors were transformed into commercially available Pichia pastoris strains (ATCC). The transformed microorganisms were grown in BMGY (buffered glycerol complex) medium and protein expression was induced by methanol addition. Pichia cells were lysed with Y-PER (yeast protein extraction reagent; Thermo Scientific), and the expressed protein was purified by immobilized metal affinity chromatography (IMAC) and desalted into a desalting buffer (20mM KPO4 pH6, 50mM NaCl). Similar to Example 1 (Table 3), the designed B13GT was expressed soluble ( Figure 3 ) and measured enzyme activity.

[0095] The polynucleotide encoding the top performing B13GT was amplified from the Pichia shuttle vector and integrated into the Pichia genome. Four integrated Pichia strains expressing the designed B13GT were grown in a 1L fermentor. The Pichia microorganism was grown with glycerol as the main carbon source for ~24 hours and then fed with methanol for ~72 hours to express the desired B13GT. The cells were collected and lysed by a French press. The expressed protein was purified by immobilized metal affinity chromatography (IMAC) and dialyzed into a desalting buffer (20mM KPO4 pH6, 50mM NaCl). All four strains successfully expressed active B13GT in fermentation.

[0096] Table 3. Purified protein concentration and Reb M conversion efficiency of the top B13GT designs expressed in Pichia pastoris

[0097]

[0098] Example 3: Computational design of improved B13GT

[0099] The results of Example 1 were used to fine-tune the previously developed protein sequence-based deep learning model. The model was used to design an improved B13GT enzyme. 80 of these computational designs were selected for experimental verification (SEQ ID NO: 55-134). Polynucleotides encoding B13GT enzymes with C-terminal His tags were synthesized (Twist Bioscience). Escherichia coli strains expressing the designed B13GT were generated as described in Example 1. The designed B13GT enzyme was expressed and purified as described in Example 1.

[0100] Each B13GT variant was assayed using the B12GT and SuSy enzymes in a one-pot reaction. B12GT and SuSy were used in excess so that the starting RA50 feed was fully converted 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. The steviol glycosides produced and consumed during the reaction were measured as described in Example 1. The designed enzymes were expressed soluble and were able to efficiently produce Reb M (Table 4).

[0101] Table 4. Purified protein concentration and Reb M conversion efficiency of the top B13GT design variants expressed in E. coli

[0102]

[0103]

[0104] The structural model of the top-ranked B13GT design was generated and analyzed. For this reason, a structure-based deep learning method designed to further improve the B13GT enzyme was used to train the protein sequence of a given protein backbone. 121 of these computational designs were selected for experimental verification (SEQ ID NO: 135-255). The machine learning model was retrained on the crystal structure and structural model of the protein from a thermophilic organism. Another 69 B13GT enzymes were selected for experimental verification (SEQ ID NO: 256-324). The polynucleotides (Twist Bioscience) encoding the B13GT enzyme (SEQ ID NO: 135-324) with a C-terminal His tag were synthesized. The Escherichia coli strain expressing the designed B13GT was produced as described in Example 1. The designed B13GT enzyme was expressed and purified as described in Example 1.

[0105] Each B13GT variant was assayed using the B12GT and SuSy enzymes in a one-pot reaction. B12GT and SuSy were used in excess so that the initial RA50 feed was fully converted 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. The steviol glycosides produced and consumed during the reaction were measured as described in Example 1. The designed enzymes were expressed soluble and were able to efficiently produce Reb M (Table 5).

[0106] Table 5. Purified protein concentration and Reb M conversion efficiency of the top B13GT design variants expressed in E. coli

[0107]

[0108]

[0109] Example 4: Computational design of further improved B13GT

[0110] The results of Examples 1 and 3 were used to fine-tune the protein-based deep learning model from Example 3. The sequence-based model was used to design an improved B13GT enzyme. One hundred of these computational designs were selected for experimental verification (SEQ ID NO: 325-424). Polynucleotides encoding B13GT enzymes (SEQ ID NO: 325-424) with C-terminal His tags were synthesized (Twist Bioscience). Escherichia coli strains expressing the designed B13GT were generated as described in Example 1. The designed B13GT enzyme was expressed and purified as described in Example 1.

[0111] Each B13GT variant was assayed using the B12GT and SuSy enzymes in a one-pot reaction. B12GT and SuSy were used in excess so that the starting RA50 feed was fully converted 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 sodium acetate at 60°C for 24 hours. The steviol glycosides produced and consumed during the reaction were measured as described in Example 1. The designed enzymes were expressed soluble and were able to efficiently produce Reb M (Table 6).

[0112] Table 6. Purified protein concentration and Reb M conversion efficiency of the top B13GT design variants expressed in E. coli

[0113]

[0114] The structural model of the top-ranked B13GT design from Example 3 was generated and analyzed. A further improved B13GT enzyme was generated using a structure-based deep learning method trained to predict the protein sequence of a given protein backbone. This method was used to design an improved B13GT enzyme. 93 of these computational designs were selected for experimental verification (SEQ ID NO: 425-517). Polynucleotides encoding the B13GT enzyme with a C-terminal His tag were synthesized (Twist Bioscience). Escherichia coli strains expressing the designed B13GT were generated as described in Example 1. The designed B13GT enzyme was expressed and purified as described in Example 1.

[0115] Each B13GT variant was assayed in a one-pot reaction using the B12GT and SuSy enzymes. B12GT and SuSy were used in excess so that the initial RA50 feed was fully converted 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 sodium acetate at 60°C for 24 hours. The steviol glycosides produced and consumed during the reaction were measured as described in Example 1. The designed enzymes were expressed soluble and were able to efficiently produce Reb M (Table 7).

[0116] Table 7. Purified protein concentration and Reb M conversion efficiency of the top B13GT design variants expressed in E. coli

[0117]

[0118] Example 5: Using PSSM to represent a successful B13GT design

[0119] Use the successful B13GT design from embodiment 1 to produce PSSM (table 8).PSSM is a concise way to represent successful design and related sequences.Sequences with a PSSM score greater than 332 are considered to be relevant to active B13GT computational design as herein described.In order to use PSSM to score sequence, the sequence must first be compared 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 wild-type B13GT SEQ ID NO: 1 has a PSSM score of only 326.7.

[0120] Use the successful B13GT design from embodiment 3 to produce PSSM (table 9).PSSM is a concise way to represent successful design and related sequences.Sequences with a PSSM score greater than 54.5 are considered to be relevant to active B13GT computational design as herein described.In order to use PSSM to score sequence, the sequence must first be compared 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 wild-type B13GT SEQ ID NO: 1 has a PSSM score of only 51.16.

[0121] Use the successful B13GT design from embodiment 4 to produce PSSM (table 10).PSSM is a concise way to represent successful design and related sequences.Sequences with a PSSM score greater than 58.3 are considered to be relevant to active B13GT computational design as herein described.In order to use PSSM to score sequence, the sequence must first be compared 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 wild-type B13GT SEQ ID NO: 1 has a PSSM score of only 51.16.

[0122] Table 8. Position-Specific Scoring Matrix (PSSM) of Successful B13GT Designs

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134] Table 9. Position-Specific Scoring Matrix (PSSM) of Successful B13GT Designs

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146] Table 10. Position-specific scoring matrix (PSSM) for successful B13GT designs

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158] Example 6: E. coli fermentation of designed B13GT

[0159] For the top-ranked B13GT from Example 3, an E. coli strain expressing B13GT with a constitutive promoter was prepared. The E. coli strain was grown in a 1-liter fermentor tank with glycerol feed for 48 hours. The fermentation of the strain expressing AA37959, AA40205, AA40232 and AA40243 produced 171, 60, 182 and 190 grams of wet cell mass / liter of fermentation broth. 20 to 30 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 purified protein samples of AA37959, AA40205, AA40232 and AA40243 obtained had protein yields of 0.47, 0.15, 1.20 and 1.42 mg / ml, respectively.

[0160] Incorporated by Reference

[0161] All references, articles, publications, patents, patent publications and patent applications cited herein are incorporated by reference in their entirety for all purposes. However, the reference to any reference, article, publication, patent, patent publication and patent application cited herein does not and should not be taken as an admission or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

Claims

1. An engineered β-1,3-glycosyltransferase polypeptide comprising an amino acid sequence having 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% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-517, 2 and 3.

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

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

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

6. A polypeptide having the following sequence: MPNTXXTXXRXXXXXXFPVPFPGHXNPMLQLANXLYXXGFXIXIXXXXFNXPKTSXYPHFXFXXXXDXXPQXXXLRNLPTXGPGXGARIPXINXHGXXXXXXXLXXXXXXXXXD XXXXCXIXDALWYFXXXVXXXLXXXXLVLXTXSLFNFXXLXXLPXFXXXGXLXXXXXXXXXXXXXXGXPXLXXXDIXXAYSHXXEXKPILXKMXXXTXXXSGXIWNSXKELEESE LETXXREIPAPSFLXPLPKHXXASSXSLLDXDXXXXXWLXXQXXXSVXYVXFGSQSXXXXXXXXXXXXGLXXSXXXXLWLVRPGFXKGXXWXXXXPXGXXGXXXRXVKSXPQQE VLAHXAIGAFWTHGGWNGTXEXVCEGVPMIXSXFGLDQPLNAXYMXXXXXXGXYLXXGXXXXXXXXAXXXXXXXXXGXXXXXXAXXLXXKXDXSXXXGGSSYESLEXLXSYISSL Where residue 5 is N or S Where residue 6 is P or T where residue 8 is T or V Where residue 9 is R or V Where residue 11 is R or D or E or H or K or S or T Where residue 12 is R or D or L or K Where residue 13 is R or H or T or W Where residue 14 is I or V Where residue 15 is A or I or V Where residue 16 is I or L or M wherein residue 25 is I or L or W or V Where residue 34 is L or V Where residue 37 is R or S Where 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 Where residue 43 is I or T or V Where residue 45 is I or L or F Where residue 46 is H or Y where residue 47 is either M or T wherein residue 48 is A or R or N or Q or E or K or S or T Where residue 51 is A or R or L or K Where residue 56 is N or L Where 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 Where residue 66 is I or L Where residue 68 is R or N or E Where residue 69 is D or E Where residue 72 is D or P or T Where residue 73 is D or E Where residue 74 is W or Y Where residue 81 is H or T Where residue 85 is A or V Where residue 91 is Q or I or L or V Where residue 94 is Q or E or K Where residue 97 is A or K Where residue 98 is A or D or E or K or P Where residue 99 is A or R or Q or E or K or P or T or V Where residue 100 is L or F Where residue 101 is R or E or L or K or Y Where 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 Where 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 I or L or M or F or Y or V Where 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 Where residue 110 is A or N or D or E or S where residue 111 is S or T Where residue 112 is A or E or G or K or P Where residue 113 is D or E or K or S Where residue 115 is E or V Wherein residue 116 is R or Q or E or K or P or T or V Where residue 117 is I or L or V Where residue 118 is A or R or S Where residue 120 is L or F or V Where residue 122 is A or T Where 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 Where residue 131 is D or E or L or P or S Where residue 133 is A or T Where residue 134 is R or D or Q or E or K Where residue 135 is R or E or K or S Where residue 137 is N or G Where residue 138 is I or L Where residue 139 is R or K or P Where residue 140 is R or L or P Where residue 144 is Q or M Where residue 146 is G or S Where residue 152 is H or L Where residue 153 is A or C Where residue 155 is A or V Where 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 Where 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 Where residue 167 is N or D Where residue 168 is L or P wherein residue 169 is A or N or D or E or S or T Where residue 170 is N or D or S or T Where residue 171 is N or Q or K or P or S or T wherein residue 172 is R or E or H 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 Where residue 174 is D or E or L or P Where residue 175 is D or E wherein residue 176 is A or D or Q or E or K or T Where residue 177 is Q or P Where residue 178 is A or V Where residue 179 is E or K or P or S or V Where 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 Where 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 Where residue 190 is R or L or K Where residue 191 is R or K or S Where residue 196 is D or I or K or S or W Where residue 197 is D or Q Where residue 199 is A or S Where residue 204 is D or E or G or M or S Where residue 207 is I or T or V Where residue 208 is E or K or T Where residue 209 is A or Q or E or M or T Where residue 211 is R or K wherein residue 212 is A or R or N or Q or E or L or K Where residue 213 is A or S Where residue 216 is I or V Where residue 221 is A or F Where residue 232 is I or V Where residue 233 is R or Q or I or W Where residue 243 is I or L Where residue 249 is L or F or Y Where residue 250 is R or T where residue 254 is S or T where residue 259 is E or T Where residue 261 is R or P Where residue 262 is S or T Where residue 263 is A or F or T or V Where residue 264 is A or F Where residue 265 is A or D or Q or E or P Where residue 268 is A or D Wherein residue 269 is R or Q or G or K or T Where residue 271 is A or P Where 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 Where residue 276 is L or V Residue 279 is either G or S Where residue 285 is Q or E or S wherein residue 286 is I or L or M or F or V Where 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 Where residue 289 is A or D or E or K Where residue 290 is D or Q or E or T Where residue 291 is A or F Where residue 292 is L or K wherein residue 293 is A or R or N or E or K or T Where residue 294 is I or L Where 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 Where residue 300 is A or R or D or E or L or K Where 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 Where residue 305 is A or F or T or V Where residue 314 is I or V wherein residue 317 is A or R or Q or E or H 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 Where residue 320 is I or L or T or V Where 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 Where residue 323 is I or L Where residue 325 is D or E or P wherein residue 327 is R or L or K or F or Y Where residue 328 is G or L or M or F Where residue 330 is D or E or P Wherein residue 331 is A or R or N or Q or L or K Where residue 332 is G or I Where residue 334 is I or V Where residue 338 is A or C Wherein residue 347 is R or E or G or K or P Where residue 362 is L or M or V Where residue 364 is A or S where residue 373 is C or F Where residue 375 is D or P Wherein residue 385 is A or R or T or V Where residue 388 is A or S or T or V wherein residue 389 is R or N or D or E or K Where residue 390 is E or I or K or V Where residue 391 is R or L or K wherein residue 392 is R or N or E or G or H or K Where residue 393 is I or V wherein residue 395 is I or V Where residue 398 is R or D or E Where residue 399 is R or N or D or E or K Where 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 Where 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 Where residue 413 is I or V Where residue 414 is L or M Wherein residue 415 is R or Q or E or L or K or T or V Where residue 416 is A or D or E or S Where 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 H or K or P or Y or V Wherein residue 422 is I or M or Y Where 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 Where 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 Where residue 430 is A or K Wherein residue 431 is A or R or D or Q or E Where residue 433 is A or L Where residue 435 is R or G or V Where residue 437 is I or L Where residue 438 is A or M Where residue 439 is K or P Where residue 449 is R or S wherein residue 451 is Q or V (SEQ ID NO: 518).

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

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

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

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

11. The polypeptide of claim 9 or 10, wherein the polypeptide is immobilized to the solid support by covalent attachment, adsorption, cross-linking, embedding or encapsulation.

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

13. The method of claim 12, wherein the β-1,3-glycosyltransferase polypeptide is an engineered β-1,3-glycosyltransferase polypeptide comprising an amino acid sequence having 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% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-517, 2 and 3.

14. The method of claim 12, wherein the sucrose synthase is an engineered sucrose synthase having a polypeptide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a sucrose synthase derived from the species Acidithiobacillus thermophilus, Nitrosomonas europaea, Denitrovibrio acetophilus, Thermosynechococcus elongatus, Oryza sativa, Arabidopsis thaliana, or Coffea arabica.

15. The method of claim 12, wherein the β-1,2-glycosyltransferase polypeptide is an engineered β-1,2-glycosyltransferase polypeptide having a polypeptide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a β-1,2-glycosyltransferase polypeptide derived from barley, rice, tomato, wolfberry, potato, or stevia.

16. The method of claim 12, wherein (a) the β-1,3-glycosyltransferase polypeptide is an engineered β-1,3-glycosyltransferase polypeptide comprising an amino acid sequence having 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% identity 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 having a polypeptide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a sucrose synthase derived from the following species: Acidithiobacillus thermophilus, Nitrosomonas europaea, Denitrificavibrio acetophilus, Thermosynechococcus elongatus, Oryza sativa, Arabidopsis thaliana, or Coffea arabica; (c) the β-1,2-glycosyltransferase polypeptide is an engineered β-1,2-glycosyltransferase polypeptide having a polypeptide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a β-1,2-glycosyltransferase polypeptide derived from the following species: barley, rice, tomato, wolfberry, potato or stevia.

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

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

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

20. 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, isomers thereof, and synthetic steviol glycosides.

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

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

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

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

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. 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. The method of any one 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%, 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% steviol glycosides by weight on an anhydrous basis.

33. The method of any one 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.

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

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

36. The microorganism of claim 35, wherein the host microorganism is Escherichia coli, Saccharomyces spp., Aspergillus spp., Pichia spp., or Bacillus spp.

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

38. The polypeptide of claim 1, 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, and wherein the position numbering is based on SEQ ID NO:

2.

39. An engineered β-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 SEQ ID NO: 4, and 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. An engineered β-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 SEQ ID NO: 3, and 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. An engineered β-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 SEQ ID NO: 219, and 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. An engineered β-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 SEQ ID NO: 208, and 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. An engineered β-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 SEQ ID NO: 181, and 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.

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