Uridine diphosphate-dependent glycosyltransferase enzymes

Abstract

In various aspects, the present invention provides uridine diphosphate-dependent glycosyltransferase (UGT) enzymes that can catalyze the transfer of a monosaccharide moiety of an NDP sugar to the 3' carbon of a sugar moiety of a substrate, such as a terpenoid glycan, thereby functioning as a "1-3UGT." In other aspects, the present invention provides polynucleotides encoding 1-3UGTs and host cells containing the same. In still other aspects, the present invention provides methods for preparing glycosylated substrates, including steviol glycosides, using the enzymes and host cells of the present disclosure.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 866,148, filed June 25, 2019, the entire disclosure of which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates to enzymes, encoding polynucleotides, host cells, and methods for producing glycosylated substrates.

[0003] Description of electronically submitted text files The entire contents of the text file submitted electronically herewith are incorporated herein by reference: Computer-readable copy of the Sequence Listing (Filename: MAN-024PC_Sequence Listing_ST25.txt, Recorded: June 24, 2020, File Size: 28,457 bytes). [Background technology]

[0004] High-intensity sweeteners have a sweetness level several times higher than that of sucrose. They are essentially non-caloric and are generally used in diet and low-calorie products, including foods and beverages. High-intensity sweeteners do not induce a glycemic response, making them suitable for use in products targeted at diabetics and other people who are concerned about controlling their carbohydrate intake.

[0005] Steviol glycosides are a class of compounds found in the leaves of Stevia rebaudiana Bertoni, a perennial shrub in the Asteraceae (Compositae) family native to certain regions of South America. They are structurally characterized by a single base, steviol, and differ by the presence of carbohydrate residues at the C13 and C19 positions. They accumulate in stevia leaves, constituting approximately 10% to 20% of the total dry weight. On a dry weight basis, the four major glycosides found in stevia leaves generally include stevioside (9.1%), rebaudioside A (3.8%), rebaudioside C (0.6–1.0%), and dulcoside A (0.3%). Other known steviol glycosides include rebaudiosides B, C, D, E, F, and M, steviolbioside, and rubusoside.

[0006] The minor glycosylation product, rebaudioside M (RebM), is estimated to be approximately 200–350 times more potent than sucrose and has been shown to have a pleasant sweet taste with a slightly bitter or licorice aftertaste. Prakash I. et al., Development of Next Generation Stevia Sweetener: Rebaudioside M, Foods 3(1), 162–175 (2014). RebM is of great interest to the global food industry.

[0007] Processes for preparing steviol glycosides from stevia plants are not sustainable and are not suitable for obtaining minor glycosylated products of stevia leaves. Therefore, there remains a need for sustainable and economical methods for preparing compositions containing steviol glycosides, including highly purified steviol glycoside compositions. Furthermore, there is a need for methods for producing large quantities of minor glycosylated products, such as RebM and others. Summary of the Invention

[0008] In various aspects, the present invention provides uridine diphosphate (UDP)-dependent glycosyltransferase (UGT) enzymes that can catalyze the transfer of a monosaccharide moiety of an NDP sugar (e.g., a UDP sugar) to the 3' carbon of a sugar moiety of a substrate, such as a terpenoid glycan, thereby functioning as a "1-3UGT." In other aspects, the present invention provides polynucleotides encoding 1-3UGTs and host cells containing the same. In still other aspects, the present invention provides methods for preparing glycosylated substrates, including steviol glycosides, using the enzymes and host cells of the present disclosure.

[0009] 1-3UGTs exhibit high glycosyltransferase activity toward terpenoid glycosides, such as steviol glycosides. For example, 1-3UGTs catalyze the transfer of a monosaccharide moiety of an NDP sugar to the 3' carbon of a sugar moiety on a terpenoid glycan, such as stevioside and RebD. That is, when the substrate is a steviol glycoside, 1-3UGTs can catalyze the NDP-dependent transfer of a monosaccharide moiety to the 3' carbon of both the C13- or C19-linked glucose moiety. In various embodiments, 1-3UGTs catalyze the biosynthesis of RebM.

[0010] In one aspect, the invention provides a 1-3 UGT enzyme comprising an amino acid sequence at least about 75% identical to the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6. In some embodiments, the enzyme comprises amino acid substitutions at positions corresponding to positions 29, 200, 357, and 414 of SEQ ID NO:1 (Stevia rebaudiana UGT76G1). Substitutions at these positions in the enzymes of SEQ ID NOs:5 and 6 (positions 183, 354, 54, and 111, respectively, of SEQ ID NO:5) can result in significantly improved activity compared to the enzyme of SEQ ID NO:1.

[0011] In some embodiments, the 1-3UGT enzyme includes an insertion of 5 to about 15 amino acids, or about 6 to about 12 amino acids (relative to SEQ ID NO:1) after a position corresponding to position 155 of SEQ ID NO:5. In some embodiments, the insertion is a flexible, hydrophilic sequence that may be mostly glycine and serine residues. In some embodiments, the sequence is GSGGSG (SEQ ID NO:7) or GSGGSGGSG (SEQ ID NO:8).

[0012] In various embodiments, the 1-3 UGT enzyme exhibits improved conversion of stevioside to RebA and improved conversion of RebD to RebM compared to UGT76G1-L200A (SEQ ID NO: 3).

[0013] In some embodiments, amino acid matches at positions 183, 354, 54, and 111 of SEQ ID NO:5 allow for further modification at other positions. For example, in some embodiments, a 1-3UGT enzyme comprises an amino acid sequence at least about 60% identical to the amino acid sequence of SEQ ID NO:5, wherein the UGT enzyme comprises glycine (G) or threonine (T) at a position corresponding to position 54 of SEQ ID NO:5, leucine (L) or isoleucine (I) at a position corresponding to position 111 of SEQ ID NO:5, methionine (M) or leucine (L) at a position corresponding to position 183 of SEQ ID NO:5, and alanine (A), glycine (G), or serine (S) at a position corresponding to position 354 of SEQ ID NO:5. In some embodiments, the 1-3UGT enzyme comprises methionine (M) at a position corresponding to position 183 of SEQ ID NO:5. In some embodiments, the 1-3UGT enzyme comprises glycine (G) at a position corresponding to position 54 of SEQ ID NO:5. In some embodiments, the 1-3UGT enzyme contains a leucine (L) at a position corresponding to position 111 of SEQ ID NO: 5. In some embodiments, the 1-3UGT has two or three methionines (M) at a position corresponding to position 183 of SEQ ID NO: 5, a glycine (G) at a position corresponding to position 54 of SEQ ID NO: 5, and a leucine (L) at a position corresponding to position 111 of SEQ ID NO: 5. These modifications can result in significant improvements in the activity of the enzyme compared to matching the amino acids at the corresponding positions of SEQ ID NO: 1.

[0014] In some embodiments, the 1-3UGT enzyme includes a deletion of amino acid residues 159 to 161, in each case relative to the amino acid sequence of SEQ ID NO:5, and one or more of a substitution at position 262 (e.g., L262Q), a substitution at position 294 (e.g., R294), and a substitution at position 413 (e.g., D413E), in each case relative to the amino acid sequence of SEQ ID NO:5.

[0015] In embodiments, the 1-3 UGT enzyme comprises a deletion of amino acid residues 159 to 161 and amino acid substitutions L262Q, R294P, and D413E, respectively, with reference to the amino acid sequence of SEQ ID NO: 5. An exemplary UGT enzyme according to these embodiments is disclosed herein as SEQ ID NO: 6.

[0016] In some embodiments, the 1-3UGT enzyme includes a deletion of one or more residues E225 to T232 with reference to the amino acid sequence of SEQ ID NO: 6. In these or other embodiments, the 1-3UGT enzyme includes one or more amino acid substitutions at a position selected from position 72 (e.g., S72Q), position 305 (e.g., A305C), position 345 (e.g., Y345F), and position 428 (e.g., L428I), in each case with reference to the amino acid sequence of SEQ ID NO: 6. In an exemplary embodiment, the 1-3UGT enzyme includes a deletion of amino residues E225 to T232 and amino acid substitutions S72Q, A305C, Y345F, and L428I, in each case with reference to the amino acid sequence of SEQ ID NO: 6. An exemplary UGT enzyme according to these embodiments is disclosed herein as SEQ ID NO: 9.

[0017] In some embodiments, the amino acid modifications described herein are instead applied to SrUGT76G1, or a circular permutant thereof.

[0018] In another aspect, the present invention provides polynucleotides encoding the 1-3UGT enzymes disclosed herein, as well as host cells containing the same. The host cells may be microorganisms, fungal cells, algal cells, or plant cells. The plants may be stevia plants, or in particular, Stevia rebaudiana plants. Stevia plants naturally express the enzymes required to synthesize steviol and steviol glycosides, but produce only trace amounts of highly glycosylated steviol glycosides, such as RebM. In contrast, the RebM content of Stevia or Stevia rebaudiana plants expressing polynucleotides encoding 1-3UGTs may result in relatively high levels of RebA, RebD, and / or RebM, or other steviol glycosides, typically containing 1-3 glycosylation, such as RebB, RebG, RebI, and Reb4, which are generally present in trace amounts. In various embodiments, the cells are microbial cells, such as E. coli.

[0019] In various embodiments, the host cell may express one or more additional UGT enzymes selected from a C-13 UGT enzyme, a C-19 UGT enzyme, and a 1-2 UGT enzyme. A "C-13 UGT" or UGTc13 is a glycosyltransferase capable of glycosylating steviol or steviol glycosides at their C13 hydroxyl group. A "C-19 UGT" or UGTc19 is a glycosyltransferase capable of glycosylation of steviol or steviol glycosides at their C19 carboxyl group. A "1-2 UGT" or UGT1-2 is a glycosyltransferase capable of β1,2 glycosylation of the C2' of 13-O-glucose and / or 19-O-glucose. "1-3UGT" or UGT1-3 is a glycosyltransferase that can β1,3 glycosylate the C3' of 13-O-glucose and / or 19-O-glucose.

[0020] In some embodiments, the host cell expresses a heterologous C-13 UGT enzyme, a heterologous C-19 UGT enzyme, and a heterologous 1-2 UGT enzyme (in addition to a 1-3 UGT enzyme) and is thereby capable of glycosylation of steviol and steviol glycoside substrates to produce RebM.

[0021] In some embodiments, host cells (e.g., bacterial or yeast cells) produce steviol substrates by expression of endogenous and / or heterologous enzymes for the biosynthesis of steviol. In these embodiments, the cells produce steviol glycosides, such as RebM, from a carbon source such as glucose, sucrose, or glycerol, among others.

[0022] In some aspects, the present invention provides methods for transferring a monosaccharide group to a substrate. The methods include contacting an NDP sugar (e.g., a UDP monosaccharide) and the substrate with a 1-3UGT enzyme described herein or a host cell or lysate thereof expressing the 1-3UGT enzyme. A variety of substrates, including but not limited to terpenoids, may be glycosylated according to the present disclosure. In some embodiments, the substrate is a terpenoid substrate, which may be a diterpenoid, such as steviol and / or a steviol glycoside. In some embodiments, the substrate includes stevioside and / or RebD. In various embodiments, the nucleotide diphosphate is UDP or ADP, or other NDPs capable of acting as glycosyl donor molecules. In various embodiments, the monosaccharide is glucose, galactose, fructose, rhamnose, or xylose. In some embodiments, the monosaccharide is glucose. The NDP sugar may be exogenously supplied for in vitro reactions or endogenously produced by the host cell for embodiments using microbial fermentation or biotransformation reactions. Various modifications may be made to the host cell to increase the available UDP glucose to support the reaction.

[0023] In some embodiments, the substrate comprises a plant extract, optionally a Stevia leaf extract. In various embodiments, microbial cells expressing 1-3UGTs may be fed steviol or lower steviol glycosides for the production of higher steviol glycosides, including RebD and RebM. Advanced intermediates from Stevia leaf extract are readily available from existing industrial extraction of steviol glycosides.

[0024] In various embodiments, the 1-3 UGT converts lower steviol glycosides to higher steviol glycosides. For example, a UGT enzyme may have 1-3' UGT activity for converting steviobioside to RebB, rubusoside to RebG, stevioside to RebA, RebA to RebI, RebG to Reb4, RebE to RebD, and / or RebD to RebM. Alternatively, a UGT may be used in combination with another 1-3 UGT enzyme(s) or enzyme with specificity preference for a particular substrate. For example, one UGT may preferentially act on C13 glycosyl substrates as a 1-3 UGT, while another UGT enzyme preferentially acts on C19 glycosyl substrates as a 1-3 UGT.

[0025] In some embodiments, the methods include growing host cells in the presence of a substrate. The substrate may be provided to the culture, and in some embodiments, the substrate is synthesized by the host cells. In some embodiments, the substrate includes steviol or a mixture of stevioside and RebA as major components, and the host cells express multiple UGT enzymes to produce target steviol glycosides, such as RebM. Item 1 A uridine diphosphate-dependent glycosyltransferase (UGT) enzyme comprising an amino acid sequence that is at least about 75% identical to the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6. Section 2 The enzyme according to Item 1, wherein the amino acid sequence is at least about 80% identical to SEQ ID NO: 5 or SEQ ID NO: 6. Section 3 Item 1, wherein the amino acid sequence is at least about 85% identical to SEQ ID NO: 5 or SEQ ID NO: 6. Section 4 The enzyme according to Item 1, wherein the amino acid sequence is at least about 90% identical to SEQ ID NO: 5 or SEQ ID NO: 6. Section 5 The enzyme according to Item 1, wherein the amino acid sequence is at least about 95% identical to SEQ ID NO: 5 or SEQ ID NO: 6. Section 6 The enzyme according to Item 1, wherein the amino acid sequence is at least about 98% identical to SEQ ID NO: 5 or SEQ ID NO: 6. Section 7 Item 1, wherein the amino acid sequence is the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6. Section 8 Item 1, wherein the amino acid sequence has 1 to 20 amino acid modifications independently selected from substitutions, deletions, and insertions relative to the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6. Section 9 9. The enzyme of claim 8, wherein the amino acid sequence has 1 to 10 amino acid modifications independently selected from substitutions, deletions, and insertions relative to the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6. Section 10 9. The enzyme of claim 8, wherein the amino acid sequence has 1 to 5 amino acid modifications independently selected from substitutions, deletions, and insertions relative to the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6. Section 11 Item 11. The enzyme of any one of items 8 to 10, wherein the enzyme comprises an amino acid substitution at a position corresponding to one or more of positions 29, 200, 357, and 414 of SEQ ID NO:1. Section 12 glycine (G) at a position corresponding to position 54 of SEQ ID NO:5; a leucine (L) at a position corresponding to position 111 of SEQ ID NO:5, and Item 12. The enzyme of any one of items 1 to 11, comprising one or more methionines (M) at a position corresponding to position 183 of SEQ ID NO:5. Section 13 13. The enzyme of paragraph 12, wherein the enzyme has a Gly at the position corresponding to position 54 of SEQ ID NO:5, a Leu at the position corresponding to position 111 of SEQ ID NO:5, and a Met at the position corresponding to position 183 of SEQ ID NO:5. Item 14 14. The enzyme of any one of paragraphs 1 to 13, wherein the enzyme comprises an insertion of about 6 to about 12 amino acids after a position corresponding to position 155 of SEQ ID NO:5. Item 15 15. The enzyme of clause 14, wherein the insertion is a flexible, hydrophilic sequence of mostly glycine and serine residues, optionally GSGGSG (SEQ ID NO: 7) or GSGGSGGSG (SEQ ID NO: 8). Section 16 Item 16. The enzyme of any one of items 1 to 15, wherein the enzyme comprises a deletion of one or more amino acids corresponding to amino acids E225 to T232 relative to the amino acid sequence of SEQ ID NO:6. Section 17 17. The enzyme of paragraph 16, wherein the enzyme comprises a deletion of the amino acids corresponding to amino acids E225 to T232 of SEQ ID NO:6. Section 18 Item 18. The enzyme of any one of items 1 to 17, wherein the enzyme comprises an amino acid substitution at one or more positions corresponding to positions 72, 305, 345, and 428 of SEQ ID NO:6. Section 19 19. The enzyme of claim 18, wherein the enzyme comprises one or more amino acid substitutions selected from glutamine (Q) at the position corresponding to position 72 of SEQ ID NO:6, cysteine ​​(C) at the position corresponding to position 305 of SEQ ID NO:6, phenylalanine (F) at the position corresponding to position 345 of SEQ ID NO:6, and isoleucine (I) at the position corresponding to position 428 of SEQ ID NO:6. Section 20 1. A uridine diphosphate-dependent glycosyltransferase (UGT) enzyme comprising an amino acid sequence that is at least about 60% identical to the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6, glycine (G) at a position corresponding to position 54 of SEQ ID NO:5; a leucine (L) at a position corresponding to position 111 of SEQ ID NO:5, and The enzyme comprising a methionine (M) at a position corresponding to position 183 of SEQ ID NO:5. Section 21 21. The enzyme of claim 20, wherein the amino acid sequence is at least about 70% identical to the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6. Section 22 21. The enzyme of claim 20, wherein the amino acid sequence is at least about 80% identical to the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6. Section 23 21. The enzyme of claim 20, wherein the amino acid sequence is at least about 90% identical to the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6. Section 24 21. The enzyme of claim 20, wherein the amino acid sequence is at least about 95% identical to the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6. Section 25 25. The enzyme of any one of paragraphs 20 to 24, wherein the 1-3UGT enzyme comprises an insertion of about 5 to about 15 amino acids after a position corresponding to position 155 of SEQ ID NO:5. Section 26 26. The enzyme of clause 25, wherein the insertion is a flexible, hydrophilic sequence of mostly glycine and serine residues, optionally GSGGSG (SEQ ID NO: 7) and GSGGSGGSG (SEQ ID NO: 8). Section 27 27. The enzyme of any one of clauses 20 to 26, wherein the enzyme comprises a deletion of one or more amino acids corresponding to amino acids E225 to T232 relative to the amino acid sequence of SEQ ID NO:6. Section 28 28. The enzyme of Clause 27, wherein the enzyme comprises a deletion of the amino acids corresponding to amino acids E225 to T232 of SEQ ID NO:6. Section 29 29. The enzyme of any one of paragraphs 20 to 28, wherein the enzyme comprises an amino acid substitution at one or more positions corresponding to positions 72, 305, 345, and 428 of SEQ ID NO:6. Item 30 30. The enzyme of claim 29, wherein the enzyme comprises one or more amino acid substitutions selected from glutamine (Q) at the position corresponding to position 72 of SEQ ID NO:6, cysteine ​​(C) at the position corresponding to position 305 of SEQ ID NO:6, phenylalanine (F) at the position corresponding to position 345 of SEQ ID NO:6, and isoleucine (I) at the position corresponding to position 428 of SEQ ID NO:6. Item 31 31. The enzyme of any one of paragraphs 20 to 30, wherein the amino acid sequence is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98% identical to the amino acid sequence of SEQ ID NO:9. Section 32 Item 32. The enzyme according to any one of Items 20 to 31, wherein the amino acid sequence has 1 to 20 amino acid alterations independently selected from substitutions, deletions, and insertions relative to the amino acid sequence of SEQ ID NO:9. Item 33 Item 32. The enzyme according to any one of Items 20 to 31, wherein the amino acid sequence has 1 to 10 amino acid alterations independently selected from substitutions, deletions, and insertions relative to the amino acid sequence of SEQ ID NO:9. Section 34 Item 32. The enzyme according to any one of Items 20 to 31, wherein the amino acid sequence has 1 to 5 amino acid alterations independently selected from substitutions, deletions, and insertions relative to the amino acid sequence of SEQ ID NO:9. Section 35 comprising an amino acid sequence having at least about 75% sequence identity with the amino acid sequence of SEQ ID NO: 1; deletion of one or more amino acids corresponding to E74 to T81; a substitution at position 357 corresponding to position 357, optionally with glycine; a substitution at a position corresponding to position 414, optionally with leucine; a substitution at position 29 corresponding to position 29, optionally with methionine; a substitution at a position corresponding to position 402, optionally with glutamine; a substitution at position 154 and the corresponding position, optionally with cysteine; a substitution at a position corresponding to position 194, optionally with phenylalanine; a substitution at position 277 corresponding to position 277, optionally with isoleucine; a substitution at a position corresponding to position 208, optionally with glutamine; a substitution at the position corresponding to position 140, optionally with proline; and Substitution at position 259 and corresponding positions, optionally with glutamic acid 1. A uridine diphosphate-dependent glycosyltransferase (UGT) enzyme comprising one or more modifications to SEQ ID NO:1 selected from: Section 36 36. The UGT enzyme of claim 35, wherein the UGT enzyme comprises a deletion of at least two or at least three amino acids corresponding to E74 to T81. Section 37 36. The UGT enzyme of paragraph 35, wherein the UGT enzyme comprises a deletion of at least four or at least five amino acids corresponding to E74 to T81. Section 38 36. The UGT enzyme of claim 35, wherein the UGT enzyme comprises a deletion of at least 6 or at least 7 amino acids corresponding to E74 to T81. Item 39 36. The UGT enzyme of claim 35, wherein the UGT enzyme comprises a deletion of the amino acids corresponding to E74 to T81. Section 40 40. The UGT enzyme of any one of paragraphs 35 to 39, wherein the enzyme further comprises an alanine substitution at a position corresponding to position 200 of SEQ ID NO:1. Section 41 41. The UGT enzyme of any one of paragraphs 35 to 40, wherein the enzyme has at least two, three, or four of 357G, 414L, 29M, 402Q, 154C, 194F, 277I, 208Q, 140P, and 259E, respectively, numbered according to SEQ ID NO:1. Section 42 42. The UGT enzyme of any one of paragraphs 35 to 41, wherein the enzyme is at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98% identical to the amino acid sequence of SEQ ID NO:1. Section 43 43. The UGT enzyme of paragraph 42, wherein the enzyme has 1 to 20, or 1 to 10, amino acid modifications independently selected from amino acid substitutions, deletions, and insertions relative to SEQ ID NO:1. Section 44 a circular permutant of SrUGT76G1, optionally having 1 to 20, or 1 to 15, or 1 to 10 amino acid modifications independently selected from amino acid substitutions, deletions, and insertions relative to the corresponding positions of SEQ ID NO: 1, wherein the circular permutant comprises: deletion of one or more amino acids corresponding to E74 to T81; a substitution at position 357 corresponding to position 357, optionally with glycine; a substitution at a position corresponding to position 414, optionally with leucine; a substitution at position 29 corresponding to position 29, optionally with methionine; a substitution at a position corresponding to position 402, optionally with glutamine; a substitution at position 154 and the corresponding position, optionally with cysteine; a substitution at a position corresponding to position 194, optionally with phenylalanine; a substitution at position 277 corresponding to position 277, optionally with isoleucine; a substitution at a position corresponding to position 208, optionally with glutamine; a substitution at the position corresponding to position 140, optionally with proline; and Substitution at position 259 and corresponding positions, optionally with glutamic acid 1. A uridine diphosphate-dependent glycosyltransferase (UGT) enzyme comprising one or more modifications to SEQ ID NO:1 selected from: Section 45 Item 45. A polynucleotide encoding the enzyme of any one of items 1 to 44. Section 46 A host cell comprising the polynucleotide of paragraph 45. Section 47 47. The host cell of paragraph 46, wherein the cell is a microorganism. Section 48 48. The host cell of paragraph 47, wherein the microorganism is a bacterium. Section 49 49. The host cell of paragraph 48, wherein the bacterium is E. coli. Section 50 50. The host cell of paragraph 49, wherein the host cell comprises a deletion or inactivation of ushA and galETKM, a deletion or inactivation of pgi, and overexpression or increased activity of pgm and galU. Section 51 48. The host cell of claim 47, wherein the microorganism is a yeast. Section 52 47. The host cell of paragraph 46, wherein the cell is a plant. Section 53 Item 53. The host cell of Item 52, wherein the plant is a stevia plant. Section 54 54. The host cell of any one of paragraphs 46 to 53, wherein the host cell expresses one or more additional UGT enzymes selected from a C-13 UGT enzyme, a C-19 UGT enzyme, and a 1-2' UGT enzyme. Section 55 55. The host cell of paragraph 54, wherein the host cell further expresses a heterologous C-13 UGT enzyme, a heterologous C-19 UGT enzyme, and a heterologous 1-2 UGT enzyme. Section 56 56. The host cell of any one of paragraphs 47 to 55, wherein the host cell produces steviol by expression of endogenous and / or heterologous enzymes for the biosynthesis of steviol. Section 57 57. A method for transferring a monosaccharide group to a substrate, comprising contacting an NDP sugar and the substrate with a UGT enzyme of any one of paragraphs 1 to 44, or a host cell or lysate thereof of any one of paragraphs 46 to 56. Section 58 58. The method of claim 57, wherein the substrate is a terpenoid. Section 59 Item 59. The method of item 58, wherein the terpenoid is a diterpenoid. Section 60 Item 60. The method of item 59, wherein the diterpenoid is steviol and / or steviol glycoside. Section 61 Item 61. The method of item 60, wherein the steviol glycoside is stevioside and / or RebD. Section 62 58. The method of claim 57, wherein the substrate is a plant extract, optionally a stevia leaf extract. Section 63 Item 59. The method of item 58, wherein the terpenoid is a monoterpenoid, sesquiterpenoid, or triterpenoid. Section 64 64. The method of claim 63, wherein the terpenoid is a triterpenoid, optionally mogrol or mogroside. Section 65 65. The method of any one of paragraphs 57 to 64, wherein the NDP sugar is UDP glucose. Section 66 66. The method of paragraph 65, wherein the reaction occurs in a host cell that has endogenous UDP glucose. Section 67 57. A method for transferring a glycosyl group to a substrate, the method comprising growing the host cell of any one of paragraphs 46 to 56 in the presence of the substrate. Section 68 68. The method of paragraph 67, wherein the substrate is synthesized by the host cell. Section 69 Item 69. The method of item 67 or 68, wherein the substrate is a terpenoid. Section 70 Item 70. The method of item 69, wherein the terpenoid is a diterpenoid. Section 71 71. The method of claim 70, wherein the diterpenoid is steviol and / or steviol glycoside. Section 72 Item 72. The method of item 71, wherein the steviol glycoside is stevioside and / or RebD. Section 73 70. The method of claim 69, wherein the terpenoid is a triterpenoid, optionally mogrol or mogroside. [Brief explanation of the drawings]

[0026] [Figure 1] The chemical structure of rebaudioside M (RebM), a minor member of the steviol glycoside family, is shown. RebM is a derivative of the diterpenoid steviol (steviol) with six glucosyl modifications. [Figure 2] The glycosylation pathway from steviol to RebM and other steviol glycosides is shown. UGTc13 is a glycosyltransferase capable of glycosylating steviol or steviol glycosides at their C13 hydroxyl group. UGTc19 is a glycosyltransferase capable of glycosylating steviol or steviol glycosides at their C19 carboxyl group. UGT1-2 is a glycosyltransferase capable of β1,2 glycosylating the C2' of 13-O-glucose and / or 19-O-glucose. UGT1-3 is a glycosyltransferase capable of β1,3 glycosylating the C3' of 13-O-glucose and / or 19-O-glucose. [Figure 3]Figure 1 shows an amino acid sequence alignment of SrUGT76G1 (SEQ ID NO: 1) and MbUGT1-3_2 (SEQ ID NO: 6), a circularly permuted form of SrUGT76G1. Panel A shows the N-terminal portion of SrUGT76G1 aligned with the C-terminal portion of MbUGT1-3_2. Panel B shows the C-terminal portion of SrUGT76G1 aligned with the N-terminal portion of MbUGT1-3_2. [Figure 4] 1 shows the percent conversion of stevioside to RebA, and the percent conversion of RebD to RebM in vitro by the following glycosyltransferases: UGT76G1-L200A (SEQ ID NO: 3), MbUGT1-3_0 (SEQ ID NO: 4), MbUGT1-3_1 (SEQ ID NO: 5), and MbUGT1-3_2 (SEQ ID NO: 6). [Figure 5] Figure 1 shows the fold improvement for the conversion of stevioside to RebA and RebD to RebM compared to UGT76G1-L200A. MbUGT1-3_1 and MbUGT1-3_2 show very significant improvements in enzyme productivity for both conversions. DETAILED DESCRIPTION OF THE INVENTION

[0027] In various aspects, the present invention provides uridine diphosphate-dependent glycosyltransferase (UGT) enzymes that can catalyze the transfer of a monosaccharide moiety of an NDP sugar (e.g., the UGT glucose) to the 3' carbon of a sugar moiety of a substrate, such as a terpenoid glycan, thereby functioning as a "1-3UGT." In other aspects, the present invention provides polynucleotides encoding 1-3UGTs and host cells containing the same. In still other aspects, the present invention provides methods for preparing glycosylated substrates, including steviol glycosides, using the enzymes and host cells of the present disclosure.

[0028] 1-3UGT enzymes exhibit high glycosyltransferase activity toward terpenoid glycosides, such as steviol glycosides. For example, 1-3UGT enzymes catalyze the transfer of a monosaccharide moiety of an NDP sugar to the 3' carbon of a sugar moiety on terpenoid glycans, such as stevioside and RebD. That is, when the substrate is a steviol glycoside, 1-3UGT can catalyze the NDP-dependent transfer of a monosaccharide moiety to the 3' carbon of both the C13- or C19-linked sugar (e.g., glucose) moiety. In some embodiments, 1-3UGT enzymes have a higher rate or productivity for glycosylation of C19-linked sugars compared to C13-linked sugars. In some embodiments, the monosaccharide is glucose, but in other embodiments, the monosaccharide may be galactose, fructose, rhamnose, xylose, or other monosaccharides.

[0029] In various embodiments, 1-3UGTs catalyze the biosynthesis of RebM. The structure of RebM is shown in Figure 1. RebM comprises a steviol backbone with six glycosylation sites: (1) C13 O-glycosylation, (2) C13 1-2 glycosylation, (3) C13 1-3 glycosylation, (4) C19 O-glycosylation, (5) C19 1-2 glycosylation, and (6) C19 1-3 glycosylation. As shown in Figure 2, 1-3UGTs can generate RebM by transferring an additional glucose to the RebD substrate. Additionally, 1-3UGT enzymes can catalyze the transfer of monosaccharides to other steviol glycoside substrates to produce products such as RebB (from steviolbioside), RebG (from rubusoside), Reb4 (from RebG), RebA (from stevioside), RebD (from RebE), and RebI (from RebA). In some embodiments, the substrate comprises a plant extract, such as a Stevia leaf extract, and 1-3UGT (together with the action of other glycosyltransferase enzymes) can glycosylate a variety of major or minor glycosylation products to produce a product that is predominantly RebM.

[0030] In one aspect, the invention provides a 1-3UGT enzyme comprising an amino acid sequence at least about 75% identical to the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6. In some embodiments, the 1-3UGT comprises an amino acid sequence at least about 80% identical to SEQ ID NO:5 or 6. In some embodiments, the amino acid sequence is at least about 85% identical to SEQ ID NO:5 or 6, or at least about 90% identical to SEQ ID NO:5 or 6, or at least about 95% identical to SEQ ID NO:5 or 6, or at least about 98% identical to SEQ ID NO:5 or 6. In some embodiments, the amino acid sequence comprises the amino acids of SEQ ID NO:5 or 6.

[0031] In some embodiments, the 1-3UGT enzyme comprises an amino acid sequence that is at least about 75% identical to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the 1-3UGT comprises an amino acid sequence that is at least about 80% identical to SEQ ID NO: 9. In some embodiments, the amino acid sequence is at least about 85% identical to SEQ ID NO: 9, alternatively at least about 90% identical to SEQ ID NO: 9, alternatively at least about 95% identical to SEQ ID NO: 9, alternatively at least about 98% identical to SEQ ID NO: 9. In some embodiments, the amino acid sequence comprises the amino acids of SEQ ID NO: 9.

[0032] For example, the amino acid sequence may have 1 to 20 amino acid modifications independently selected from substitutions, deletions, and insertions relative to the amino acid sequence SEQ ID NO: 5 or 6. In some embodiments, the amino acid sequence has 1 to 10 amino acid modifications (e.g., 1 to 5) independently selected from substitutions, deletions, and insertions relative to the amino acid sequence of SEQ ID NO: 5 or 6. In some embodiments, the UGT amino acid sequence has 1 to 20 amino acid modifications independently selected from substitutions, deletions, and insertions relative to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the amino acid sequence has 1 to 10 amino acid modifications (e.g., 1 to 5) independently selected from substitutions, deletions, and insertions relative to the amino acid sequence of SEQ ID NO: 9. Amino acid modifications to the amino acid sequence of SEQ ID NO: 5, 6, or 9 may be guided by available enzyme structures and homology model construction. Exemplary structures are described, for example, in Li, et al., "Crystal Structure of Medicago truncatula UGT85H2—Insights into the Structural Basis of a Multifunctional (iso) Flavonoid Glycosyltransferase," J. of Mol. Biol. 370.5 (2007): 951-963 and Lee et al., "Molecular Basis for Branched Steviol Glucoside Biosynthesis," PNAS 116:13131-13136 (2019). Publicly available crystal structures (e.g., PDB entry: 2PQ6) can be used to inform amino acid modifications. For example, one or more amino acid modifications can be made in or around the active site to improve substrate binding and / or the reaction geometry of these substrates with the catalytic side chains.

[0033] In some embodiments, the enzyme contains amino acid substitutions at positions corresponding to positions 29, 200, 357, and 414 of SEQ ID NO:1 (Stevia rebaudiana UGT76G1). Substitutions at these positions in the enzymes of SEQ ID NOs:5, 6, and 9 (positions 183, 354, 54, and 111, respectively, of SEQ ID NO:5) can result in significantly improved activity. In some embodiments, matching amino acids at positions corresponding to positions 183, 354, 54, and 111 of SEQ ID NO:5 allows for further modifications at other positions. For example, in some embodiments, the 1-3UGT enzyme comprises an amino acid sequence at least about 60% identical to the amino acid sequence of SEQ ID NO:5, 6, or 9, wherein the UGT enzyme comprises glycine (G) or threonine (T) at a position corresponding to position 54 of SEQ ID NO:5, leucine (L) or isoleucine (I) at a position corresponding to position 111 of SEQ ID NO:5, methionine (M) or leucine (L) at a position corresponding to position 183 of SEQ ID NO:5, and alanine (A), or glycine (G), or serine (S) at a position corresponding to position 354 of SEQ ID NO:5. In some embodiments, the 1-3UGT enzyme comprises methionine (M) at a position corresponding to position 183 of SEQ ID NO:5. In some embodiments, the 1-3UGT enzyme comprises glycine (G) at a position corresponding to position 54 of SEQ ID NO:5. In some embodiments, the 1-3UGT enzyme comprises leucine (L) at a position corresponding to position 111 of SEQ ID NO:5. In some embodiments, the 1-3UGT has two or three methionines (M) at a position corresponding to position 183 of SEQ ID NO: 5, a glycine (G) at a position corresponding to position 54 of SEQ ID NO: 5, and a leucine (L) at a position corresponding to position 111 of SEQ ID NO: 5. These modifications can result in significant improvements in the activity of the enzyme.

[0034] The 1-3UGT enzyme may include a substitution other than the serine at the corresponding position (357) of SEQ ID NO:1 at a position corresponding to position 54 of SEQ ID NO:5. Thus, in some embodiments, the substitution at the position corresponding to position 54 of SEQ ID NO:5 is a hydrophobic amino acid, such as alanine (A), valine (V), leucine (L), isoleucine (I), phenylalanine (F), or methionine (M). In some embodiments, the amino acid at the position corresponding to position 54 of SEQ ID NO:5 has a side chain that is incapable of forming a hydrogen bond. In other embodiments, the amino acid at the position corresponding to position 54 of SEQ ID NO:5 is glycine (G), asparagine (N), cysteine ​​(C), glutamine (Q), threonine (T), or tyrosine (Y).

[0035] In various embodiments, the 1-3UGT enzyme includes a leucine (L) amino acid at a position corresponding to position 111 of SEQ ID NO:5. In some embodiments, the 1-3UGT enzyme may include an amino acid other than leucine at this position. In various embodiments, the position corresponding to position 111 of SEQ ID NO:5 is not valine, the amino acid at the corresponding position in SEQ ID NO:1. Other suitable substitutions for position 111 may include glycine (G), alanine (A), isoleucine (I), or methionine (M). In some embodiments, the amino acid at position 111 of SEQ ID NO:5 has a less hydrophobic and / or less bulky side chain than valine.

[0036] In various embodiments, the UGT comprises a methionine (M) amino acid at a position corresponding to position 183 of SEQ ID NO: 5. In various embodiments, the UGT comprises another suitable amino acid at this position other than isoleucine, which is at the corresponding position of SEQ ID NO: 1. For example, the amino acid at this position may be less hydrophobic than isoleucine and / or may have a side chain that may provide for hydrogen bonding. Some exemplary substitutions at the position corresponding to position 183 of SEQ ID NO: 5 include alanine (A), valine (V), leucine (L), cysteine ​​(C), serine (S), threonine (T), tyrosine (Y), asparagine (N), glutamine (Q), aspartic acid (D), and glutamic acid (E).

[0037] In various embodiments, the 1-3UGT contains an alanine (A) or a glycine (G) at a position corresponding to position 354 of SEQ ID NO: 5. In various embodiments, the amino acid at this position has a less hydrophobic and / or less bulky side chain than the leucine at the corresponding position in SEQ ID NO: 1.

[0038] In some embodiments, the 1-3 UGT enzyme includes an insertion of 5 to about 15 amino acids, e.g., 6 to 12 amino acids, or about 6 or about 11 amino acids, after a position corresponding to position 155 of SEQ ID NO:5 (relative to UGT76G1, SEQ ID NO:1). In some embodiments, the insertion is a flexible, hydrophilic sequence consisting mostly of glycine and serine residues. In some embodiments, the sequence is GSGGSG (SEQ ID NO:7) or GSGGSGGSG (SEQ ID NO:8).

[0039] In various embodiments, the 1-3UGT enzymes exhibit improved conversion of stevioside to RebA and improved conversion of RebD to RebM compared to UGT76G1-L200A (SEQ ID NO: 3). This improved conversion is demonstrated in bioconversion assays in which stevioside or RebD substrates are fed to microbial cells expressing the disclosed 1-3UGT enzymes. The improved conversion can be demonstrated in reactions using cell lysates containing recombinantly expressed 1-3UGTs or in in vitro reactions using purified or partially purified 1-3UGTs. Such reactions are well known in the art. Alternatively, whole-cell assays can be used. For example, an E. coli strain expressing a 1-3UGT enzyme (ΔushA, ΔgalETKM, Δpgi, overexpressing pgm, galU) is grown overnight in a 96-well plate at 250 rpm and 37°C. The cells are then transferred to a new production culture to make up 10% of the total volume. The production culture contains 0.5 mM substrate (e.g., stevioside or rebaudioside D). The production culture is then grown in a 96-well plate at 250 rpm and 37° C. for 48 hours. The product can be quantified using LC-MS QQQ.

[0040] In some embodiments, amino acid matches at positions 183, 354, 54, and 111 of SEQ ID NO:5 allow for additional modifications at other positions. For example, in some embodiments, a 1-3UGT enzyme comprises an amino acid sequence at least about 60% identical to the amino acid sequence of SEQ ID NO:5, 6, or 9, wherein the UGT enzyme comprises glycine (G) or threonine (T) at a position corresponding to position 54 of SEQ ID NO:5, leucine (L) or isoleucine (I) at a position corresponding to position 111 of SEQ ID NO:5, methionine (M) or leucine (L) at a position corresponding to position 183 of SEQ ID NO:5, and alanine (A), glycine (G), or serine (S) at a position corresponding to position 354 of SEQ ID NO:5. In some embodiments, the 1-3UGT enzyme comprises methionine (M) at a position corresponding to position 183 of SEQ ID NO:5. In some embodiments, the 1-3UGT enzyme comprises glycine (G) at a position corresponding to position 54 of SEQ ID NO:5. In some embodiments, the 1-3UGT enzyme contains a leucine (L) at a position corresponding to position 111 of SEQ ID NO:5. In some embodiments, the 1-3UGT has two or three methionines (M) at a position corresponding to position 183 of SEQ ID NO:5, a glycine (G) at a position corresponding to position 54 of SEQ ID NO:5, and a leucine (L) at a position corresponding to position 111 of SEQ ID NO:5. These modifications can result in significant improvements in the activity of the enzyme. In some embodiments, the amino acid sequence is at least about 70% identical to the amino acid sequence of SEQ ID NO:5, 6, or 9, or at least about 80% identical to the amino acid sequence of SEQ ID NO:5, 6, or 9, or at least about 90% identical to the amino acid sequence of SEQ ID NO:5, 6, or 9, or at least about 95% identical to the amino acid sequence of SEQ ID NO:5, 6, or 9.

[0041] In some embodiments, the 1-3UGT enzyme includes a glycine (G) amino acid at a position corresponding to position 54 of SEQ ID NO:5, a leucine (L) amino acid at a position corresponding to position 111 of SEQ ID NO:5, and a methionine (M) amino acid at a position corresponding to position 183 of SEQ ID NO:5.

[0042] In some embodiments, the 1-3UGT enzyme comprises a glycine (G) amino acid at a position corresponding to position 54 of SEQ ID NO:5 and a leucine (L) amino acid at a position corresponding to position 111 of SEQ ID NO:5.

[0043] In some embodiments, the 1-3UGT enzyme comprises a glycine (G) amino acid at a position corresponding to position 54 of SEQ ID NO:5 and a methionine (M) amino acid at a position corresponding to position 183 of SEQ ID NO:5.

[0044] In some embodiments, the 1-3UGT enzyme comprises a leucine (L) amino acid at a position corresponding to position 111 of SEQ ID NO:5 and a methionine (M) amino acid at a position corresponding to position 183 of SEQ ID NO:5.

[0045] In these or other embodiments, the 1-3 UGT enzyme has one or more amino acid deletions at positions E225 through T232 relative to the amino acid sequence of SEQ ID NO: 6. For example, the UGT may have at least two, at least three, at least four, at least five, at least six, at least seven, or eight amino acid deletions corresponding to amino acids E225 through T232 of SEQ ID NO: 6.

[0046] In these or other embodiments, the 1-3UGT enzyme further comprises one or more amino acid substitutions at positions corresponding to position 72, position 305, position 345, and position 428. For example, the 1-3UGT enzyme may have a substitution of glutamine (Q) or asparagine (N) at a position corresponding to position 72 of SEQ ID NO:6. In some embodiments, the 1-3UGT enzyme has a substitution of a neutral hydrophilic amino acid, such as cysteine ​​(C), serine (S), or threonine (T), at a position corresponding to position 305 of SEQ ID NO:6. In some embodiments, the 1-3UGT enzyme has a substitution of phenylalanine (F) or tryptophan (W) at a position corresponding to position 345 of SEQ ID NO:6. In some embodiments, the 1-3UGT enzyme has a substitution of isoleucine (I), valine (V), or alanine (A) at a position corresponding to position 428 of SEQ ID NO:6. In some embodiments, the 1-3UGT enzyme has two, three, or four of the following substitutions relative to SEQ ID NO:6: S72Q, A305C, Y345F, and L428I.

[0047] In some embodiments, the 1-3UGT enzyme includes an insertion of 5 to about 15 amino acids, e.g., about 6 to about 12 amino acids, or about 6 or about 11 amino acids (relative to 76G1) after a position corresponding to position 155 of SEQ ID NO:5. In some embodiments, the insertion is a flexible, hydrophilic sequence, such as an amino acid sequence of mostly glycine and serine residues. In some embodiments, the sequence is GSGGSG (SEQ ID NO:7) or GSGGSGGSG (SEQ ID NO:8).

[0048] In still other aspects and embodiments, the 1-3UGT enzyme is not a circular permutant of UGT76G1, i.e., the enzyme comprises an amino acid sequence having at least about 75% sequence identity to the amino acid sequence of SEQ ID NO: 1. In such embodiments, the enzyme may comprise one or more amino acid modifications described herein. Exemplary modifications of UGT76G1 (SEQ ID NO:1) include modifications to SEQ ID NO:1 selected from the following: (i) a deletion of one or more amino acids corresponding to E74 to T81, (ii) a substitution at a position corresponding to position 357, optionally with a glycine, (iii) a substitution at a position corresponding to position 414, optionally with a leucine, (iv) a substitution at a position corresponding to position 29, optionally with a methionine, (v) a substitution at a position corresponding to position 402, optionally with a glutamine, (vi) a substitution at a position corresponding to position 154, optionally with a cysteine, (vii) a substitution at a position corresponding to position 194, optionally with a phenylalanine, (viii) a substitution at a position corresponding to position 277, optionally with an isoleucine, (ix) a substitution at a position corresponding to position 208, optionally with a glutamine, (x) a substitution at a position corresponding to position 140, optionally with a proline, and (xi) a substitution at a position corresponding to position 259, optionally with a glutamic acid.

[0049] For example, in some embodiments, the UGT enzyme includes a deletion of at least two, or at least three, at least four, at least five, at least six, at least seven, or all eight amino acids corresponding to E74 through T81 of SEQ ID NO: 1. In various embodiments, the enzyme further includes an alanine substitution at a position corresponding to position 200 of SEQ ID NO: 1. In some embodiments, the enzyme has at least two, three, or four of 357G, 414L, 29M, 402Q, 154C, 194F, 277I, 208Q, 140P, and 259E, respectively, numbered according to SEQ ID NO: 1.

[0050] In such embodiments, the enzyme may have at least about 80% sequence identity, or at least about 85% sequence identity, or at least about 90% sequence identity, or at least about 95% sequence identity, or at least about 98% sequence identity to the amino acid sequence of SEQ ID NO: 1. For example, the enzyme may have 1 to 20, or 1 to 10 amino acid modifications independently selected from amino acid substitutions, deletions, and insertions relative to SEQ ID NO: 1.

[0051] In still other aspects and embodiments, the 1-3UGT enzyme is a circular permutant of SrUGT76G1 (see US 2017 / 0332673, incorporated herein by reference in its entirety), optionally having 1 to 20, or 1 to 15, or 1 to 10 amino acid modifications independently selected from amino acid substitutions, deletions, and insertions relative to the corresponding positions in SEQ ID NO:1. Exemplary modifications to positions in SEQ ID NO: 1 may be selected from the following: (i) a deletion of one or more amino acids corresponding to E74 to T81 (as described herein); (ii) a substitution at a position corresponding to position 357, optionally with a glycine; (iii) a substitution at a position corresponding to position 414, optionally with a leucine; (iv) a substitution at a position corresponding to position 29, optionally with a methionine; (v) a substitution at a position corresponding to position 402, optionally with a glutamine; (vi) a substitution at a position corresponding to position 154, optionally with a cysteine; (vii) a substitution at a position corresponding to position 194, optionally with a phenylalanine; (viii) a substitution at a position corresponding to position 277, optionally with an isoleucine; (ix) a substitution at a position corresponding to position 208, optionally with a glutamine; (x) a substitution at a position corresponding to position 140, optionally with a proline; and (xi) a substitution at a position corresponding to position 259, optionally with a glutamic acid. In some embodiments, the UGT enzyme comprises a deletion of amino acids corresponding to E74 to T81.

[0052] Changes in the amino acid sequence of an enzyme may alter its activity or may have no measurable effect. Silent changes without measurable effects are most likely conservative substitutions and small insertions or deletions in solvent-exposed surfaces located away from the active site and substrate binding site. In contrast, enzyme activity is more likely to be affected by non-conservative substitutions, large insertions or deletions, and changes in buried positions within the active site, substrate binding site, and important for protein folding or conformation. Changes that alter enzyme activity may increase or decrease the reaction rate, or may increase or decrease the affinity or specificity for a particular substrate. For example, a change that increases the size of the substrate binding site may allow the enzyme to act on larger substrates, and a change that positions the catalytic amino acid side chain closer to the substrate target site may increase the enzyme's rate.

[0053] Knowledge of the enzyme's three-dimensional structure and the location of the relevant active site, substrate-binding site, and other interaction sites can facilitate the rational design of mutations and provide mechanistic insight into the phenotype of specific alterations. Plant UGTs share highly conserved secondary and tertiary structures while possessing relatively low amino acid sequence identity. Osmani et al., Substrate specificity of plant UDP-dependent glycosyltransferases predicted from crystal structures and homology modeling, Phytochemistry 70 (2009) 325-347. The sugar acceptor and donor substrates of UGTs are accommodated in a cleft formed between the N- and C-terminal domains. Several regions of the primary sequence contribute to the formation of the substrate-binding pocket, including structurally conserved domains and loop regions that differ in amino acid sequence and sequence length.

[0054] The construction of UGT derivatives may be guided by structural analysis and homology modeling (see Soon Goo Lee, et al., Molecular Basis for Branched Steviol Glucoside Biosynthesis, PNAS, June 19, 2019).

[0055] For example, based on the independent crystal structure generation and analysis of SrUGT76G1_L200A and the amino acid sequence alignment of SrUGT76G1 to MbUGT3-1_1, the steviol core of stevioside is predicted to be close (within 4 Å) to the following residues of MbUGT3-1_1: I244, L280, W351, A354, I357, M362, and T438. Furthermore, the C19 1-2 glycosylation is predicted to be close (within 4 Å) to T438. The steviol core of RebD is predicted to be close (within 4 Å) to the following hydrophobic side chains of MbUGT3-1_1: L239, M242, I244, L280, I353, A354, and I357. The C13 1-2' glycosylation is predicted to be close (within 4 Å) to the following hydrogen-bonded side chains of MbUGT3-1_1: S301 and D77. Because the L200A and corresponding mutations are located in the loops between the V341-Q352 and K355-A367 helices of MbUGT3-1_1, the positioning and amino acid content of these helices in MbUGT3-1_1 may be important for catalysis. Positions L76 and / or D77 of MbUGT3-1_1 may also interact with the C13 glycosylation of stevioside.

[0056] Amino acid substitutions may be conservative or non-conservative. A conservative substitution is defined when the old and new amino acids have similar properties, such as size and charge. Naturally occurring residues are divided into groups based on the following general side chain properties: (Group 1) Hydrophobic (aliphatic): methionine (Met), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), (Group 2) Neutral hydrophilic: cysteine ​​(Cys), serine (Ser), threonine (Thr), asparagine (Asn), glutamine (Gln), (Group 3) Acidic: Aspartic acid (Asp), Glutamic acid (Glu), (Group 4) Basic: histidine (His), lysine (Lys), arginine (Arg), (Group 5) residues that influence chain orientation: glycine (Gly), proline (Pro), and (Group 6) Aromatic: tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe).

[0057] Non-conservative substitutions would involve exchanging a member of one of these classes for another amino acid from another class.

[0058] The amino acid sequence of the UGT enzyme can optionally contain an alanine inserted or substituted at position 2 to reduce turnover in cells. In various embodiments, the 1-3 UGT enzyme contains an alanine amino acid residue inserted or substituted at position 2 relative to SEQ ID NO: 5, 6, or 9 to provide additional stability in vivo.

[0059] The identity of amino acid sequences, i.e., the percentage of sequence identity, can be determined by sequence alignment. Such alignment can be performed using several known algorithms, such as those described by Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877), using hmmalign (HMMER package), or using the CLUSTAL algorithm (Thompson, JD, Higgins, DG & Gibson, TJ (1994) Nucleic Acids Res. 22, 4673-80). The grade of sequence identity (sequence matching) can be calculated, for example, using BLAST, BLAT, or BlastZ (or BlastX). Similar algorithms are incorporated into the BLASTN and BLASTP programs of Altschul et al. (1990) J. Mol. Biol. 215:403-410. BLAST protein alignments may be performed using the BLASTP program, score = 50, word length = 3. To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Altschul et al (1997) Nucleic Acids Res. 25: 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs are used.

[0060] In another aspect, the present invention provides polynucleotides encoding the 1-3UGT enzymes disclosed herein, as well as host cells containing the same. The host cells may be microorganisms, fungal cells, algae cells, or plant cells. The plant may be a stevia plant, or in particular a Stevia rebaudiana plant. Stevia plants naturally express the enzymes required to synthesize steviol and steviol glycosides, but produce only trace amounts of highly glycosylated steviol glycosides, such as RebM. In contrast, the RebM content of a Stevia or Stevia rebaudiana plant expressing a polynucleotide encoding a 1-3UGT may be greater than about 1%, or greater than about 2%, or greater than about 5%, or greater than about 10% of the steviol glycoside content of the plant's leaves. Additionally, the RebA content of a Stevia or Stevia rebaudiana plant expressing a polynucleotide encoding a 1-3UGT may be greater than about 5%, or greater than about 10%, or greater than about 15% of the steviol glycoside content of the plant's leaves.

[0061] Microbial host cells in various embodiments can be prokaryotic or eukaryotic. In some embodiments, the microbial host cell is a bacterium selected from the genera Escherichia, Bacillus, Corynebacterium, Rhodobacter, Zymomonas, Vibrio, and Pseudomonas. For example, in some embodiments, the bacterial host cell is a species selected from Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Rhodobacter capsulatus, Rhodobacter sphaeroides, Zymomonas mobilis, Vibrio natriegens, or Pseudomonas putida. In some embodiments, the bacterial host cell is E. coli. Alternatively, the microbial cell may be a yeast cell, such as, but not limited to, a species of Saccharomyces, Pichia, or Yarrowia, including Saccharomyces cerevisiae, Pichia pastoris, and Yarrowia lipolytica.

[0062] The polynucleotide encoding the 1-3UGT enzyme may be integrated into the chromosome of the microbial cell or may be expressed extrachromosomally, for example, the 1-3UGT enzyme may be expressed from a bacterial artificial chromosome (BAC) or a plasmid.

[0063] Expression of UGT enzymes may be tuned for optimal activity using, for example, gene modules (e.g., operons) or for independent expression of UGT enzymes. For example, gene or operon expression can be regulated by selecting promoters with different strengths (e.g., strong, medium, or weak), such as inducible or constitutive promoters. Some non-limiting examples of promoters of different strengths include Trc, T5, and T7. Furthermore, gene or operon expression can be regulated by manipulating the copy number of the gene or operon in the cell. In some embodiments, the cell expresses a single copy of each UGT enzyme. In some embodiments, gene or operon expression can be regulated by manipulating the order of genes within a module, where genes that are transcribed first are generally expressed at higher levels. In some embodiments, gene or operon expression is regulated by integrating one or more genes or operons into a chromosome.

[0064] Optimization of UGT expression can be achieved by selecting appropriate promoters and ribosome binding sites. In some embodiments, this may involve selecting high copy number plasmids, or single copy, low copy, or medium copy number plasmids. The step of transcription termination can also be targeted to regulate gene expression by introducing or eliminating structures such as stem loops.

[0065] In some embodiments, the cell is a plant cell. For example, the polynucleotide may be heterologously expressed in a stevia plant under the control of a suitable promoter, such as a constitutive or inducible promoter.

[0066] In various embodiments, the host cell may express one or more additional UGT enzymes selected from a C-13 UGT enzyme, a C-19 UGT enzyme, and a 1-2 UGT enzyme. In some embodiments, the host cell expresses a heterologous C-13 UGT enzyme, a heterologous C-19 UGT enzyme, and a heterologous 1-2 UGT enzyme (in addition to the 1-3 UGT enzyme) and is thereby capable of glycosylation of steviol and steviol glycoside substrates to produce RebM.

[0067] Figure 2 shows the structures of steviol and various steviol glycosides and identifies the enzyme activities in the biosynthetic pathway from steviol to RebM. Similarly, Table 1 identifies the substrates and products for the biosynthetic pathway from steviol to RebM. For each substrate and product pair, Table 1 indicates the type of glycosylation and identifies the enzymes with the required activity. Four types of glycosylation activity are required for the production of RebM: a primary glycosylation at the C13 and C19 carbons, and a secondary glycosylation at the 2' or 3' position of the primary glycan. Glycans are added one six-carbon monosaccharide unit at a time. Therefore, the primary glycan must be attached to C13 or C19 of steviol or a steviol glycoside before the secondary glycan can be attached to the primary glycan. However, the order of glycosylation of steviol glycosides is not otherwise limited. Table 2 identifies enzymes from various sources with the activities required for the biosynthetic pathway from steviol to RebM and provides references to the nucleotide and amino acid sequences. See U.S. Patent Application Publication No. 20170332673, which is incorporated herein by reference in its entirety.

[0068] [Table 1]

[0069] [Table 2]

[0070] In some embodiments, host cells produce steviol by expression of endogenous and / or heterologous enzymes for steviol biosynthesis. Host cells may be engineered to co-express 1-3 UGTs with other UGT enzymes active on terpenoids and terpenoid glycosides, such as C-13 UGT enzymes, C-19 UGT enzymes, and 1-2 UGT enzymes (see Tables 1-2). Microbial cells may further be engineered to co-express enzymes for steviol biosynthesis, such as enzymes of the MEP or MVA pathway, copalyl synthase and kaurene synthase (which may exist as a bifunctional enzyme in some embodiments), P450 enzymes, such as kaurene oxidase and kaurenoic acid hydroxylase, and P450 reductase enzymes. Table 3. Pathways and enzymes for steviol biosynthesis are disclosed in US20170332673, which is incorporated herein by reference in its entirety.

[0071] [Table 3]

[0072] In some aspects, the present invention provides methods for transferring a monosaccharide group to a substrate. The methods include contacting an NDP sugar (e.g., a UDP monosaccharide) and the substrate with a 1-3UGT enzyme described herein, or a host cell or lysate thereof expressing the 1-3UGT enzyme. A variety of substrates, including but not limited to terpenoids, may be glycosylated according to the present disclosure. In some embodiments, the substrate is a terpenoid substrate, which may be a diterpenoid, such as steviol and / or a steviol glycoside. In some embodiments, the substrate includes stevioside and / or RebD. In various embodiments, the monosaccharide is glucose, galactose, fructose, rhamnose, or xylose. In some embodiments, the monosaccharide is glucose. In some embodiments, the methods are performed in vitro using recombinant 1-3UGT and include an exogenously added NDP sugar (e.g., UDP-glucose or ADP-glucose) reagent. In other embodiments, the 1-3UGT is recombinantly expressed in the cell, and the NDP-glucose is endogenously available. In these embodiments, the substrate may be supplied to the cell and is available in the cell for the glycosyltransferase reaction.

[0073] Whole-cell conversion (i.e., "biotransformation") requires that the substrate (e.g., glycosidic intermediate) and product be transported into or out of the cell, respectively, and that the cell provide for UDP-glucose coenzyme regeneration. This contrasts with processes that use enzymes from cell lysis or extracellular secretion, which require an exogenous NDP-glucose supply, or an NDP-glucose precursor or NDP-glucose regeneration mechanism, or an NDP-glucose regeneration enzyme system. In embodiments of the present invention, the catalytic reaction (glycosylation) occurs within living microbial cells. UDP-glucose coenzyme recycling is carried out using native cellular metabolism without the need for externally provided enzymes or expensive substrates. According to some embodiments, the glycosidic intermediate is transported into the cell, and the product is transported out of the cell.

[0074] US2017 / 0332673 describes E. coli strains overexpressing MEP pathway enzymes, along with the downstream steviol biosynthetic pathway and UGT enzymes that drive the production of RebM from glucose. However, these strains do not biocatalyze the fed steviol glycoside intermediates to RebM. This may be due, in part, to the inability of the host cells to take up the steviol glycoside substrate. In some embodiments, genetic modifications to the microbial cells allow for the translocation of glycosylated intermediates into the cell while the highly glycosylated products are secreted into the culture medium.

[0075] In some embodiments, the microbial cells have one or more genetic modifications that increase UDP-glucose availability. In some embodiments, without wishing to be bound by theory, these modifications may also stress the cells for glucose availability, leading to increased expression of endogenous transporters that import steviol glycosides into the cells. Wild-type UDP-glucose levels in exponentially growing E. coli are approximately 2.5 mM (Bennett BD, et al., Absolute metabolite concentrations and implied enzyme active site occupancy in Escherichia coli. Nat Chem Biol. 2009;5(8):593-9). In some embodiments, the genetic modifications to the host cells are engineered to increase UDP-glucose in exponentially growing cells (e.g., without recombinant expression of UGT enzymes), for example, to at least 5 mM, or at least 10 mM.

[0076] In some embodiments, the microbial cells have a deletion, inactivation, or reduced activity or expression of a gene encoding an enzyme that consumes UDP-glucose. For example, the microbial cells may have a deletion, inactivation, or reduced activity of one or more of ushA (a UDP-sugar hydrolase) and / or galE, galT, galK, and galM (involved in UDP-galactose biosynthesis from UDP-glucose), or their orthologs in various microbial species. In some embodiments, the galETKM genes are inactivated, removed, or have substantially reduced expression.

[0077] In these or other embodiments, the microbial cells have a deletion, inactivation, or reduced activity or expression of a gene encoding an enzyme that consumes a precursor of UDP-glucose. For example, in some embodiments, the microbial cells have a deletion, inactivation, or reduced activity or expression of pgi (glucose-6-phosphate isomerase) or its orthologs in various microbial species of the host cell.

[0078] In these or other embodiments, the cells have overexpression or increased activity of one or more genes encoding enzymes involved in the conversion of glucose-6-phosphate to UDP-glucose. For example, pgm (phosphoglucomutase) and / or galU (UTP glucose-1-phosphate uridylyltransferase) (or orthologs or derivatives thereof) may be overexpressed or modified to increase enzyme productivity. See US 2020 / 0087692, incorporated herein by reference in its entirety.

[0079] In some embodiments, the substrate is a plant extract, optionally a Stevia leaf extract. In various embodiments, microbial cells expressing 1-3UGTs may be fed steviol or lower steviol glycosides for the production of higher steviol glycosides, including RebD and RebM. Advanced intermediates from Stevia leaf extract are readily available from existing industrial extraction of steviol glycosides. As shown in Table 4, available leaf extracts primarily contain the pathway intermediates stevioside and rebaudioside A (RebA). In various embodiments, the Stevia leaf extract is an extract of steviol glycosides. In some embodiments, the extract contains one or more of stevioside, steviolbioside, and rebaudioside A as major components. The major component generally accounts for at least about 10% of the steviol glycosides in the extract, but in some embodiments may account for at least about 20%, or at least about 25%, or at least about 30% of the steviol glycosides in the extract.

[0080] [Table 4]

[0081] In various embodiments, the 1-3 UGT converts lower steviol glycosides to higher steviol glycosides. For example, a UGT enzyme may have 1-3' UGT activity for converting steviobioside to RebB, rubusoside to RebG, stevioside to RebA, RebA to RebI, RebG to Reb4, RebE to RebD, and / or RebD to RebM. Alternatively, a UGT may be used in combination with another 1-3 UGT enzyme(s) or enzyme with specificity preference for a particular substrate. For example, one UGT may preferentially act on C13 glycosyl substrates as a 1-3 UGT, while another UGT enzyme preferentially acts on C19 glycosyl substrates as a 1-3 UGT.

[0082] In some embodiments, microbial cells expressing a 1-3UGT enzyme are fed RebD and convert at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 50%, or at least about 75%, or at least about 90% of the RebD to RebM. In various embodiments, such conversion is allowed to occur for at least about 8 hours, or in some embodiments, at least about 24 hours. For example, conversion may be allowed to occur in culture for 8 to about 72 hours. In some embodiments, conversion may be allowed to occur for about 24 to about 60 hours. In some embodiments, the microbial cells convert at least about 40%, or at least about 50%, or at least about 75%, or at least about 90% of the RebD to RebM in about 48 hours or less, or in about 24 hours or less.

[0083] In some embodiments, the microbial cells are fed stevioside, express a 1-3UGT, and convert at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 50% of the stevioside to RebA. In some embodiments, the enzyme converts at least about 75% or at least about 90% of the stevioside to RebA. In various embodiments, such conversion is allowed to occur for at least about 8 hours, or in some embodiments, at least about 24 hours. For example, conversion may be allowed to occur in culture for 8 to about 72 hours. In some embodiments, conversion may be allowed to occur for about 24 to about 60 hours. In some embodiments, the microbial cells convert at least about 40%, or at least about 50%, or at least about 75% of the stevioside to RebA in about 48 hours or less, or in about 24 hours or less.

[0084] While naturally occurring UGT enzymes are generally plant enzymes or derived from plant enzymes (often having temperature optima in the range of 20-24° C.), in some embodiments, the present disclosure enables production of glycosylated products in high yields in microbial cells (e.g., bacterial cells such as E. coli) that have enzyme productivity above 24° C., such as 24° C. to 37° C., or 27° C. to 37° C., or 30° C. to 37° C. In some embodiments, cultivation is performed at 30-34° C.

[0085] In some embodiments, the process is scalable for large-scale production, for example, in some embodiments, the culture size is at least about 100 L, at least about 200 L, at least about 500 L, at least about 1,000 L, or at least about 10,000 L, or at least about 100,000 L, or at least about 500,000 L.

[0086] In various embodiments, the method further includes recovering the glycosylation product from the cell culture or cell lysate. In some embodiments, the culturing produces at least about 100 mg / L, or at least about 200 mg / L, or at least about 500 mg / L, or at least about 1 g / L, or at least about 2 g / L, or at least about 5 g / L, or at least about 10 g / L, or at least about 20 g / L, or at least about 30 g / L, or at least about 40 g / L, or at least about 50 g / L of glycosylation product, which in some embodiments is extracted from the culture medium.

[0087] In some embodiments, the substrate is a terpenoid, such as a monoterpenoid, sesquiterpenoid, or triterpenoid. In some embodiments, the terpenoid is a triterpenoid, optionally mogrol or mogroside. In some embodiments, the 1-3 UGT enzymes of the present disclosure have broad substrate activity toward glycosides, aliphatic and branched alcohols, substituted phenols, flavonoids, and gallates. See, e.g., Dewitt, G. et al., "Screening of recombinant glycosyltransferases reveals the broad acceptor specificity of stevia UGT-76G1," J. Biotechnology 233 (2016) 49-55. Substrates for UGT enzymes can include terpenoid glycosides (isoprenoids), including diterpenoid glycosides, such as steviol glycosides, and triterpenoid glycosides, such as mogrosides.

[0088] In some embodiments, the method includes growing host cells in the presence of a substrate. The substrate may be provided to the culture, and in some embodiments, the substrate is synthesized by the host cells. In some embodiments, the substrate is steviol, and the host cells express multiple UGT enzymes to produce a target steviol glycoside, such as RebM.

[0089] In some embodiments, the glycosylated product (e.g., RebM) is purified from media components. Thus, in some embodiments, the method comprises separating the growth medium from the E. coli cells and isolating the desired glycosylated product (e.g., RebM) from the growth medium. In some embodiments, the product, such as RebM, is further extracted from the cellular material.

[0090] In some embodiments, the present invention provides methods for producing products containing glycosylated products such as RebM. The methods include incorporating target steviol glycosides (produced according to the present disclosure) into products such as foods, beverages, oral care products, sweeteners, flavorings, or other products. Purified steviol glycosides prepared according to the present invention may be used in a variety of products, including, but not limited to, foods, beverages, texturants (e.g., starches, fibers, gums, fats and fat mimetics, and emulsifiers), pharmaceutical compositions, tobacco products, nutraceutical compositions, oral hygiene compositions, and cosmetic compositions. Non-limiting examples of flavors with which RebM can be used in combination include lime, lemon, orange, fruit, banana, grape, pear, pineapple, mango, bitter almond, cola, cinnamon, sugar, cotton candy, and vanilla flavors. Non-limiting examples of other food ingredients include flavors, acidulants, and amino acids, colorants, bulking agents, modified starches, gums, texturizers, preservatives, antioxidants, emulsifiers, stabilizers, thickeners, and gelling agents.

[0091] In some embodiments, the present invention provides a method for producing a sweetener product comprising a plurality of high-intensity sweeteners, the plurality including two or more of steviol glycosides, mogrosides, sucralose, aspartame, neotame, advantame, acesulfame potassium, saccharin, cyclamate, neohesperidin dihydrochalcone, gnetifolin E, and / or piceatannol 4'-O-β-D-glucopyranoside. The method may further include incorporating the sweetener product into a food, beverage, oral care product, sweetener, flavoring, or other product containing the foregoing.

[0092] Target steviol glycoside(s), such as RebM, and sweetener compositions containing the same, can be used in combination with various physiologically active substances or functional ingredients. Functional ingredients are generally classified into categories such as carotenoids, dietary fiber, fatty acids, saponins, antioxidants, nutraceuticals, flavonoids, isothiocyanates, phenols, plant sterols and stanols (phytosterols and phytostanols), polyols, prebiotics, probiotics, phytoestrogens, soy proteins, sulfides / thiols, amino acids, proteins, vitamins, and minerals. Functional ingredients may also be classified based on health benefits, such as cardiovascular, cholesterol-lowering, and anti-inflammatory properties.

[0093] Furthermore, the target steviol glycoside(s), such as RebM, and sweetener compositions obtained by the present invention may be utilized as high-intensity sweeteners to produce zero-calorie, low-calorie, or diabetic beverages and foods with improved taste characteristics. They may also be used in beverages, foods, pharmaceuticals, and other products where sugar cannot be used. Furthermore, RebM and sweetener compositions may be used as sweeteners in beverages, foods, and other products intended for human consumption with improved characteristics, as well as in animal diets and feeds.

[0094] Examples of products in which the target steviol glycoside(s) and sweetener compositions can be used include alcoholic beverages such as vodka, wine, beer, distilled spirits, and sake; natural juices; carbonated soft drinks; diet drinks; zero-calorie drinks; low-calorie drinks and foods; yogurt drinks; instant juices; instant coffee; powdered instant drinks; canned goods; syrups; fermented miso paste; soy sauce; vinegar; dressings; mayonnaise; ketchup; curry; soups; instant bouillon; powdered soy sauce; powdered vinegar; and types of biscuits. , rice crackers, crackers, bread, chocolate, caramel, candy, chewing gum, jelly, pudding, preserved fruits and vegetables, fresh cream, jam, marmalade, flower paste, powdered milk, ice cream, sherbet, bottled vegetables and fruits, canned boiled beans, meat and food stewed in sweet sauces, agricultural vegetable foods, seafood, ham, sausage, fish ham, fish sausage, fish paste, fried fish products, dried seafood products, frozen foods, preserved seaweed, preserved meat, tobacco, medicines, and many others.

[0095] Conventional methods such as mixing, kneading, dissolving, soaking, infusing, extracting, sprinkling, spraying, infusing, and other methods may be used during the manufacture of products such as food, beverages, pharmaceuticals, cosmetics, tableware, and chewing gum.

[0096] Aspects and embodiments of the present invention will now be illustrated with reference to the following examples. [Example]

[0097] Aspects and embodiments of the present invention will now be illustrated with reference to the following examples.

[0098] Example 1: Screening of mutants of MbUGT1-3_1 (SEQ ID NO: 5) Plasmids containing the polynucleotide encoding the UGT MbUGT1-3_1 (SEQ ID NO: 5) and 95 selected mutants were transformed into E. coli cells (ΔushA, ΔgalETKM, Δpgi, overexpressing pgm, galU). The resulting strains were plated in rich seed medium and incubated overnight at 37°C at 250 rpm in 96-well plates to generate seed cultures.

[0099] The seed culture was inoculated into a production culture by adding 40 μL of seed to 360 μL of fresh production medium containing 0.5 mM stevioside and 0.5 mM rebaudioside D. The production culture was then grown in a 96-well plate at 250 rpm and 37° C. for 48 hours. Products were quantified using LC-MS QQQ.

[0100] The 95 mutants screened include the following (amino acid positions of these mutants are relative to MbUGT1-3_1 (SEQ ID NO: 5)): i) Substitution: F11L, K13R, T16E, V18L, E19A, G48T, S62A, D73P, L89W, V93L, Y94E, W99F, E100C, N 106R, E115K, Y119T, Q122E, K130W, V133A, V133S, M136K, S153L, G164R, R165N, R1 66G, I169L, F200L, F204H, F204P, K206A, K208D, K208N, F219D, F219S, L221P, R22 9A, I230F, L233I, G241A, R243M, P245S, I246L, R257D, E260A, L261K, L262Q, L264 S, D269E, E271P, S273A, R294P, L296I, S301N, F304S, H309F, V310A, T327S, A333V , F336L, V341I, Y348T, Q352A, Q352D, Q352E, A354S, A354T, K355Y, L358I, M361Q, I362V, S375T, E383F, V387L, I388E, I388S, R389Q, L400F, L404F, A406S, D413E, V418C, P426A, S428K, S428G, V441I, D445E, D455N, Q457G, Q457K / S458Q, or Q457K. ii) Substitutions and deletions: Q457K and S458 deletion (ΔS458). iii) Deletion: ΔG159, ΔG159ΔG161, or ΔG159ΔS160ΔG161. iv) Insertion: Insertion of S between positions 158 and 159 or insertion of K between 456 and 457.

[0101] All 95 mutants of MbUGT1-3_1 were screened for their ability to produce RebA, RebI, and RebM from stevioside (substrate). Table 5 shows the fold improvement of selected mutants of MbUGT1-3_1 in producing RebA and RebI (by 1-3 glycosylation at C-13-glucose) and RebM (by 1-3 glycosylation at C-19-glucose). In Table 5, column 2 generally corresponds to glycosylation at C-13-glucose of the substrate, and column 3 generally corresponds to glycosylation at C-19-glucose of the substrate. As shown, C-19 glycosylation is improved over C-13 glycosylation. The constructed mutant with the mutations in Table 5 is referred to herein as MbUGT1-3_2 (SEQ ID NO: 6).

[0102] [Table 5]

[0103] Example 2: Biotransformation with 1-3' glycosylation enzymes Plasmids containing polynucleotides encoding three UGTs, SrUGT76G1-L200A (SEQ ID NO: 1), MbUGT1-3_0 (SEQ ID NO: 4), MbUGT1-3_1 (SEQ ID NO: 5), and MbUGT1-3_2 (SEQ ID NO: 6), were individually transformed into E. coli cells (ΔushA, ΔgalETKM, Δpgi, overexpressing pgm, galU). The resulting strains were plated in culture medium containing 1 mM stevioside or 1 mM RebD. After growth, steviol glycosides were recovered from the culture medium. The strains were grown overnight in 96-well plates at 250 rpm and 37°C. The cells were then transferred to a new production culture at 10% of the total volume. Stevioside (0.5 mM) and rebaudioside D (0.5 mM) were each included in the production culture. The production cultures were then grown in 96-well plates for 48 hours at 250 rpm and 37° C. Products were quantified using LC-MS QQQ.

[0104] Figure 4 shows the percent conversion of stevioside to RebA and the percent conversion of RebD to RebM. The percent conversion in both reactions was highest for MbUGT1-3_1 and MbUGT1-3_2, which have the amino acid sequences of SEQ ID NO: 5 and SEQ ID NO: 6, respectively. Figure 5 shows the fold improvement of MbUGT1-3_1 and MbUGT1-3_2 compared to SrUGT76G1-L200A.

[0105] Example 3: Screening of mutants of MbUGT1-3_2 (SEQ ID NO: 6) A plasmid containing a polynucleotide encoding the UGT MbUGT1-3_2 (SEQ ID NO: 6) and 96 selected MbUGT1-3_2 mutants was transformed into E. coli cells (ΔushA, ΔgalETKM, Δpgi, ΔaraA, overexpressing pgm, galU, and UGT1-2 enzymes). The resulting strain was plated on rich seed medium and incubated overnight at 37°C at 250 rpm in a 96-well plate to generate seed cultures.

[0106] The seed culture was then inoculated into a production culture by adding 40 μL of seed to 360 μL of fresh production medium containing 1 g / L of stevioside / RebA leaf extract. The production culture was then grown in a 96-well plate at 250 rpm and 37° C. for 48 hours. Products were quantified using UPLC-DAD.

[0107] Mutants screened include the following (amino acid positions are relative to MbUGT1-3_1 (SEQ ID NO: 5)): i) Substitution: T16G, V18L, E19A, D23E, R31K, C64S, I70V, L76N, P79M, W99F, D114E, R125L, V133A, S1 50D, T202M, F204E, Y212F, Y212H, R218L, D227S, E228S, I230F, I230Y, L233I, G237S / P238A, P238S, L239E, L239G, A240S, A240V, M242A, M242I, I246Y, I247F, A252I, D253 E, L261K, L264E, A265D, E271P, S273A, C274G, C274L, Y282W, N292K, S302G, L303M, F30 6W, L312M, Q314L, L318W, P323L, E331A, K340R, S346V, Y348F / S349D / N350T / W351L / Q 352E / I353N, Y348G / S349E / N350F / W351G / Q352E / I353K, Y348T / S349N / N350E / W351P / Q352E / I353E, W351F / Q352E / A354G, W351P, Q352D / A354G, Q352E, A354S, I362L, A367N, E383S, E385A, H403Y, L404F, F419K, S428K, L431I, D445E, D445I, A450L, or Q457G. ii) Substitutions and deletions: D227T and Δ228 to 234 iii) Deletion: ΔG158 to G161, ΔG159 to G161, ΔS160 to ΔG161, ΔG161, Δ228, Δ228 to 229, Δ228 to 230, Δ228 to 231, Δ228 to 232, Δ228 to 233, Δ228 to 234, Δ228 to 235, Δ228 to 236, Δ228 to 237, ΔY320 to P323, ΔD325 to K326, or ΔK326. iv) Insertion: Insert LEA between F25 and L26. iv) Substitution: Exchange Y348~I353 for INPQG

[0108] Table 6 shows the ability of selected MbUGT1-3_2 (SEQ ID NO: 6) mutants to produce RebM. Column 2 of Table 6 shows the fold improvement in the percentage of RebM in the reaction product.

[0109] [Table 6]

[0110] Deletion of amino acids E225 to T232 resulted in a remarkable 11-fold improvement in the production of RebM. Based on homology modeling, amino acids E225 to T232 appear to form a loop near the bound substrate. Deletion of amino acids E225 to T232 may shift the substrate specificity of the UGT enzyme in favor of the reaction from RebD to RebM. The sequence with the deletion of the E225 to T232 loop and the S72Q, A305C, Y345F, and L428I mutations relative to MbUGT1-3_2 (SEQ ID NO: 6) is referred to as MbUGT1-3_3 (SEQ ID NO: 9).

[0111] Several deletions in the region from E225 to T232 were tested and also showed increased RebM production. Table 7 shows various beneficial deletions in the region from E225 to T232 (numbered according to SEQ ID NO: 6).

[0112] [Table 7]

[0113] array SEQ ID NO: 1 UGT76G1 [Stevia rebaudiana] (SEQ ID NO: 1) MENKTETTVRRRRRIILFPVPFQGHINPILQLANVLYSKGFSITIFHTNFNKPKTSNYPHFTFRFILDNDPQDERISNLPTHGPLAGMRIPIINEHGADELRRELELLMLASEE DEEVSCLITDALWYFAQSVADSLNLRRLVLMTSSLFNFHAHVSLPQFDELGYLDPDDKTRLEEQASGFPMLKVKDIKSAYSNWQILKEILGKMIKQTKASSGVIWNSFKELEESE LETVIREIPAPSFLIPLPKHLTASSSSLLDHDRTVFQWLDQQPPSSVLYVSFGSTSEVDEKDFLEIARGLVDSKQSFLWVVRPGFVKGSTWVEPLPDGFLGERGRIVKWVPQQE VLAHGAIGAFWTHSGWNSTLESVCEGVPMIFSDFGLDQPLNARYMSDVLKVGVYLENGWERGEIANAAIRRVMVDEEGEYIRQNARVLKQKADVSLMKGGSSYESLESLVSYISSL

[0114] SEQ ID NO: 2 MbUGT1-3 Reference: US2017 / 0332673 MANWQILKEILGKMIKQTKASSGVIWNSFKELEESELETVIREIPAPSFLIPLPKHLTASSSSLLDHDRTVFQWLDQQPPSSVLYVSFGSTSEVDEKDFLEIARGLVDSKQSFL WVVRPGFVKGSTWVEPLPDGFLGERGRIVKWVPQQEVLAHGAIGAFWTHSGWNSTLESVCEGVPMIFSDFGLDQPLNARYMSDVLKVGVYLENGWERGEIANAIRRVMVDEEGEY IRQNARVLKQKADVSLMKGGSSYESLESLVSYISSLENKTETTVRRRRRIILFPVPFQGHINPILQLANVLYSKGFSITIFHTNFNKPKTSNYPHFTFRFILDNDPQDERISNLP THGPLAGMRIPIINEHGADELRRELELLMLASEEDEEVSCLITDALWYFAQSVADSLNLRRLVLMTSSLFNFHAHVSLPQFDELGYLDPDDKTRLEEQASGFPMLKVKDIKSAYS

[0115] SEQ ID NO: 3 UGT76G1_L200A Reference: US2017 / 0332673 MAENKTETTVRRRRRIILFPVPFQGHINPILQLANVLYSKGFSITIFHTNFNKPKTSNYPHFTFRFILDNDPQDERISNLPTHGPLAGMRIPIINEHGADELRRELELLMLASE EDEEVSCLITDALWYFAQSVADSLNLRRLVLMTSSLFNFHAHVSLPQFDELGYLDPDDKTRLEEQASGFPMLKVKDIKSAYSNWQIAKEILGKMIKQTKASSGVIWNSFKELEES ELETVIREIPAPSFLIPLPKHLTASSSSLLDHDRTVFQWLDQQPPSSVLYVSFGSTSEVDEKDFLEIARGLVDSKQSFLWVVRPGFVKGSTWVEPLPDGFLGERGRIVKWVPQQE VLAHGAIGAFWTHSGWNSTLESVCEGVPMIFSDFGLDQPLNARYMSDVLKVGVYLENGWERGEIANAAIRRVMVDEEGEYIRQNARVLKQKADVSLMKGGSSYESLESLVSYISSL

[0116] SEQ ID NO:4 MbUGT1-3_0 MAKQSFLWVVRPGFVKGSTWVEPLPDGFLGERGRIVKWVPQQEVLAHGAIGAFWTHSGWNSTLESVCEGVPMIFSDFGLDQPLNARYMSDVLKVGVYLENGWERGEIANAIRRV MVDEEGEYIRQNARVLKQKADVSLMKGGSSYESLESLVSYISSLENKTETTVRRRRRIILFPVPFQGHINPILQLANVLYSKGFSITIFHTNFNKPKTSNYPHFTFRFILDDNDPQ DERISNLPTHGPLAGMRIPIINEHGADELRRELELLMLASEEDEEVSCLITDALWYFAQSVADSLNLRRLVLMTSSLFNFHAHVSLPQFDELGYLDPDDKTRLEEQASGFPMLKV KDIKSAYSNWQIAKEILGKMIKQTKASSGVIWNSFKELEESELETVIREIPAPSFLIPLPKHLTASSSSLLDHDRTVFQWLDQQPPSSVLYVSFGSTSEVDEKDFLEIARGLVDS

[0117] SEQ ID NO:5 MbUGT1-3_1 MAFLWVVRPGFVKGSTWVEPLPDGFLGERGRIVKWVPQQEVLAHGAIGAFWTHGGWNSTLESVCEGVPMIFSDFGLDQPLNARYMSDVLKVGVYLENGWERGEIANAIRRLMVD EEGEYIRQNARVLKQKADVSLMKGGSSYESLESLVSYISSLGSGGSGGSGRRRRIILFPVPFQGHINPMLQLANVLYSKGFSITIFHTNFNKPKTSNYPHFTFRFILDNDPQDER ISNLPTHGPLAGMRIPIINEHGADELRRELELLMLASEEDEEVSCLITDALWYFAQSVADSLNLRRLVLMTSSLFNFHAHVSLPQFDELGYLDPDDKTRLEEQASGFPMLKVKD IKSAYSNWQIAKEILGKMIKQTKASSGVIWNSFKELEESELETVIREIPAPSFLIPLPKHLTASSSSLLDHDRTVFQWLDQQPPSSVLYVSFGSTSEVDEKDFLEIARGLVDSQS

[0118] SEQ ID NO:6 MbUGT1-3_2 MAFLWVVRPGFVKGSTWVEPLPDGFLGERGRIVKWVPQQEVLAHGAIGAFWTHGGWNSTLESVCEGVPMIFSDFGLDQPLNARYMSDVLKVGVYLENGWERGEIANAIRRLMV DEEGEYIRQNARVLKQKADVSLMKGGSSYESLESLVSYISSLGSGGSGRRRRIILFPPVPFQGHINPMLQLANVLYSKGFSITIFHTNFNKPKTSNYPHFTFRFILDNDPQDERI SNLPTHGPLAGMRIPIINEHGADELRRELELQMLASEEDEEVSCLITDALWYFAQSVADSLNLPRLVLMTSSLFNFHAHVSLPQFDELGYLDPDDKTRLEEQASGFPMLKVKDI KSAYSNWQIAKEILGKMIKQTKASSGVIWNSFKELEESELETVIREIPAPSFLIPLPKHLTASSSSLLEHDRTVFQWLDQQPPSSVLYVSFGSTSEVDEKDFLEIARGLVDSQS

[0119] SEQ ID NO:7 Linker GSGGSG

[0120] SEQ ID NO:8 Linker GSGGSGGSG

[0121] SEQ ID NO:9 MbUGT1-3_3 MAFLWVVRPGFVKGSTWVEPLPDGFLGERGRIVKWVPQQEVLAHGAIGAFWTHGGWNSTLESVCEGVPMIFQDFGLDQPLNARYMSDVLKVGVYLENGWERGEIANAIRRLMVDEEGEYIRQNARVLKQKADVSLMKGGSSYESLESLVSYISSLGSGGSGRRRRIILFPVPFQGHINPMLQLANVLYSKGFSITIFHTNFNKPKTSNYPHFTFRFILDNDPQDHGPLAGMRIPIINEHGADELRRELELQMLASEEDEEVSCLITDALWYFAQSVADSLNLPRLVLMTSSLFNFHCHVSLPQFDELGYLDPDDKTRLEEQASGFPMLKVKDIKSAFSNWQIAKEILGKMIKQTKASSGVIWNSFKELEESELETVIREIPAPSFLIPLPKHLTASSSSLLEHDRTVFQWLDQQPPSSVIYVSFGSTSEVDEKDFLEIARGLVDSQS

Claims

1. 1. A uridine diphosphate-dependent glycosyltransferase (UGT) enzyme comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6, the enzyme has a flexible, hydrophilic sequence of 6 to 12 amino acids, mostly glycine and serine residues, after a position corresponding to position 155 of SEQ ID NO:5; The enzyme is a glycine (G) at a position corresponding to position 54 of SEQ ID NO:5; a leucine (L) at a position corresponding to position 111 of SEQ ID NO:5, and A methionine (M) at a position corresponding to position 183 of SEQ ID NO:5 holding one or more of The enzyme.

2. The enzyme of claim 1 , wherein the amino acid sequence is at least about 95% identical to SEQ ID NO:5 or SEQ ID NO:

6.

3. 2. The enzyme of claim 1, wherein the enzyme has a Gly at a position corresponding to position 54 of SEQ ID NO:5, a Leu at a position corresponding to position 111 of SEQ ID NO:5, and a Met at a position corresponding to position 183 of SEQ ID NO:

5.

4. The enzyme of claim 1 , wherein the flexible, hydrophilic sequence is GSGGSG (SEQ ID NO: 7) or GSGGSGGGSG (SEQ ID NO: 8).

5. The enzyme of claim 1 , wherein the enzyme comprises a deletion of at least three amino acids corresponding to amino acids E225 to T232 relative to the amino acid sequence of SEQ ID NO:

6.

6. The enzyme of claim 5 , wherein the enzyme comprises a deletion of amino acids corresponding to amino acids E225 to T232 of SEQ ID NO:

6.

7. 2. The enzyme of claim 1, wherein the enzyme comprises amino acid substitutions at one or more positions corresponding to positions 72, 305, 345, and 428 of SEQ ID NO:

6.

8. 8. The enzyme of claim 7, wherein the enzyme comprises one or more amino acid substitutions selected from glutamine (Q) at a position corresponding to position 72 of SEQ ID NO:6, cysteine ​​(C) at a position corresponding to position 305 of SEQ ID NO:6, phenylalanine (F) at a position corresponding to position 345 of SEQ ID NO:6, and isoleucine (I) at a position corresponding to position 428 of SEQ ID NO:

6.

9. 1. A uridine diphosphate-dependent glycosyltransferase (UGT) enzyme comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6, glycine (G) at a position corresponding to position 54 of SEQ ID NO:5; leucine (L) at a position corresponding to position 111 of SEQ ID NO:5, and A methionine (M) at a position corresponding to position 183 of SEQ ID NO:5 The enzyme comprising:

10. 6. The enzyme of claim 5, comprising a deletion of at least 5 amino acids corresponding to amino acids E225 to T232 relative to the amino acid sequence of SEQ ID NO:

6.

11. 8. The enzyme of claim 7, wherein the enzyme comprises a glutamine (Q) or asparagine (N) substitution at a position corresponding to position 72 of SEQ ID NO:

6.

12. The enzyme of claim 7, wherein the enzyme comprises a neutral hydrophilic amino acid selected from cysteine ​​(C), serine (S), or threonine (T) at a position corresponding to position 305 of SEQ ID NO:

6.

13. 8. The enzyme of claim 7, wherein the enzyme comprises a phenylalanine (F) or tryptophan (W) substitution at a position corresponding to position 345 of SEQ ID NO:

6.

14. 8. The enzyme of claim 7, wherein the enzyme comprises a substitution of isoleucine (I), valine (V), or alanine (A) at a position corresponding to position 428 of SEQ ID NO:

6.

15. 8. The enzyme of claim 7, wherein the enzyme comprises two, three or four of the following substitutions relative to SEQ ID NO: 6: S72Q, A305C, Y345F, and L428I.

16. A uridine diphosphate-dependent glycosyltransferase (UGT) enzyme comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6, wherein the enzyme contains a deletion of at least three amino acids corresponding to amino acids E225 to T232 of SEQ ID NO:

6.

17. 17. The UGT enzyme of claim 16, comprising a deletion of at least five amino acids corresponding to amino acids E225 to T232 of SEQ ID NO:

6.

18. 17. The UGT enzyme of claim 16, comprising a deletion of amino acids corresponding to amino acids E225 to T232 of SEQ ID NO:

6.

19. 17. The UGT enzyme of claim 16, wherein the enzyme is at least 95% identical to SEQ ID NO:5 or SEQ ID NO:

6.

20. A polynucleotide encoding the enzyme of claim 1, 9 or 16.

21. 21. An isolated recombinant microorganism comprising the polynucleotide of claim 20.

22. 22. A method for transferring a monosaccharide group to a steviol glycoside substrate, comprising contacting the NDP sugar and the substrate with the isolated recombinant microorganism of claim 21.

23. 22. A method for transferring a glycosyl group to a steviol glycoside substrate, comprising growing the isolated recombinant microorganism of claim 21 in the presence of the substrate.

Images