Production of steviol glycosides in recombinant hosts

Abstract

The invention relates to recombinant microorganisms and methods for producing steviol glycosides and steviol glycoside precursors.

1 . A recombinant host cell capable of producing a steviol glycoside in a cell culture, wherein the host cell comprises:
 (a) a gene encoding a Sugar Efflux Transporter (SET) polypeptide;
 wherein the SET polypeptide comprises a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:20 or 22; and/or 
   (b) a gene encoding a Sugar Transporter SWEET (SWEET) polypeptide;
 wherein the SWEET polypeptide comprises a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:24, 26, or 28; 
   wherein at least one of the genes is a recombinant gene.   
     
     
         2 . The recombinant host cell of  claim 1 , wherein the host cell further comprises a deletion of a gene encoding a trafficking adapter polypeptide having at least 50% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 166, 168, 170, 172, or 174. 
     
     
         3 . The recombinant host cell of  claim 2 , wherein the deletion of the one or more genes encoding the one or more polypeptides having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NOs:30, 42, 46, or 166 increases excretion of Rebaudioside D (RebD). 
     
     
         4 . The recombinant host cell of  claim 2 , wherein the deletion of the one or more genes encoding the one or more polypeptides having at least 50% sequence identity to the amino acid sequence set forth in any one of SEQ ID Nos:30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 166, 170, 172, 174 increases excretion of Rebaudioside M (RebM). 
     
     
         5 . The host cell of  claim 1 , wherein the host cell further comprises a deletion of one or more genes encoding one or more transporter polypeptides having the amino acid sequence set forth in any one of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 54, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, or a functional homolog thereof. 
     
     
         6 . The recombinant host cell of  claim 1 , further comprising:
 (a) a gene encoding a polypeptide capable of synthesizing geranylgeranyl pyrophosphate (GGPP) from farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP);
 wherein the polypeptide comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:182, 208, 210, 212, 214, 216, 218, or 220; 
   (b) a gene encoding a polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP;
 wherein the polypeptide comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:184, 222, 224, 226, 228, or 230; 
   (c) a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid from ent-kaurene;
 wherein the polypeptide comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:188, 206, 246, 249, 251, 253, 255, 257, 259, or 261; 
   (d) a gene encoding a polypeptide capable of synthesizing ent-kaurene from ent-copalyl pyrophosphate;
 wherein the polypeptide comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:186, 232, 234, 236, or 238; 
   (e) a gene encoding a polypeptide capable of synthesizing steviol from ent-kaurenoic acid;
 wherein the polypeptide comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:192, 266, 269, 270, 271, 272, 273, 275, 277, 279, 281, or 283; 
   (f) a gene encoding a polypeptide capable of reducing cytochrome P450 complex;
 wherein the polypeptide comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:190, 194, 263, 265, 287, 289, 291, or 293; or 
   (g) a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group;
 wherein the polypeptide comprises a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:196; 
   (h) a gene encoding a polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside;
 wherein the polypeptide comprises a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:200; 
   (i) a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group;
 wherein the polypeptide comprises a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:198; and/or; 
   (j) a gene encoding a polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside;
 wherein the polypeptide comprises a polypeptide having at least 65% sequence identity to the amino acid sequence set forth in SEQ ID NO:202 or 204; 
 wherein at least one of the genes in items (a)-(j) is a recombinant gene. 
   
     
     
         7 . The recombinant host cell of  claim 1 , wherein the host cell excretes a decreased amount of steviol-13-O-glucoside (13-SMG) relative to a steviol glycoside-producing host cell that does not have the deletion of the one or more genes encoding the one or more transporter polypeptides. 
     
     
         8 . The recombinant host cell of  claim 1 , wherein the host cell excretes an increased amount of RebA, RebB, RebD, and/or RebM relative to a steviol glycoside-producing host cell that does not have the deletion of the one or more genes encoding the one or more transporter polypeptides. 
     
     
         9 . The recombinant host cell of  claim 1 , wherein the host cell produces an increased amount of RebA, RebB, RebD, and/or RebM relative to a steviol glycoside-producing host cell that does not have the deletion of the one or more genes encoding the one or more transporter polypeptides. 
     
     
         10 . The recombinant host of  claim 1 , wherein the steviol glycoside is Rebaudioside A (Reb A), Rebaudioside B (Reb B), Reb D and/or Reb M or an isomer thereof. 
     
     
         11 . The recombinant host cell of  claim 1 , wherein the host is a fungal cell or a bacterial cell. 
     
     
         12 . The recombinant host cell of  claim 11 , wherein the bacterial cell comprises  Escherichia  bacteria cells,  Lactobacillus  bacteria cells,  Lactococcus  bacteria cells,  Cornebacterium  bacteria cells,  Acetobacter  bacteria cells,  Acinetobacter  bacteria cells, or  Pseudomonas  bacterial cells. 
     
     
         13 . The recombinant host cell of  claim 10 , wherein the fungal cell is a yeast cell. 
     
     
         14 . The recombinant host cell of  claim 13 , wherein the yeast cell is a cell from  Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata, Ashbya gossypii, Cyberlindnera jadinii, Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, Candida boidinii, Arxula adeninivorans, Xanthophyllomyces dendrorhous , or  Candida albicans  species. 
     
     
         15 . The recombinant host cell of  claim 1 , wherein the host cell is a  Yarrowia lipolytica  cell. 
     
     
         16 . A method of increasing production of a steviol glycoside in a recombinant host cell or increasing excretion of a steviol glycoside into a culture medium, comprising culturing the host cell of  claim 1  in a cell culture, under conditions in which one or more of the genes are expressed; wherein the steviol glycoside is produced by the host cell. 
     
     
         17 . The method of  claim 16 , wherein excretion of 13-SMG from the host cell into the cell culture is decreased relative to RebA, RebB, RebD, and/or RebM-producing host cell that does not have the deletion of the one or more genes encoding the one or more transporter polypeptides and wherein RebA, RebB, RebD, and/or RebM are produced by the host cell. 
     
     
         18 . The method of  claim 16 , that further comprises isolating RebA, RebB, RebD, and/or RebM, alone or in combination from the cell culture; wherein the isolating step comprises separating a liquid phase of the cell culture from a solid phase of the cell culture to obtain a supernatant comprising RebA, RebB, RebD, and/or RebM, alone or in combination, and:
 (a) contacting the supernatant with one or more adsorbent resins in order to obtain at least a portion of RebA, RebB, RebD, and/or RebM, alone or in combination; or   (b) contacting the supernatant with one or more ion exchange or reversed-phase chromatography columns in order to obtain at least a portion of RebA, RebB, RebD, and/or RebM, alone or in combination; or   (c) crystallizing or extracting RebA, RebB, RebD, and/or RebM, alone or in combination;   
       thereby isolating RebA, RebB, RebD, and/or RebM, alone or in combination. 
     
     
         19 . The method of  claim 16 , that further comprises recovering RebA, RebB, RebD, and/or RebM alone or a composition comprising RebA, RebB, RebD, and/or RebM from the cell culture; wherein the recovered composition is enriched for RebA, RebB, RebD, and/or RebM, relative to a steviol glycoside composition of  Stevia  plant and has a reduced level of  Stevia  plant-derived components relative to a steviol glycoside composition obtained from a plant-derived  Stevia  extract. 
     
     
         20 . The method of  claim 16 , wherein the cell culture comprises:
 (a) RebA, RebB, RebD, and/or RebM produced by the host cell of  claim 1 ,   (b) glucose, fructose, sucrose, xylose, rhamnose, uridine diphosphate (UDP)-glucose, UDP-rhamnose, UDP-xylose, and/or N-acetyl-glucosamine; and   (c) supplemental nutrients comprising trace metals, vitamins, salts, yeast nitrogen base (YNB), and/or amino acids.   
     
     
         21 . The method of  claim 16 , wherein:
 (a) Reb A is produced in the recombinant host cell expressing the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group; the polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group; and the polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside;   (b) Reb B is produced in the recombinant host cell expressing the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group; the polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; and the polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-0-glucose of a steviol glycoside;   (c) Reb D is produced in the recombinant host cell expressing the polypeptide capable of glycosylating steviol or the steviol glycoside at its C-13 hydroxyl group; the polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group; and the polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; and/or   (d) Reb M is produced in the recombinant host cell expressing the polypeptide capable of glycosylation of the 13-OH of steviol; the polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group; and the polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside.   
     
     
         22 . A cell culture, comprising the host cell of  claim 1 , the cell culture further comprising:
 (a) RebA, RebB, RebD, and/or RebM produced by the host cell;   (b) glucose, fructose, sucrose, xylose, rhamnose, uridine diphosphate (UDP)-glucose, UDP-rhamnose, UDP-xylose, and/or N-acetyl-glucosamine; and   (c) supplemental nutrients comprising trace metals, vitamins, salts, YNB, and/or amino acids;   wherein RebA, RebB, RebD, and/or RebM are present at a concentration of at least 1 mg/liter of the cell culture.   
     
     
         23 . A cell lysate, comprising RebA, RebB, RebD, and/or RebM produced by the host cell of  claim 1 , and the cell lysate further comprising glucose, fructose, sucrose, xylose, rhamnose, uridine diphosphate (UDP)-glucose, UDP-rhamnose, UDP-xylose, and/or N-acetyl-glucosamine, supplemental nutrients comprising trace metals, vitamins, salts, YNB, and/or amino acids. 
     
     
         24 . RebA, RebB, RebD, and/or RebM produced by the method of  claim 16 . 
     
     
         25 . A sweetener composition, comprising RebA, RebB, RebD, and/or RebM of  claim 24 . 
     
     
         26 . A food product, a beverage, or a beverage concentrate comprising, comprising the sweetener composition of  claim 25 .