Production of steviol glycosides in recombinant hosts

Abstract

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

1 . A method for producing one or more steviol glycosides and/or glycosylated steviol precursors, or a composition thereof, comprising whole cell bioconversion of a plant-derived or a synthetic steviol, steviol precursors, glycosylated steviol precursors, steviol glycosides, and/or a mixture thereof in a cell culture medium of a recombinant host cell expressing a gene encoding:
 (a) a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:4, and further having at least one amino acid substitution corresponding to residues 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 184, 260, 286, or 377 of SEQ ID NO:4;   (b) a bifunctional polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group and glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group, having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:132, 134, 136, 138, 140, 142, 144, 146, or 148; and/or   (c) a tagged polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group, having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:174, 176, 178, or 180;   wherein at least one of the polypeptides is a recombinant polypeptide; and producing the one or more steviol glycosides and/or the glycosylated steviol precursors, or the composition thereof, thereby.   
     
     
         2 . The method of  claim 1 , wherein the one or more steviol glycosides and/or the glycosylated steviol precursors are, or the composition thereof comprises steviol-13-O-glucoside (13-SMG), steviol-19-O-glucoside (19-SMG), steviol-1,2-bioside, steviol-1,3-bioside, 1,2-stevioside, 1,3-stevioside, rubusoside, Rebaudioside A (RebA), Rebaudioside B (RebB), Rebaudioside C (RebC), Rebaudioside D (RebD), Rebaudioside E (RebE), Rebaudioside F (RebF), Rebaudioside M (RebM), Rebaudioside Q (RebQ), Rebaudioside I (RebI), dulcoside A, a mono-glycosylated ent-kaurenoic acid, a di-glycosylated ent-kaurenoic acid, a tri-glycosylated ent-kaurenoic acid, a mono-glycosylated ent-kaurenols, a di-glycosylated ent-kaurenol, a tri-glycosylated ent-kaurenol, a tri-glycosylated steviol glycoside, a tetra-glycosylated steviol glycoside, a penta-glycosylated steviol glycoside, a hexa-glycosylated steviol glycoside, a hepta-glycosylated steviol glycoside, or an isomer thereof. 
     
     
         3 . The method of  claim 2 , wherein:
 (a) the di-glycosylated ent-kaurenoic acid comprises ent-kaurenoic acid+2Glc (# 7 );   (b) the tri-glycosylated ent-kaurenoic acid comprises ent-kaurenoic acid+3Glc (isomer 1) or ent-kaurenoic acid+3Glc (isomer 2);   (c) the di-glycosylated ent-kaurenol comprises ent-kaurenol+2Glc (#8);   (d) the tri-glycosylated ent-kaurenol comprises ent-kaurenol+3Glc (isomer 1) or ent-kaurenol+3Glc (#6);   (e) the steviol glycoside comprises 13-SMG, 19-SMG, steviol-1,2-bioside, steviol-1,3-bioside, rubusoside, RebA, RebE, RebD, or RebM;   (f) the tetra-glycosylated steviol comprises steviol+4Glc (#26) or steviol+4Glc (#33);   (g) the penta-glycosylated steviol comprises steviol+5Glc (#24) or steviol+5Glc (#25);   (h) the hexa-glycosylated steviol comprises steviol+6Glc (isomer 1) or steviol+6Glc (#23); and/or   (i) the hepta-glycosylated steviol comprises steviol+7Glc (isomer 2) or steviol+7Glc (isomer 5).   
     
     
         4 . The method of  claim 1 , wherein the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group comprises a M79V, M79E, S80C, A81W, E83K, A81W and E83K, H184V, H184T, N260T, K286C, N260T and K286C, K286E, K286N, K286T, and/or S377Q amino acid substitution of SEQ ID NO:4. 
     
     
         5 . The method of  claim 1 , wherein the whole cell used in the whole cell method further comprises:
 (a) a gene encoding a polypeptide capable of synthesizing geranylgeranyl pyrophosphate (GGPP) from farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP) and having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:20, 22, 24, 26, 28, 30, 32, or 116;   (b) a gene encoding a polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP and having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:34, 36, 38, 40, 42, or 120;   (c) a gene encoding an a polypeptide capable of synthesizing ent-kaurene from ent-copalyl diphosphate and having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:44, 46, 48, 50, or 52;   (d) a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid from ent-kaurene and having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:60, 62, 66, 68, 70, 72, 74, 76, or 117;   (e) a gene encoding a polypeptide capable of reducing cytochrome P450 complex and having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:78, 80, 82, 84, 86, 88, 90, or 92;   (f) a gene encoding a polypeptide capable of synthesizing steviol from ent-kaurenoic acid and having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:94, 97, 100-104, 106, 108, 110, 112, or 114;   (g) a gene encoding a polypeptide capable of transferring a sugar moiety to the C-13 hydroxyl group of a steviol or a steviol glycoside when contacted with the steviol or the steviol glycoside and one or more UDP-sugars under suitable reaction conditions for the transfer of the additional sugar moiety to the steviol or the steviol glycoside and having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:7;   (h) a gene encoding a polypeptide capable of transferring a sugar moiety to the C3′ of a glucose in a steviol glycoside by beta 1,3 glycosylating C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of the steviol glycoside when contacted with the steviol glycoside and one or more UDP-sugars under suitable reaction conditions for the transfer of the additional sugar moiety to the steviol glycoside and having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:9; and/or   (i) a gene encoding a polypeptide capable of transferring a sugar moiety to the C2′ of a glucose in a steviol glycoside by beta 1,2 glycosylating C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of the steviol glycoside when contacted with the steviol glycoside and one or more UDP-sugars under suitable reaction conditions for the transfer of the additional sugar moiety to the steviol glycoside and having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:11, 13, or 16;   wherein at least one of the genes is a recombinant gene.   
     
     
         6 . The method of  claim 5 , further comprising supplying the one or more UDP-sugars or supplying a cell-free system for regeneration of the one or more UDP-sugars. 
     
     
         7 . The method of  claim 6 , further comprising feeding raw materials, comprising Stevia extracts, comprising the one or more UDP-sugars and the steviol, the steviol precursors, the glycosylated steviol precursors, the steviol glycosides, and/or the mixture thereof to the whole cell. 
     
     
         8 . The method of  claim 7 , further comprising feeding raw materials during or after the whole cell growth. 
     
     
         9 . The method of  claim 8 , wherein the whole cell is:
 (a) in suspension or immobilized;   (b) entrapped in a calcium or sodium alginate bead;   (c) linked to a hollow fiber tube reactor system;   (d) concentrated and entrapped within a membrane reactor system; or   (e) in fermentation broth or in a reaction buffer.   
     
     
         10 . The method of  claim 9 , further comprising permeabilizing the whole cell by using a permeabilizing agent, wherein the permeabilizing agent is a solvent, a detergent, or a surfactant, by a mechanical shock, an electroporation, or an osmotic shock. 
     
     
         11 . The method of  claim 5 , wherein the steviol glycoside is steviol-13-O-glucoside, steviol-19-O-glucoside, rubusoside, steviol-1,2-bioside, stevioside, RebA, RebB, or RebE and/or a mixture thereof, the one or more UDP-sugars is UDP-glucose, and RebD, RebM, and/or a composition thereof is produced upon transfer of the one or more sugar moieties from the one or more UDP-glucose to the steviol, the steviol glycoside, and/or the mixture thereof by the polypeptide capable of transferring a sugar moiety to the C-13 hydroxyl group of a steviol or a steviol glycoside, the polypeptide capable of transferring a sugar moiety to the C3′ of a glucose in a steviol glycoside by beta 1,3 glycosylating C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of the steviol glycoside, and/or the polypeptide capable of transferring a sugar moiety to the C2′ of a glucose in a steviol glycoside by beta 1,2 glycosylating C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of the steviol glycoside. 
     
     
         12 . The method of  claim 1 , further comprising isolating the produced one or more steviol glycosides and/or the glycosylated steviol precursors, or the composition thereof. 
     
     
         13 . The method of  claim 12 , wherein the isolating step comprises separating a liquid phase of the cell culture medium from a solid phase of the cell culture medium to obtain a supernatant comprising the produced one or more steviol glycosides and/or the glycosylated steviol precursors, or the composition thereof, and:
 (a) contacting the supernatant with one or more adsorbent resins in order to obtain at least a portion of the produced one or more steviol glycosides and/or the glycosylated steviol precursors, or the composition thereof; 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 the produced one or more steviol glycosides and/or the glycosylated steviol precursors, or the composition thereof; or   (c) crystallizing or extracting the produced one or more steviol glycosides and/or the glycosylated steviol precursors, or the composition thereof;   thereby isolating the produced one or more steviol glycosides and/or the glycosylated steviol precursors, or the composition thereof.   
     
     
         14 . The method of  claim 1 , further comprising recovering the one or more steviol glycosides and/or the glycosylated steviol precursors alone or as a composition comprising the one or more steviol glycosides and/or the glycosylated steviol precursors. 
     
     
         15 . The method of  claim 14 , wherein the recovered composition is enriched for the one or more steviol glycosides and/or the glycosylated steviol precursors 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. 
     
     
         16 . The method of  claim 1 , wherein the whole cell used in the whole cell method is a fungal cell from  Aspergillus  genus or a yeast 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, an algal cell or a bacterial cell from  Escherichia coli  species or  Bacillus  genus. 
     
     
         17 . The method of  claim 1 , wherein the whole cell used in the whole cell method is a yeast cell from  Yarrowia lipolytica  species.