Microorganisms for diterpene production
The invention disclosed herein relates generally to the field of recombinant production of a steviol glycoside, to the field of bioconversion of steviol into a steviol glycoside and to the field of bioconversion of a steviol glycoside into a further steviol glycoside. Particularly, the invention provides a process for recombinant production of a steviol glycoside, a process of bioconversion of steviol into a steviol glycoside, a process for bioconversion of a steviol glycoside into a further steviol glycoside and a composition comprising a steviol glycoside. More particularly, the invention relates to a microorganism that has a deficiency of a serine/threonine protein kinase and comprises a polynucleotide encoding a polypeptide having uridine diphosphate-dependent glucosyltransferase (UGT) activity.
1 . A recombinant yeast comprising one or more polynucleotide(s) encoding one or more polypeptide(s) having uridine diphosphate-dependent glycosyltransferase (UGT) activity, wherein said recombinant yeast comprises a mutation, insertion, substitution, or deletion in a serine/threonine protein kinase gene selected from serine/threonine protein kinase 1 (PSK1) and serine/threonine protein kinase 2 (PSK2) that reduces the kinase activity relative to the corresponding yeast lacking the mutation, insertion, substitution, or deletion.
2 . The recombinant yeast according to claim 1 , wherein the serine/threonine protein kinase 1 or serine/threonine protein kinase 2 activity is reduced by at least 40%.
3 . The recombinant yeast according to claim 1 , comprising one or more polynucleotide expression constructs selected from the group consisting of:
(a) a polynucleotide expression construct encoding a functional UGT1 polypeptide,
(b) a polynucleotide expression construct encoding a functional UGT3 polypeptide,
(c) a polynucleotide expression construct encoding a functional UGT4 polypeptide,
(d) a polynucleotide expression construct encoding a first functional UGT2 polypeptide, and
(e) a polynucleotide expression construct encoding a second functional UGT2 polypeptide.
4 . The recombinant yeast according to claim 1 , comprising one or more polynucleotide expression constructs selected from the group consisting of:
(a) a polynucleotide expression construct encoding a UGT1 polypeptide capable of glycosylating steviol or a precursor steviol glycoside at a C-13 hydroxyl group present in said steviol or precursor steviol glycoside, wherein the glycosylation is a beta-glycosylation;
(b) a polynucleotide expression construct encoding a UGT3 polypeptide capable of glycosylating steviol or a precursor steviol glycoside at a C-19 carboxyl group present in said steviol or precursor steviol glycoside, wherein the glycosylation is a beta-glycosylation;
(c) a polynucleotide expression construct encoding a UGT4 polypeptide capable of beta 1,3 glycosylation of the C3′ of a 13-O-glucose, of a 19-O-glucose or both the 13-O-glucose and the 19-O-glucose of a precursor steviol glycoside having a 13-O-glucose, a 19-O-glucose, or both a 13-O-glucose and a 19-O-glucose;
(d) a polynucleotide expression construct encoding a first UGT2 polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, of the 19-O-glucose or both the 13-O-glucose and the 19-O-glucose of a precursor steviol glycoside having a 13-O-glucose, a 19-O-glucose, or both the 13-O-glucose and the 19-O-glucose; and
(e) a polynucleotide expression construct encoding a second UGT2 polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, of the 19-O-glucose or both the 13-O-glucose and the 19-O-glucose of the precursor steviol glycoside having a 13-O-glucose, a 19-O-glucose, or both the 13-O-glucose and the 19-O-glucose, wherein the second UGT2 polypeptide has an higher beta 1,2 glycosylation activity at the C2′ of the 19-O-glucose in the precursor steviol glycoside if compared with the same activity in the first UGT2 polypeptide;
wherein the recombinant yeast produces a steviol glycoside selected from the group consisting of: steviol-13-O-glucoside, steviol-19-O-glucoside, steviol-1,2-bioside, steviol-1,3-bioside, stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside I, rebaudioside Q, rebaudioside M, rubusoside, and dulcoside A.
5 . The recombinant yeast according to claim 1 , additionally comprising a polynucleotide selected from the group consisting of:
(f) a polynucleotide expression construct encoding a geranyl-geranyl pyrophosphate synthase (GGPPS),
(g) a polynucleotide expression construct encoding an ent-copalyl diphosphate synthase (CDPS),
(h) a polynucleotide expression construct encoding a kaurene oxidase (KO),
(i) a polynucleotide expression construct encoding a kaurene synthase (KS), and
(j) a polynucleotide expression construct encoding a kaurenoic acid 13-hydroxylase (KAH); and
wherein the yeast produces a steviol glycoside, selected from the group consisting of: steviol-13-O-glucoside, steviol-19-O-glucoside, steviol-1,2-bioside, steviol-1,3-bioside, stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside I, rebaudioside Q, rebaudioside M, rubusoside, and dulcoside A.
6 . The recombinant yeast according to claim 1 , wherein the recombinant yeast additionally comprises, a polynucleotide encoding a cytochrome P450 reductase (CPR).
7 . The recombinant yeast according to claim 1 , wherein the ability of the recombinant yeast to produce geranylgeranyl diphosphate (GGPP) is upregulated.
8 . The recombinant yeast according to claim 7 , comprising one or more polynucleotide(s) encoding hydroxymethylglutaryl-CoA reductase, farnesyl-pyrophosphate synthetase and geranylgeranyl diphosphate synthase, whereby expression of the polynucleotide(s) confer(s) on the recombinant yeast the ability to produce elevated levels of GGPP.
9 . The recombinant yeast according to claim 1 , wherein the recombinant yeast belongs to one of the genera Saccharomyces, Pichia, Kluyveromyces, Candida, Hansenula, Trichosporon, Brettanomyces, Pachysolen, Yarrowia , or Yamadazyma.
10 . The recombinant yeast according to claim 9 , wherein the recombinant yeast is a Saccharomyces cerevisiae cell, or a Yarrowia lipolytica cell.
11 . A process for producing a steviol glycoside which process comprises culturing the recombinant yeast according to claim 2 under conditions conducive to production of the steviol glycoside, and recovering the steviol glycoside.
12 . A process for producing a steviol glycoside comprising contacting steviol or steviol glycosides with the recombinant yeast according to claim 1 , a fermentation broth comprising such recombinant yeast, and recovering the steviol glycoside.
13 . The process according to claim 12 , wherein the process is a whole cell bioconversion process.
14 . The process according to claim 13 , wherein
steviol is converted to steviol-13-O-glucoside by a UGT1, wherein the UGT1 is a UGT85C2,
steviol-19-O-glucoside is converted to rubusoside by a UGT1, wherein the UGT1 is a UGT85C2,
steviol-13-O-glucoside is converted to rubusoside by a UGT3, wherein the UGT3 is a UGT74G1,
steviol-1,2-bioside is converted to 1,2-stevioside by a UGT3, wherein the UGT3 is a UGT74G1,
rebaudioside B is converted to rebaudioside A by a UGT3, wherein the UGT3 is a UGT74G1,
steviol-1,2-bioside is converted to rebaudioside B by a UGT 4, wherein the UGT 4 is a UGT76G1,
1,2-stevioside is converted to rebaudioside A by a UGT 4, wherein the UGT 4 is a UGT76G1,
rebaudioside E is converted to rebaudioside D by a UGT 4, wherein the UGT 4 is a UGT76G1,
rebaudioside D is converted to rebaudioside M by a UGT 4, wherein the UGT 4 is a UGT76G1,
steviol 13-O-glucoside is converted to steviol-1,2-bioside by a UGT2, wherein the UGT2 is a UGT91 D2e,
rubusoside is converted to 1,2-stevioside by a UGT2, wherein the UGT2 is a UGT91 D2e,
stevioside is converted to rebaudioside E, by a UGT2, wherein the UGT2 is a UGT91 D2e and a EUGT11, and
rebaudioside A is converted to rebaudioside D by a UGT2, wherein the UGT2 is a EUGT11.
15 . The recombinant yeast of claim 4 , wherein the one or more polynucleotide expression constructs is selected from the group consisting of:
(a) the polynucleotide expression construct encoding a UGT1 polypeptide, wherein the polynucleotide encodes for a UGT85C2 polypeptide;
(b) the polynucleotide expression construct encoding a UGT3 polypeptide, wherein the polynucleotide encodes for a UGT74G1 polypeptide;
(c) the polynucleotide expression construct encoding a UGT4 polypeptide, wherein the polynucleotide encodes for a UGT76G1 polypeptide;
(d) the polynucleotide expression construct encoding a UGT2 polypeptide, wherein the polynucleotide encodes for a UGT91 d2 polypeptide or a UGT2 polypeptide having at least uridine 5′-diphosphoglucosyl: steviol-13-O-glucoside transferase activity; and
(e) the polynucleotide expression construct encoding a second UGT2 polypeptide, wherein the polynucleotide encodes for a EUGT11 polypeptide.