Recombinant Production of Steviol Glycosides

摘要

Recombinant microorganisms, plants, and plant cells are disclosed that have been engineered to express recombinant genes encoding UDP-glycosyltransferases (UGTs). Such microorganisms, plants, or plant cells can produce steviol glycosides, e.g., Rebaudioside A and/or Rebaudioside D, which can be used as natural sweeteners in food products and dietary supplements.

主权项

1. A recombinant host cell comprising a recombinant gene encoding a polypeptide having at least 80% identity to the amino acid sequence set forth in SEQ ID NO:152;
said host optionally comprising one or more of the following:
(a) a recombinant gene encoding a UGT91d2e or a UGT91d2m polypeptide, wherein the UGT91d2e polypeptide comprises a UGT91d2e polypeptide having a substitution at residues 211 and 286 of SEQ ID NO:5 or a UGT91D2e-b polypeptide having a methionine at residue 211 and an alanine at residue 286 of SEQ ID NO:5;(b) a recombinant gene encoding a steviol synthase polypeptide (KAH) having at least 80% identity to the amino acid sequence set forth in (SEQ ID NO:164);(c) a recombinant gene encoding an ent-copalyl diphosphate synthaseCDPS) polypeptide lacking a chloroplast transit peptide;
(d) one or more exogenous nucleic acids encoding a sucrose transporter (SUC1) and a sucrose synthase (SUS1) wherein expression of the one or more exogenous nucleic acids encoding a sucrose transporter and expression of a glucosyltransferase results in increased levels of uridine diphosphate glucose (UDP-glucose) in the host;(e) a recombinant gene encoding a cytochrome P450 reductase (CPR) polypeptide having at least 80% sequence identity to the S. rebaudiana CPR amino acid sequence set forth in (SEQ ID NO: 170;(f) a nucleic acid sequence, said nucleic acid comprising a heterologous insert sequence operably linked to an open reading frame, wherein the heterologous insert sequence has a formula (I):
-X1-X2-X3-X4-X5-  (I) wherein X2 comprises at least 4 consecutive nucleotides complementary to at least 4 consecutive nucleotides of X4, wherein X3 comprises zero nucleotides or one or more nucleotides forming a hairpin loop, wherein X1 and X5 each individually consists of zero nucleotides or one or more nucleotides, wherein the open reading frame encodes a squalene synthase (SQS);
(g) one or more exogenous nucleic acids encoding an S. rebaudiana kaurene oxidase KO) polypeptide, the polypeptide comprising a sequence having at least 80% identity to SEQ ID NO:138, and an A. thaliana CPR polypeptide, the polypeptide comprising a sequence having at least 80% identity to SEQ ID NO:168;(h) a recombinant gene encoding an A. thaliana kaurene synthase (KS) polypeptide, the polypeptide comprising a sequence having at least 80% identity to SEQ ID NO:156;(i) a codon optimized recombinant gene encoding a UGT85C polypeptide; or(j) a recombinant gene encoding a geranylgeranyl diphosphate synthase (GGPPS).