Efficient production of steviol glycosides in recombinant hosts

A recombinant microorganism capable of producing a steviol glycoside in a cell culture,

• wherein the microorganism has a reduced expression of at least one endogenous transporter gene encoding a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:
• 107;
• wherein the reduced expression is produced in the microorganism by disrupting or deleting a gene locus to by disrupting or deleting the gene locus for the at least one endogenous transporter gene;
• wherein the microorganism further comprises:
• (a) a gene encoding a polypeptide capable of synthesizing steviol from ent-kaurenoic acid;
• wherein the polypeptide comprises a polypeptide having at least 90% sequence identity to one of the amino acid sequences set forth in any one of SEQ ID NOs:
• 11-17 or 19;
• and further comprises:
• (b) 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 90% sequence identity to one of the amino acid sequences set forth in any one of SEQ ID NOs:
• 30 or 91;
• (c) 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 90% sequence identity to one of the amino acid sequences set forth in any one of SEQ ID NOs:
• 85or 89;
• (d) 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 90% sequence identity to one of the amino acid sequences set forth in any one of SEQ ID NOs:
• 29 or 88;
• and/or
• (e) 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 gene has a copy number of 2 or more;
• and
• wherein the polypeptide comprises a polypeptide having at least 90% sequence identity to one of the amino acid sequences set forth in any one of SEQ ID NOs:
• 51, 54, 55, 86, or 90;
• wherein at least one of the genes in items (a)-(e) is a recombinant gene;
• and
• wherein the steviol glycoside is Rebaudioside A, Rebaudioside B, Rebaudioside D, Rebaudioside E, Rebaudioside M, or an isomer thereof.