Steviol glycoside transport

Abstract

A recombinant host capable of producing a steviol glycoside which overexpresses a polypeptide which mediates steviol glycoside transport and which polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 35 or SEQ ID NO: 38 or an amino acid sequence having at least about 50% sequence identity to either thereto. A recombinant host capable of producing a steviol glycoside which has been modified, preferably in its genome, to result in a deficiency in the production of a polypeptide which mediates steviol glycoside transport and which polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 35 or SEQ ID NO: 38 or an amino acid sequence having at least about 50% sequence identity to either thereto.

1 . A recombinant host capable of producing a steviol glycoside which overexpresses a polypeptide which mediates steviol glycoside transport and which polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 35 or SEQ ID NO: 38 or an amino acid sequence having at least about 50% sequence identity to either thereto. 
     
     
         2 . A recombinant host capable of producing a steviol glycoside which has been modified, optionally in its genome, to result in a deficiency in the production of a polypeptide which mediates steviol glycoside transport and which polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 35 or SEQ ID NO: 38 or an amino acid sequence having at least about 50% sequence identity to either thereto. 
     
     
         3 . The recombinant host according to  claim 1 , which comprises a recombinant nucleic acid encoding a polypeptide which comprises the amino acid sequence set forth in SEQ ID NO: 35 or SEQ ID NO: 38 or an amino acid sequence having at least about 50% sequence identity to either thereto. 
     
     
         4 . The recombinant host according to  claim 1 , which further comprises one or more recombinant nucleotide sequence(s) encoding:
 a polypeptide having ent-copalyl pyrophosphate synthase activity;   a polypeptide having ent-Kaurene synthase activity;   a polypeptide having ent-Kaurene oxidase activity; and   a polypeptide having kaurenoic acid 13-hydroxylase activity.   
     
     
         5 . The recombinant host according to  claim 1 , which further comprises a recombinant nucleic acid sequence encoding a polypeptide having NADPH-cytochrome p450 reductase activity. 
     
     
         6 . The recombinant host according to  claim 1 , which further comprises a recombinant nucleic acid sequence encoding one or more of:
 (i) a polypeptide having UGT74G1 activity;   (ii) a polypeptide having UGT2 activity;   (iii) a polypeptide having UGT85C2 activity; and   (iv) a polypeptide having UGT76G1 activity.   
     
     
         7 . The recombinant host according to  claim 1 , wherein the host belongs to one of the genera  Saccharomyces, Aspergillus, Pichia, Kluyveromyces, Candida, Hansenula, Humicola, Issatchenkia, Trichosporon, Brettanomyces, Pachysolen, Yarrowia, Yamadazyma , or  Escherichia.    
     
     
         8 . The recombinant host according to  claim 7 , wherein the recombinant host is a  Saccharomyces cerevisiae  cell, a  Yarrowia  lipolitica cell, a  Candida krusei  cell, an  Issatchenkia orientalis  cell or an  Escherichia coli  cell. 
     
     
         9 . The recombinant host according to  claim 1 , wherein the ability of the host to produce geranylgeranyl diphosphate (GGPP) is upregulated. 
     
     
         10 . The recombinant host according to  claim 1 , which further comprises a nucleic acid sequence encoding one or more of:
 a polypeptide having hydroxymethylglutaryl-CoA reductase activity; or
 a polypeptide having farnesyl-pyrophosphate synthetase activity. 
   
     
     
         11 . A recombinant host capable of producing a steviol glycoside which overexpresses a heterologous polypeptide which mediates steviol glycoside transport. 
     
     
         12 . A process for the preparation of a steviol glycoside which comprises fermenting the recombinant host according to  claim 1  in a suitable fermentation medium and, optionally, recovering the steviol glycoside. 
     
     
         13 . The process according to  claim 12  for the preparation of a steviol glycoside, wherein the process is carried out on an industrial scale. 
     
     
         14 . A fermentation broth comprising a steviol glycoside obtainable by the process according to  claim 12 . 
     
     
         15 . A steviol glycoside obtained by the process according to  claim 12 . 
     
     
         16 . A composition obtainable by the process according to  claim 12 . 
     
     
         17 . A foodstuff, feed or beverage which comprises the steviol glycoside according to  claim 15 .