Biosynthetic production of steviol glycoside rebaudioside d4 from rebaudioside e

Abstract

The present invention relates, in some aspects, to the production of steviol glycoside rebaudioside D4 through the use of rebaudioside E. In some aspects, the invention relates to mutant CP1 enzymes, mutant HV1 enzymes as well as host cells and methods utilizing such enzymes, such as to produce rebaudioside D4.

1 .- 44 . (canceled) 
     
     
         45 . A recombinant polypeptide comprising an amino acid sequence at least 80% identical to any one of SEQ ID NOs: 3, 5, 7, 9, 11, and 13. 
     
     
         46 . The recombinant polypeptide of  claim 45 , comprising an amino acid sequence at least 95% identical to any one of SEQ ID NOs: 3, 5, 7, 9, 11, and 13. 
     
     
         47 . The recombinant polypeptide of  claim 45 , comprising the amino acid sequence of any one of SEQ ID NOs: 3, 5, 7, 9, 11 and 13. 
     
     
         48 . A nucleic acid encoding the recombinant polypeptide of  claim 45 . 
     
     
         49 . The nucleic acid of  claim 48 , comprising a nucleotide sequence at least 80% identical to the nucleotide sequence of any one of SEQ ID NOs: 4, 6, 8, 10, 12, and 14. 
     
     
         50 . The nucleic acid of  claim 48 , comprising a nucleotide sequence at least 95% identical to the nucleotide sequence of any one of SEQ ID NOs: 4, 6, 8, 10, 12, and 14. 
     
     
         51 . The nucleic acid of  claim 47 , comprising the nucleotide sequence of any one of SEQ ID NOs: 4, 6, 8, 10, 12, and 14. 
     
     
         52 . A vector comprising the nucleic acid of  claim 48 . 
     
     
         53 . The vector of  claim 52 , wherein the vector is a plasmid. 
     
     
         54 . A host cell comprising the nucleic acid of  claim 48 . 
     
     
         55 . The host cell of  claim 54 , wherein the host cell is selected from the group consisting of: a bacteria, a yeast, a filamentous fungi, a cyanobacteria algae and a plant cell. 
     
     
         56 . The host cell of  claim 54 , wherein the host cell is a bacterium selected from the group consisting of:  Escherichia; Salmonella; Bacillus; Acinetobacter; Streptomyces; Corynebacterium; Methylosinus; Methylomonas; Rhodococcus; Pseudomonas; Rhodobacter; Synechocystis; Saccharomyces; Zygosaccharomyces; Kluyveromyces; Candida; Hansenula; Debaryomyces; Mucor; Pichia; Torulopsis; Aspergillus; Arthrobotlys; Brevibacteria; Microbacterium; Arthrobacter; Citrobacter; Escherichia; Klebsiella; Pantoea; Salmonella Corynebacterium; Clostridium;  and  Clostridium acetobutylicum.    
     
     
         57 . The host cell of  claim 54 , wherein the host cell is a cell isolated from plants selected from the group consisting of soybean; rapeseed; sunflower; cotton; corn; tobacco; alfalfa; wheat; barley; oats; sorghum; rice; broccoli; cauliflower; cabbage; parsnips; melons; carrots; celery; parsley; tomatoes; potatoes; strawberries; peanuts; grapes; grass seed crops; sugar beets; sugar cane; beans; peas; rye; flax; hardwood trees; softwood trees; forage grasses; Arabidopsis thaliana; rice ( Oryza sativa );  Hordeum yulgare;  switchgrass ( Panicum vigratum );  Brachypodium  spp.;  Brassica  spp.; and  Crambe abyssinica.    
     
     
         58 . A composition comprising the recombinant polypeptide of  claim 45 . 
     
     
         59 . The composition of  claim 58 , further comprising a steviol glycoside. 
     
     
         60 . The composition of  claim 58 , further comprising a sucrose synthase. 
     
     
         61 . The composition of  claim 60 , wherein the sucrose synthase is selected from the group consisting of an  Arabidopsis  sucrose synthase 1, an  Arabidopsis  sucrose synthase 3 and a  Vigna radiate  sucrose synthase. 
     
     
         62 . The composition of  claim 60 , wherein the sucrose synthase is an  Arabidopsis thaliana  sucrose synthase 1. 
     
     
         63 . The composition of  claim 58 , further comprising uridine diphosphate (UDP), uridine diphosphate glucose (UDP-glucose), or any combinations thereof.