Compositions and methods for the biosynthesis of vanillin or vanillin beta-d-glucoside
Recombinant microorganisms, plants, and plant cells are disclosed that have been engineered to express a mutant AROM polypeptide and/or mutant catechol-O-methyltransferase polypeptide alone or in combination with one or more vanillin biosynthetic enzymes or UDP-glycosyltransferases (UGTs). Such microorganisms, plants, or plant cells can produce vanillin or vanillin beta-D-glucoside.
1. A method for producing vanillin and/or vanillin beta-D-glucoside comprising
(a) providing a recombinant host capable of producing vanillin, wherein said recombinant host harbors a heterologous nucleic acid encoding a mutant Catechol-O-Methyl Transferase (COMT) polypeptide, wherein the mutant COMT polypeptide comprises 1 to 10 amino acid substitutions in the amino acid sequence set forth in SEQ ID NO:27;
(b) cultivating said recombinant host for a time sufficient for said recombinant host to produce vanillin and/or vanillin glucoside; and
(c) isolating vanillin and/or vanillin glucoside from said recombinant host or from the cultivation supernatant, thereby producing vanillin and/or vanillin beta-D-glucoside.
2. The method of claim 1 , wherein the mutant COMT polypeptide is capable of catalyzing methylation of an —OH group of protocatechuic acid, wherein said methylation results in generation of at least 4 times more vanillic acid compared to iso-vanillic acid.
3. The method of claim 1 , wherein the mutant COMT polypeptide comprises an amino acid having a lower hydropathy index than leucine at position 198 of SEQ ID NO:27.
4. The method of claim 1 , wherein the mutant COMT polypeptide comprises Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Lys, Met, Phe, Pro, Ser, Thr, Trp and Tyr at position 198 of SEQ ID NO:27.
5. The method of claim 1 , wherein the mutant COMT polypeptide comprises an amino acid which has either a neutral or positive side-chain charge at pH 7.4 at position 199 of SEQ ID NO:27.
6. The method of claim 1 , wherein the mutant COMT polypeptide comprises Ala, Arg, Asn, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val at position 199 of SEQ ID NO:27.
7. The method of claim 1 , wherein the mutant COMT polypeptide further comprises a purification tag, a chloroplast transit peptide, a mitochondrial transit peptide, an amyloplast peptide, signal peptide, or a secretion tag at the N or C-terminus of said polypeptide.
8. The method of claim 1 , wherein said host is a microorganism.
9. The method of claim 1 , wherein said host is Saccharomyces cerevisiae, Schizosaccharomyces pombe or Escherichia coli.
10. The method of claim 1 , wherein said host is S. cerevisiae comprising a deletion of pdc1, a deletion of gdh1, and overexpressing glutamate dehydrogenase 2 (GHD2).
11. The method of claim 1 , wherein said host is a plant or plant cell.
12. The method of claim 1 , wherein said host is a Physcomitrella or tobacco plant or plant cell.
13. The method of claim 1 , wherein said host further comprises a gene encoding a 3-dehydroshikimate dehydratase (3DSD), a gene encoding an aromatic carboxylic acid reductase (ACAR), a gene encoding a uridine 5′-diphosphoglucosyl transferase (UGT), a gene encoding a phosphopantetheine transferase (PPTase) and/or a gene encoding a vanillyl alcohol oxidase (VAC)).
14. The method of claim 1 , wherein said host further comprises a gene encoding a wild-type O-methyltransferase (OMT).