Biosynthesis of eriodictyol
The present invention relates to methods and transformed host cells for the production of eriodictyol from naringenin via bioconversion.
1 . A method of producing a 3′-hydroxylated flavonoid, the method comprising incubating a transformed host cell in a suitable medium comprising a flavonoid for a sufficient time to convert said flavonoid to a 3′-hydroxylated flavonoid, wherein the transformed host cell comprises a synthetic or recombinant nucleic acid molecule comprising a first polynucleotide sequence that encodes a flavonoid 3′-hydroxylase, said flavonoid 3′-hydroxylase comprising: i) an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 12, ii) the amino acid sequence of SEQ ID NO: 20, or iii) an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 12 and the amino acid sequence of SEQ ID NO: 20.
2 . The method of claim 1 , wherein said flavonoid 3′-hydroxylase comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 12.
3 . The method of claim 1 , wherein said flavonoid 3′-hydroxylase comprises the amino acid sequence of SEQ ID NO: 20.
4 . The method of claim 1 , wherein said flavonoid 3′-hydroxylase comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10.
5 . The method of claim 4 , wherein the first polynucleotide sequence comprises a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9.
6 . The method of claim 1 , wherein the nucleic acid molecule further comprises a second polynucleotide sequence that encodes a flavin reductase.
7 . The method of claim 6 , wherein the flavin reductase is a polypeptide comprising the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 18.
8 . The method of claim 1 , wherein the flavonoid has the generic structure of one of the following:
wherein R 3′ is H, and each of R 2′ , R 4′ , R 5′ , R 3 , R 5 , R 6 , R 7 , and R 8 independently, is selected from the group consisting of H, OH, and OCH 3 , and wherein R 3′ is OH in the 3′-hydroxylated flavonoid and each of R 2′ , R 4′ , R 5′ , R 3 , R 5 , R 6 , R 7 , and R 8 in the 3′-hydroxylated flavonoid is identical to the flavonoid.
9 . The method of claim 8 , wherein the flavonoid is naringenin and the 3′-hydroxylated flavonoid is eriodictyol.
10 . The method of claim 4 , wherein the flavonoid 3′-hydroxylase encoded by the first polynucleotide sequence comprises the amino acid sequence of SEQ ID NO: 4.
11 . The method of claim 7 , wherein the flavin reductase encoded by the second polynucleotide sequence comprises the amino acid sequence of SEQ ID NO: 14.
12 . The method of claim 1 , wherein the host cell is selected from the group of microbial species 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; Klebsiella; Pantoea ; and Clostridium.
13 . An isolated recombinant host cell transformed with a nucleic acid construct comprising a first polynucleotide sequence encoding a flavonoid 3′-hydroxylase comprising: i) an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 12, ii) the amino acid sequence of SEQ ID NO: 20, or iii) an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 12 and the amino acid sequence of SEQ ID NO: 20.
14 . The host cell of claim 13 , wherein said flavonoid 3′-hydroxylase comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 12.
15 . The host cell of claim 13 , wherein said flavonoid 3′-hydroxylase comprises the amino acid sequence of SEQ ID NO: 20.
16 . The host cell of claim 13 , wherein said flavonoid 3′-hydroxylase comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10.
17 . The host cell of claim 16 , wherein the first polynucleotide sequence comprises a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9.
18 . The host cell of claim 13 , wherein the nucleic acid construct further comprises a second polynucleotide sequence encoding a flavin reductase.
19 . The host cell of claim 18 , wherein the flavin reductase is a polypeptide comprising the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 18.
20 . The host cell of claim 13 , wherein the nucleic acid construct comprises a first polynucleotide sequence encoding a flavonoid 3′-hydroxylase comprising the amino acid sequence of SEQ ID NO: 4 and a second polynucleotide sequence encoding a flavin reductase comprising the amino acid sequence of SEQ ID NO: 14.
21 . The host cell of claim 13 , wherein the host cell is selected from the group of microbial species 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; Klebsiella; Pantoea ; and Clostridium.