Production of hydroxylated and methoxylated flavonoids in yeast by expression specific enzymes
Recombinant yeast that are genetically modified to contain and express genes or gene pathways that produce hydroxylated and/or methoxylated flavonoids are provided. The genes or gene pathways are derived from plants, for example, from sweet basil ( Ocimum basilicum ) and the hydroxylated and/or methoxylated flavonoids include, for example, 6-methoxylated naringenin, 6-methoxylated luteolin and 6-methoxylated kaempferol.
1. A recombinant yeast that has been transformed with
at least one heterologous plant gene encoding a flavonoid ring A hydroxylase having at least 70% sequence identity to SEQ ID NO: 6 with flavonoid hydroxylase activity; and
at least one heterologous plant gene encoding a flavonoid O-methyltransferase having at least 70% sequence identity to SEQ ID NO: 1 with flavonoid O-methyltransferase activity.
2. The recombinant yeast of claim 1 , wherein the flavonoid ring A hydroxylase is a flavonoid 6-hydroxylase (F6H) and the flavonoid O-methyltransferase is a flavonoid 7-O-methyltransferase (F7OMT).
3. The recombinant yeast of claim 2 , wherein the F6H is ObF6H-1 as set forth in SEQ ID NO: 6.
4. The recombinant yeast of claim 2 , wherein the F70MT is ObFOMT1 as set forth in SEQ ID NO: 1.
5. The recombinant yeast of claim 1 , wherein the recombinant yeast is also transformed with at least one heterologous plant gene encoding one or more of:
a flavonoid 4-O-methyl transferase (F4OMT);
a flavonoid 5-O-methyl transferase (F5OMT);
a flavonoid 6-O-methyl transferase (F6OMT);
a flavonoid 8-O-methyltransferase (F8OMT);
a bifunctional enzyme with both flavonoid 4-O-methyl transferase and 6-O-methyl transferase activity; or
a flavone 8-hydroxylase (F8H).
6. The recombinant yeast of claim 5 , wherein the bifunctional enzyme is ObFOMT3 as set forth in SEQ ID NO: 2.
7. The recombinant yeast of claim 5 , wherein the at least one heterologous gene encodes a flavone 8-hydroxylase (F8H) and the recombinant yeast is further transformed with a heterologous gene encoding an Fdx-NADP + reductase.
8. The recombinant yeast of claim 1 , wherein the at least one heterologous plant gene encoding a flavonoid ring A hydroxylase and/or the at least one heterologous plant gene encoding at least one flavonoid O-methyltransferase are from sweet basil ( Ocimum basilicum ).
9. The recombinant yeast of claim 1 , wherein the recombinant yeast is a recombinant Saccharomyces cerevisiae.
10. The recombinant yeast of claim 1 , wherein the recombinant yeast is selected from the group consisting of:
SALV-1 deposited with NRRL under deposit number NRRL Y-67759;
8HS-1 deposited with NRRL under deposit number NRRL Y-67760;
GB-1 deposited with NRRL under deposit number NRRL Y-67761;
CIRM-1 deposited with NRRL under deposit number NRRL Y-67762; and
LAD-1 deposited with NRRL under deposit number NRRL Y-67763.
11. A method for producing at least one hydroxylated flavonoid and/or at least one methoxylated flavonoid, comprising
culturing the recombinant yeast of claim 1 with at least one precursor of either or both the at least one hydroxylated flavonoid and/or the at least one methoxylated flavonoid, wherein the step of culturing is performed under conditions suitable to produce the at least one hydroxylated flavonoid and/or methoxylated flavonoid; and
recovering the at least one hydroxylated and/or the at least one methoxylated flavonoid produced during culturing.
12. The method of claim 11 , wherein the at least one precursor is apigenin (API), naringenin (NAR), luteolin (LUT) or kaempferol (KAEM).
13. The method of claim 11 , wherein the at least one hydroxylated flavonoid and/or methoxylated flavonoid is:
i) scutellarein-7-methyl ether, cirismaritin, ladanein, salvigenin, 8-hydroxysalvigenin, gardenin B and/or genkwanin if API is fed;
ii) 2,3-dihydro-cirsimaritin, 2,3,-dihydro-ladanein, sakuranetin and/or carthamidin-7-methyl ether if naringenin is fed;
iii) pedalitin, cirsiliol, L7Me and/or eupatorine if luteolin is fed; and
iv) one or more 7-methyl- and/or 7,4′-dimethylated derivatives when kaempferol is fed.
14. The method of claim 11 , wherein the at least one heterologous plant gene encoding a flavonoid ring A hydroxylase and/or the at least one heterologous plant gene encoding at least one flavonoid O-methyltransferase are from sweet basil ( Ocimum basilicum ).
15. The method of claim 11 , wherein the recombinant yeast is a recombinant Saccharomyces cerevisiae.
16. The method of claim 11 , wherein the recombinant yeast is selected from the group consisting of:
SALV-1 deposited with NRRL under deposit number NRRL Y-67759;
8HS-1 deposited with NRRL under deposit number NRRL Y-67760;
GB-1 deposited with NRRL under deposit number NRRL Y-67761;
CIRM-1 deposited with NRRL under deposit number NRRL Y-67762; and
LAD-1 deposited with NRRL under deposit number NRRL Y-67763.