Methods for producing dicarboxylic acids
The present invention provides methods of producing dicarboxylic acids. The methods involve incubating a fatty acid or hydrocarbon substrate with an enzyme to produce a dicarboxylic acid product. The enzyme acts on the substrate to produce a product that has been both over-oxidized and has undergone cleavage of a C—C bond. In some embodiments the enzymes having these useful characteristics are mutants of a cytochrome P450 enzyme, for example an enzyme of the class CYP102 or a mutant thereof. The invention provides enzymes where these desirable characteristics can be found in a single enzyme, and thus in some embodiments the methods can be performed through the action of a single enzyme.
1. A method of producing a dicarboxylic acid comprising:
contacting a saturated fatty acid substrate having 15-20 carbon atoms and not bound to or by an acyl carrier protein with a CYP102 sub-family enzyme that comprises a ferredoxin reductase and a ferredoxin and that oxidizes the fatty acid substrate and breaks a C—C bond of the fatty acid substrate to produce a dicarboxylic acid product, wherein the dicarboxylic acid product has 3 less carbon atoms than the substrate and the enzyme has at least 95% sequence identity with a sequence selected from the group consisting of: SEQ ID NOs: 2, 4, 6 and 8; and
detecting the dicarboxylic acid product.
2. The method of claim 1 further comprising that hydroxylated fatty acid products are produced in addition to the dicarboxylic acid product when the enzyme is incubated with the fatty acid.
3. The method of claim 1 wherein the enzyme overoxidizes the fatty acid substrate.
4. The method of claim 3 wherein the reaction comprises the over-oxidation of adjacent diols.
5. The method of claim 1 wherein oxidizing the fatty acid substrate and breaking the C—C bond comprises the reaction mechanism:
wherein m+n=9-14.
6. The method of claim 5 further comprising contacting the substrate with an oxidizing agent.
7. The method of claim 1 wherein
the substrate is tridecanedioic acid (C19) and the dicarboxylic acid is palmitic acid (C16).
8. The method of claim 1 wherein the enzyme has at least one amino acid residue different from CYP102A1 wt (BM3) and/or CYP102A2 wt (YetO) and/or CYP102A3 wt (yrhJ) enzymes.
9. The method of claim 1 wherein the enzyme is selected from the group consisting of: BM3 (F87A), YetO (F89A), YetO (F89I), YetO (F89S), YetO (F89V).
10. The method of claim 9 wherein the substrate is palmitic acid or heptadecanoic acid.
11. The method of claim 9 wherein the enzyme is BM3-F87A.
12. The method of claim 1 wherein the enzyme has at least one amino acid residue different from CYP102A1 wt (BM3) or CYP102A2 wt (YetO).
13. The method of claim 1 wherein the substrate is palmitic acid or heptadecanoic acid.
14. The method of claim 13 wherein the substrate is palmitic acid.
15. The method of claim 1 wherein the enzyme has the sequence of SEQ ID NO: 2 or SEQ ID NO: 4.