Method for the microbial production of 8-methyl nonanoic acid
The present disclosure relates to the production of 8-methyl nonanoic acid and medium chain branched fatty acids, e.g., via microbial fermentation.
1. A biosynthetic method of producing 8-methyl nonanoic acid (8M) comprising:
a) expressing a KASIIIa gene, a KASIIIb gene and one or more genes encoding an acyl-acyl carrier protein (ACP) thioesterase in a transformed cellular system;
b) feeding or producing isobutyryl-CoA in said transformed cellular system; and,
c) producing 8-methyl nonanoic acid;
wherein the KASIIIa gene comprises a nucleic acid sequence that is at least 90% identical to SEQ ID NO. 6 and the KASIIIb gene comprises a nucleic acid sequence that is at least 90% identical to SEQ ID NO. 7.
2. The biosynthetic method of claim 1 , wherein the one or more genes encoding an acyl-(ACP) thioesterase comprise at least one fatty acyl-ACP thioesterase B is (FATB) gene cloned from a plant of the Capsicum genus.
3. The biosynthetic method of claim 2 , wherein the one or more genes encoding an acyl-(ACP) thioesterase comprise a fatty acyl-ACP thioesterase B (FATB2) gene, said FATB2 gene comprising a nucleic acid sequence that is at least 90% identical to SEQ ID NO. 5.
4. The biosynthetic method of claim 3 , wherein the one or more genes encoding an acyl-(ACP) thioesterase further comprise a fatty acyl-ACP thioesterase B gene comprising a nucleic acid sequence that is at least 90% identical to SEQ ID NO. 4.
5. The biosynthetic method of claim 1 , wherein said transformed cellular system is selected from the group including yeast, non-capsaicinoid producing plants, algae and bacteria.
6. The biosynthetic method of claim 5 , wherein said cellular system is E. Coli.
7. The biosynthetic method of claim 1 , further comprising purifying said 8M to at least 70% purity.
8. The biosynthetic method of claim 1 , wherein isobutyryl-CoA is produced in said transformed cellular system by feeding isobutyric acid and co-expressing an acyl-CoA synthetase (ACS) gene in said transformed cellular system.
9. The biosynthetic method of claim 8 , wherein said ACS gene encodes an isobutyryl-CoA synthetase which converts isobutyric acid into isobutyryl-CoA.
10. The biosynthetic method of claim 8 , wherein ACS gene is CCL4 gene comprising a nucleic acid sequence that is at least 90% identical to SEQ ID NO. 8.
11. The biosynthetic method of claim 1 , wherein isobutyryl-CoA is produced in said transformed cellular system by feeding isobutyric acid with acetyl-CoA and co-expressing a propionate-CoA transferase (PCT) gene in said transformed cellular system.
12. The biosynthetic method of claim 11 , wherein said PCT gene encodes a propionate-CoA transferase that converts isobutyric acid and acetyl-CoA into isobutyryl-CoA and acetate.
13. The biosynthetic method of claim 12 , wherein said acetate-CoA transferase gene is a PCT gene comprising a nucleic acid sequence that is at least 90% identical to SEQ ID NO. 9.
14. The biosynthetic method of claim 1 , wherein isobutyryl-CoA is produced in said transformed cellular system by feeding glucose and co-expressing alsS, ilvC, ilvD and bkd genes in said transformed cellular system.
15. The biosynthetic method of claim 14 , wherein the alsS and bkd genes are cloned from Bacillus subtilis.
16. The biosynthetic method of claim 14 , wherein the ilvC and ilvD genes are clone from E. coli.