The production of ω-hydroxy palmitic acid using fatty acid metabolism and cofactor optimization in Escherichia coli.

Hydroxylated fatty acids (HFAs) are used as important precursors for bulk and fine chemicals in the chemical industry. Here, to overproduce long-chain (C16–C18) fatty acids and hydroxy fatty acid, their biosynthetic pathways including thioesterase (Lreu_0335) from Lactobacillus reuteri DSM20016, β-hydroxyacyl-ACP dehydratase (fabZ) from Escherichia coli, and a P450 system (i.e., CYP153A from Marinobacter aquaeolei VT8 and camA/camB from Pseudomonas putida ATCC17453) were overexpressed. Acyl-CoA synthase (fadD) involved in fatty acid degradation by β-oxidation was also deleted in E. coli BW25113. The engineered E. coli FFA4 strain without the P450 system could produce 503.0 mg/l of palmitic (C16) and 508.4 mg/l of stearic (C18) acids, of which the amounts are ca. 1.6- and 2.3-fold higher than those of the wild type. On the other hand, the E. coli HFA4 strain including the P450 system for ω-hydroxylation could produce 211.7 mg/l of ω-hydroxy palmitic acid, which was 42.1 ± 0.1 % of the generated palmitic acid, indicating that the hydroxylation reaction was the rate-determining step for the HFA production. For the maximum production of ω-hydroxy palmitic acid, NADH, i.e., an essential cofactor for P450 reaction, was overproduced by the integration of NAD+-dependent formate dehydrogenase (FDH) from Candida boidinii into E. coli chromosome and the deletion of alcohol dehydrogenase (ADH). Finally, the NADH-level-optimized E. coli strain produced 610 mg/l of ω-hydroxy palmitic acid (ω-HPA), which was almost a threefold increase in its yield compared to the same strain without NADH overproduction.