Overproduction of w-Hydroxypalmitic Acid from Glucose in Escherichia coli

Fatty acid and its dervatives are good starting materials for various functional chemicals. Among the compounds, hydroxyfatty acids, especially ω-hydroxyfatty acids, are becoming highly significant ingredients of ceramides, lubricants, therapeutics, and plastic monomers in cosmetic, food, and chemical industry. Here, we have produced ω-hydroxypalmitic acid from glucose in E. coli BW25113 by introducing biosysthesis pathway of ω-hydroxypalmitic acid and applying strategies for overproduction of fatty acid and cofactor optimization. To introduce biosynthesis pathway of ω-hydroxypalmitic acid, CYP153A from Marinobacter aquaeolei was expressed with camA/camB from Pseudomonas putida. β-hydroxyacyl-ACP dehydratase (fabZ) and acyl-ACP thioesterase from Lactobacillus reuteri were expressed and acyl-CoA synthase (fadD) was deleted in order to overproduce long-chain fatty acids and block the degradation of fatty acid, respectively. The engineered E. coli without CYP153A produced 1.01 g /L of free fatty acid (C16 503.0mg/L, C18 508.4mg/L) which were 1.8-fold higher than that of wild type strain. Transaldolase A (talA), which connects pentose phosphate pathway and hexose diphosphate pathway, was deleted to increase intracellular NADPH concentration, which is required in biosynthesis of fatty acid. The strategy slightly increased the production free fatty acid to 1.08 g/L (C16 503.0mg/L, C18 508.4mg/L). CYP153A and CamA/CamB were applied into the engineered E. coli showing similar amount of production of ω-hydroxypalmitic acid (211.7 mg/L). Increase in precursor concentration did not improved increase of ω-hydroxypalmitic acid which indicates the hydroxylation step was the rate-limiting step. To maximize production of ω-hydroxypalmitic acid, NADH was overproduced by replacing alcohol dehydrogenase (adh)in E. coli chromosome with NAD⁺-dependent formate dehydrogenase (fdh) from Candida boidinii. Such strategy showed 2.8-fold increase in production of ω-hydroxypalmitic acid (610.8mg/L). Quantification of NADH and NAD+ showed that expression of fdh and deletion of adh increase the ratio of NADH/NAD⁺. Throughout this study, the ω-hydroxypalmitic acid overproduction was achieved by increasing production of free fatty acid and enhancing activity of P450 by increasing NADH pool. Further increment would be achieved by engineering P450 by increasing activity or electron transfer efficiency to release the rate-limiting step.