Metabolic engineering for overproduction of fatty acids from glucose, and its application to biosynthesis of ω-hydroxy palmitic acid in Escherichia coli

Abstract

Metabolic engineering of fatty acid biosynthesis pathway combined with cofactor regeneration system was performed to overproduce palmitic acid (PA) and stearic acid (SA) for bioenergy and biomaterials such as bio-diesel and bio-degradable plastics. To produce fatty acids, thioesterase from Lactobacillus reuteri, and acyl-CoA dehydratase from Escherichia coli, the two key enzymes for fatty acid biosynthesis, were over-expressed to enhance PA biosynthesis. Additionally, acyl-CoA transferase was deleted to block fatty acid β-oxidation pathway. To direct metabolic flux into fatty acid synthesis, and control energy consumption, succinyl-CoA synthetase of TCA cycle, and transaldolase A of pentose phosphate pathway were also deleted. The engineered E. coli FFA4 strain without a P450 system could produce 503.0 mg/L of PA (C16) and 508.4 mg/L of SA (C18), of which the amounts are ca. 1.6 and 2.3 fold higher than those of the wild-type. To hydroxylate palmitic acid (HPA) produced, CYP153A monooxygenase from Marinobacter aqueolei and redox partners CamA/B from Pseudomonas putida were over-expressed. However, the production yield of hydroxyl palmitic acid did not increase as much as free fatty acid production did, suggesting that the hydroxylation was a rate determining step (RDS) of HPA production. For the maximum production of HPA, 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). Ultimately, the E.coli strain with NADH-level optimization produced 610 mg/L of HPA, which was almost a three-fold increase in its yield compared to the same strain without NADH overproduction.