Construction of artificial micro-aerobic metabolism for energy- and carbon-efficient synthesis of medium chain fatty acids in Escherichia coli

Medium-chain (C-6-C-10) chemicals are important components of fuels, commodities and fine chemicals. Numerous exciting achievements have proven reversed beta-oxidation cycle as a promising platform to synthesize these chemicals. However, under native central carbon metabolism, energetic and redox constraints limit the efficient operation of reversed beta-oxidation cycle. Current fermentative platform has to use different chemically and energetically inefficient ways for acetyl-CoA and NADH biosynthesis, respectively. The characteristics such as supplementation of additional acetate and formate or high ATP requirement makes this platform incompatible with large-scale production. Here, an artificial micro-aerobic metabolism for energy and carbon-efficient conversion of glycerol to MCFAs was constructed to present solutions towards these barriers. After evaluating numerous bacteria pathways under micro-aerobic conditions, one synthetic metabolic step enabling biosynthesis of acetyl-CoA and NADH simultaneously, without any energy cost and additional carbon requirement, and reducing loss of carbon to carbon dioxide-emitting reactions, was conceived and successfully constructed. The pyruvate dehydrogenase from Enterococcus faecalis was identified and biochemically characterized, demonstrating the most suitable characteristics. Furthermore, the carbon and energy metabolism in Escherichia coli was rewired by the clustered regularly interspaced short palindromic repeats interference system, inhibiting native fermentation pathways outcompeting this synthetic step. The present engineered strain exhibited a 15.7-fold increase in MCFA titer compared with that of the initial strain, and produced 15.67 g/L MCFAs from the biodiesel byproduct glycerol in 3-L bioreactor without exogenous feed of acetate or formate, representing the highest MCFA titer reported to date. This work demonstrates this artificial micro-aerobic metabolism has the potential to enable the cost-effective, large-scale production of fatty acids and other value-added reduced chemicals.