Engineering an in vivo EP-bifido pathway in Escherichia coli for high-yield acetyl-CoA generation with low CO emission.

The low carbon yield from native metabolic machinery produces unfavorable process economics during the biological conversion of substrates to desirable bioproducts. To obtain higher carbon yields, we constructed a carbon conservation pathway named EP-bifido pathway in Escherichia coli by combining Embden-Meyerhof-Parnas Pathway, Pentose Phosphate Pathway and "bifid shunt", to generate high yield acetyl-CoA from glucose. C-Metabolic flux analysis confirmed the successful and appropriate employment of the EP-bifido pathway. The CO release during fermentation significantly reduced compared with the control strains. Then we demonstrated the in vivo effectiveness of the EP-bifido pathway using poly-β-hydroxybutyrate (PHB), mevalonate and fatty acids as example products. The engineered EP-bifido strains showed greatly improved PHB yield (from 26.0 mol% to 63.7 mol%), fatty acid yield (from 9.17% to 14.36%), and the highest mevalonate yield yet reported (64.3 mol% without considering the substrates used for cell mass formation). The synthetic pathway can be employed in the production of chemicals that use acetyl-CoA as a precursor and can be extended to other microorganisms.