Production of propionic acid through biotransformation of glucose and d-lactic acid by construction of synthetic acrylate pathway in metabolically engineered E. coli

Construction of an efficient synthetic acrylate pathway in recombinant hosts such as E. coli and lactic acid bacteria should lead to synthesis of an array of products such as propionic acid, β-alanine, α-amino butyric acid and other products. The major bottlenecks impeding the titre of propionic acid, from d-lactate via the acrylate pathway in Escherichia coli and Lactococcus lactis, mainly include regulatory hurdles, inefficiency of enzymes involved in production and inability to overexpress multiple enzymes in soluble functional form along with other factors. In this work, the three enzymes, propionyl-CoA transferase (Pct) and acryloyl-CoA reductase (Acr) from E. coli, and lactoyl-CoA dehydratase (Lcd) from Megasphaera elsdenii, that possess better kinetic parameters and reduced size, have been recruited based on the insights gained from kinetic modelling of the acrylate pathway. Secondly, a common strategy for functional expression of these pathway enzymes has been demonstrated to improve their specific activities. The expression levels of Pct, Acr and Lcd were enhanced by sorbitol-induced native folding, with exposure to heat and low expression temperature, resulting in 11-, 4- and 4-fold higher yield of soluble protein than in the control. The specific activities of Pct and Acr were 39- and 34-fold higher than Clostridium propionicum counterparts. Also, the enzyme activity of Lcd was equivalent to that in the native producer, C. propionicum. The recombinant strains exhibited 11% and 20% lesser growth rates than in the control with propionate titre of 240 mg/L and 320 mg/L when grown in glucose and d-lactate, respectively. The yields of propionic acid from glucose and lactic acid were 5% and 32%, respectively. Further improvement in yields should be achieved by expressing all the enzymes in sufficient amount and appropriate ratios, overcoming all the regulatory hurdles.