Engineering Escherichia coli for propionic acid production through the Wood‐Werkman cycle

Native to propionibacteria, the Wood-Werkman cycle enables propionate production via succinate decarboxylation. Current limitations in engineering propionibacteria strains have re-directed attention towards the heterologous production in model organisms. Here, we report the functional expression of the Wood-Werkman cycle in Escherichia coli to enable propionate and 1-propanol production. The initial proof of concept attempt showed that the cycle can be used for production. However, production levels were low (0.17 mM). In silico optimisation of the expression system by operon rearrangement and RBS tuning improved performance by five fold. Adaptive laboratory evolution further improved performance redirecting almost 30% of total carbon through the Wood-Werkman cycle, achieving propionate and propanol titres of 9 mM and 5 mM, respectively. Rational engineering to reduce the generation of by-products showed that lactate (∆ldhA) and formate (∆pflB) knockout strains exhibit an improved propionate and 1-propanol production, while the ethanol (∆adhE) knockout strain only showed improved propionate production. This article is protected by copyright. All rights reserved