AbstractsEngineering

Biological platforms for propionate production have been limited to anaerobic native microbial producers, such as Propionibacterium and Clostridium. In this work, we demonstrated high-level heterologous production of propionate under microaerobic conditions in engineered Escherichia coli (E. coli). Activation of the native Sleeping beauty mutase (Sbm) operon not only transformed E. coli to be propionogenic (i.e. propionate-producing) but also introduced an intracellular “flux competition” between the traditional C2-fermentative pathway (forming acetate and ethanol) and the novel C3-fermentative pathway (forming propionate and 1-propanol). The propionogenic E. coli was further engineered by inactivation or overexpression of various genes involved in the glycerol dissimilation pathways and their individual genetic effects on propionate production were investigated. Generally, knocking out genes involved in glycerol dissimilation (except glpA) can minimize levels of solventogenesis and shift more dissimilated carbon flux toward the C3-fermentative pathway. For effective propionate production, glycerol dissimilation should be channeled through the respiratory pathway and, upon suppressed solventogenesis with minimal production of highly reduced alcohols, the alternative NADH-consuming route associated with propionate synthesis can be critical for more flexible redox balancing. With the implementation of various biochemical and genetic strategies, high propionate titres of more than 11 g/L with high yields up to 0.4 g-propionate/g-glycerol (accounting for ~50% of dissimilated glycerol) were achieved, implying the potential for industrial application. To our knowledge, this represents the most effective non-native engineered microbial system for propionate production.