High-Level Microbial Production of Propionate in Engineered Escherichia coli

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.1 yea

Manipulating the sleeping beauty mutase operon for the production of 1-propanol in engineered Escherichia coli

Background: While most resources in biofuels were directed towards implementing bioethanol programs, 1-propanol has recently received attention as a promising alternative biofuel. Nevertheless, no microorganism has been identified as a natural 1-propanol producer. In this study, we manipulated a novel metabolic pathway for the synthesis of 1-propanol in the genetically tractable bacterium Escherichia coli. Results: E. coli strains capable of producing heterologous 1-propanol were engineered by extending the dissimilation of succinate via propionyl-CoA. This was accomplished by expressing a selection of key genes, i.e. (1) three native genes in the sleeping beauty mutase (Sbm) operon, i.e. sbm-ygfD-ygfG from E. coli, (2) the genes encoding bifunctional aldehyde/alcohol dehydrogenases (ADHs) from several microbial sources, and (3) the sucCD gene encoding succinyl-CoA synthetase from E. coli. Using the developed whole-cell biocatalyst under anaerobic conditions, production titers up to 150 mg/L of 1-propanol were obtained. In addition, several genetic and chemical effects on the production of 1-propanol were investigated, indicating that certain host-gene deletions could abolish 1-propanol production as well as that the expression of a putative protein kinase (encoded by ygfD/argK) was crucial for 1-propanol biosynthesis. Conclusions: The study has provided a novel route for 1-propanol production in E. coli, which is subjected to further improvement by identifying limiting conversion steps, shifting major carbon flux to the productive pathway, and optimizing gene expression and culture conditions.Natural Sciences and Engineering Research Council (NSERC); Canada Research Chair (CRC) program of Canad

for the production of 1-propanol in engineered Escherichia coli

Manipulating the sleeping beauty mutase opero