Engineer Escherichia coli for 1-propanol production by integrating novel biotechnological and bioprocessing strategies

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Advances in modern biotechnology have significantly extended the capacity of the bacterium Escherichia coli (E. coli) so that it is now one of the most popular cell factories for biomanufacturing. Specifically, genetic engineering based on recombinant DNA technology allows the introduction of foreign genes for episomal expression. Genomic engineering, based on site-specific gene knock-in and knock-out technology, enables optimum genomic editing. Metabolic engineering enables both control and tuning of key metabolic fluxes to overproduce target metabolites. Synthetic biology allows one to graft foreign pathways to produce non-natural metabolites. The proposed Discovery Grant program will pursue a novel approach that integrates these biotechnological strategies to realize the ultimate goal of “genetic tailoring” of E. coli strains for industrial purposes. 1-Propanol is a fine chemical with various industrial applications and has strong potential to replace ethanol as an alternative biofuel. However, up to now, 1-propanol production primarily relies on chemical synthesis and no microorganisms have been identified as a natural 1-propanol producer. This non-native metabolite of 1-propanol for wild-type E. coli will be used as the target product for technological demonstration and the developed strategies can be generically applied to produce other high-value metabolites. Recently, my research group identified a novel biosynthesis of 1-propanol in E. coli by manipulating the sleeping beauty mutase (Sbm) operon. This four-gene operon (sbm-ygfD-ygfG-ygfH) encodes various enzymes involved in a cobalamin-dependent metabolic pathway for decarboxylation of succinate into propionate. By expressing certain genes within the Sbm operon for extensive dissimilation of succinate along with key genes for increasing the availability of various precursors, heterologous production of 1-propanol in engineered E. coli strains was successfully demonstrated. To enhance 1-propanol production, the proposed research program will target a new systematic approach based on biotechnological and bioprocessing strategies to address various issues to optimize this biotransformation system. Fundamental biotechnological issues associated with strain construction include: (1) search and identification of novel genes involved in biosynthesis of 1-propanol from various microorganisms, (2) metabolic engineering of E. coli to drive the central carbon flux towards 1-propanol production pathway, (3) genomic engineering of E. coli to knock in and knock out various key genes affecting 1-propanol production on the genome. On the other hand, applied bioprocessing issues associated with the cultivation system include: (1) generic characterization of cultivation conditions, such as pH, temperature, medium recipe, aerobic or anaerobic cultivation, alternative cheap carbon sources, (2) development of operating protocols and control strategies for batch, fedbatch, and chemostat cultivations, (3) mathematical modeling and analysis of metabolic fluxes under various genetic and bioprocessing backgrounds to identify potential steps limiting 1-propanol production. The proposed Discovery research program provides a unique training program in novel scientific and industrial biotechnologies. Trainees will obtain a wide range of advanced skills required for future careers in biomanufacturing. In addition to extensive scientific understanding of various novel biotechnologies associated with biomanufacturing, the developed biological strains and bioprocess for 1-propanol production can be readily transferred to the Canadian bio-industry for commercialization, boosting Canada’s technological leadership in biomanufacturing and biofuels.