Microbial synthesis of terpene and fatty acid biofuel precursors

Abstract

Our dependence on petroleum-derived fuels has accelerated the need for renewable fuel production. Microbial engineering is a promising avenue for production of advanced biofuels that can be dropped into existing transportation infrastructure. In this thesis, Escherichia coli was engineered to produce the terpene pinene, which is a precursor to a biosynthetic tactical fuel. The combinatorial approach of screening and fusing pinene-producing enzymes in E. coli improved pinene titers to 32 mg/L. This work also led to the discovery that pinene isomer ratios depend not only on the pinene synthase, but also the geraniol diphosphate synthase. In a similar light, medium-chain chemicals (MCCs) are also used as precursors to biofuels in addition to specialty chemicals. The engineering strategies, applications, and challenges surrounding the microbial production of MCCs are also explored in this thesis. To date, MCCs have generally been produced in mg/L levels; yet engineering of MCC precursor and tailoring enzymes can help improve microbial MCC titers to reach g/L levels. Medium-chain fatty acids (MCFAs) are key intermediates for MCCs but have low natural availability, which hinders their microbial production. In this thesis, E. coli MCFA production was improved over 3-fold by engineering the Acinetobacter baylyi medium-chain specific thioesterase (TE) for an improved interface with the E. coli acyl carrier protein. Finally, a phylogenetic-guided approach was used to engineer the A. baylyi TE for improved E. coli MCFA titers. The best phylogenetic-derived mutants improved MCFAs 1.3-fold with one mutant shifting the profile toward long-chain fatty acids. In conclusion, this work encompassed a combination of pathway and enzyme engineering to develop microbial strains capable of producing biofuel precursors. The accomplishments in this thesis work contribute engineered strains and improved enzymes to scientific community and sets essential groundwork for developing industrially relevant strains in the future.