Efficient synthesis of hydrocarbons using an engineered reversal of the B-oxidation cycle: A new paradigm for the production of advanced biofuels

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PI: GonzalezProposal Number: 1134541Intellectual MeritAdvanced liquid biofuels based on long-chain hydrocarbons offer several advantages compared to alcohol biofuels, including less hygroscopicity and volatility, higher energy density, and compatibility with current infrastructure for storage, distribution and usage. Many microorganisms possess biosynthetic pathways for production of alkene and alkane hydrocarbons. For alkenes, the ?head-to-head? condensation of acyl-CoA thioesters has been proposed as the primary pathway, whereas alkanes are synthesized through a two-step pathway that involves conversion of acyl-CoA thioesters to fatty aldehydes, which are then decarbonylated to alkanes. To date, the fatty acid biosynthesis pathway has been used as the exclusive means to generate the acyl-CoA thioesters required for the synthesis of hydrocarbons. However, the operation of this pathway is not efficient because it consumes ATP in the synthesis of malonyl-ACP, which is the donor of two-carbon units for chain elongation. As a consequence, the ATP yield associated with the production of hydrocarbon through the fatty acid synthesis pathway is very low. This, in turn, greatly limits cell growth and hydrocarbon production.The overall goal of the proposed research is to metabolically engineer a functional reversal of the beta-oxidation cycle as a new metabolic platform for the efficient biosynthesis of long-chain hydrocarbons in E. coli host strains. Unlike the fatty acid biosynthesis pathway, the reversal of the beta-oxidation cycle operates with coenzyme-A (CoA) thioester intermediates and uses acetyl-CoA directly for acyl-chain elongation, rather than first requiring ATP-dependent activation to malonyl-CoA. These characteristics enable product synthesis at maximum carbon and energy efficiency. To achieve this goal, four objectives are proposed: 1) engineer a functional reversal of the beta-oxidation cycle with a minimal set of enzymes, 2) engineer pathways for the synthesis of alkanes and alkenes from acyl-CoA intermediates generated in the functional reversal of the beta-oxidation cycle, 3) improve the efficiency of the engineered reversal of the beta-oxidation cycle during the synthesis of hydrocarbons, and 4) perform system-wide characterization of wild-type and engineered strains.Broader ImpactsThe proposed research extends beyond the confines of advanced biofuel applications, as the ubiquitous nature of beta-oxidation enzymes has the potential to enable the combinatorial synthesis of a variety of non-native products in industrial organisms with a minimum number of foreign genes, an approach that has the potential to lead to efficient functioning of the engineered pathways. The proposed research will train post-doctoral research associates in metabolic engineering of these pathways.The proposed outreach and education activities will focus on sustainable energy topics, and work with school districts serving predominantly Hispanic populations. In particular, collaborative efforts with the Houston Harmony Science Academy will expose middle and high schools students to renewable energy concepts and associated career opportunities in this field.