(569g) Engineering Anaerobic Metabolism of Short-Chain Alkanes in E. coli (Invited Speaker)

Natural gas liquids (NGLs) are abundant carbon sources for fuels and chemicals, but their efficient utilization is met with many technical hurdles relating to their low energy density, high cost of transportation and storage, and most notably the catalytic challenges associated with selective and controlled functionalization of these alkanes. While biological routes to alkane functionalization are promising, those requiring oxygen still suffer from significant energy and carbon inefficiencies. In contrast, a variety of anaerobic alkane-degrading bacteria (largely nitrate and sulfate reducers) activate sub-terminal C‑H bonds via fumarate addition. Through metabolic engineering, the “alkylsuccinate” products of this reaction can be converted into biofuels or other value-added chemicals, with co-regeneration of fumarate in a cyclic pathway.

Alkylsuccinate synthase enzymes require activation by a partner “activase”, which utilizes an iron-sulfur cluster to reductively cleave AdoMet, generating a 5’-dAdo· radical that in turn generates a catalytic glycyl radical on the synthase. Functional, heterologous expression of these finicky enzyme systems has proven difficult. With E. coli as the host microorganism, chosen for its ease of genetic manipulation, we have established anaerobic growth and gene expression conditions that support continued biosynthesis of alkylsuccinates using the mas gene system from Azoarcus sp. str. HxN1. I will describe some of the key experimental considerations and results that led to production of compounds such as 1-methyl-pentylsuccinate (from hexane) and 1-methyl-ethylsuccinate (from propane). I will also discuss other enzyme systems being developed for smaller alkane substrates, steps toward the directed evolution of alkylsuccinate synthases for improved activity and altered substrate specificity, and how these enzymes / intermediates will be interfaced with downstream pathways for biosynthesis of diverse products.