Engineering Escherichia coli for Anaerobic Alkane Activation: Biosynthesis of (1-methylalkyl) succinates

Short-chain alkanes are abundant carbon sources of potential value for producing fuels and chemicals. However, their efficient utilization is met with many technical and economic hurdles relating to their low energy density, high cost of transportation, and the catalytic challenges associated with selective and controlled functionalization of these hydrocarbons. While biological routes to alkane functionalization using microorganisms are promising, those requiring oxygen still suffer from energy and carbon inefficiencies due to aerobic respiration. Therefore, oxygen-independent alkane utilization has the potential to offer greater biocatalytic efficiency. In anoxic environments, microbial activation of alkanes for subsequent metabolism occurs most commonly through the addition of fumarate to a sub-terminal carbon, producing an alkyl succinate. Alkyl succinate synthases are complex, multi-subunit enzymes that utilize a catalytic glycyl radical and require a partner, activating enzyme for hydrogen abstraction. While many genes encoding putative alkyl succinate synthases have been identified, primarily from nitrate- and sulfate-reducing bacteria, few have been characterized and none have been reported to be functionally expressed in a heterologous host organism. This thesis described the functional expression of the (1-methylalkyl) succinate synthase (Mas) enzyme system from Azoarcus sp. strain HxN1 in recombinant Escherichia coli. In detail, an anaerobic enzyme system was established, different media, buffer and host strains were compared to determine suitable culturing conditions for Mas system expression. The anaerobic biosynthesis of the expected products of fumarate addition to hexane, butane, and propane was confirmed and quantified by mass spectrometry. Maximum production of (1-methylpentyl) succinate was observed when masC, masD, masE, masB, and masG are all present on the expression plasmid; omitting masC reduces production by 66%, while omitting any other gene eliminates production. Meanwhile, deleting iscR (encoding the repressor of the E. coli iron-sulfur cluster operon) improves product titer, as did performing the biotransformation at reduced temperature (18o C), both suggesting alkyl succinate biosynthesis is largely limited by functional expression of this enzyme system. Protein engineering was investigated and protein purification was prepared for further enzyme assay works. Functional, heterologous expression of alkyl succinate synthases which catalyze anaerobic alkane activation has proven difficult. As the first case of functionally expressing alkyl succinate synthase in E. coli, our work should prove useful for further enzymatic alkane utilization.Chemical and Biomolecular Engineering, Department o