Bioproduction of commodity chemicals from petroleum-derived feedstocks is not sustainable, and production from lignocelluloses has not progressed at the projected rate. Organisms capable of growth on methane (CH4) or carbon dioxide (CO2) require extensive optimization with respect to enzyme engineering, metabolic pathway manipulation, and culturing on a large scale in bioreactors. We present the utilization of acetylene dicarboxylic acid (ADCA) as a promising alternative carbon source for the platform organism Escherichia coli. ADCA can be synthesized in two steps from CH4 and CO2, two of the most potent greenhouse gases whose atmospheric concentrations continue to increase. We report that E. coli adapts to growth on ADCA as the exclusive carbon source using an adaptive laboratory evolution-type approach, where lag-phase reductions and maximum growth rate improvement occurred across successive generations. The expression levels of 22 genes were monitored in glucose, fumarate, and ADCA separately as the sole carbon source. Transcriptomic analysis and subsequent gene knockout studies revealed that DctA is the primary transporter for ADCA. We propose that two unique two-component signal transduction systems mediate the transport of ADCA and fumarate. We hypothesized that FumA catalyzes the hydration of ADCA in the cytoplasm, resulting in oxaloacetate, which subsequently enters the TCA cycle for biomass. We knocked out 15 genes differentially expressed and monitored the growth of the resulting strains on glucose, fumarate, and ADCA. We observed that strains lacking FumA fail to grow on ADCA. Consonant with reports in the literature, FumA catalyzes hydration of ADCA with a substrate specificity of 0.7 relative to fumarate. To identify mutations that benefit growth in ADCA media, whole-genome sequencing of strains is in progress. Upon completing this investigation, we anticipate engineering E. coli to synthesize a range of chemicals from CH4- and CO2-derived ADCA.

Acetylene dicarboxylate as a feedstock for E. coli growth: Identification of proteins implicated in transport and metabolism

ACS Spring 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.