Engineering formate assimilation in E.coli using stable isotope tracers and thermodynamic analysis
- Author(s): Xu, Jimmy
- Advisor(s): Park, Junyoung O
- et al.
Metabolite concentrations, fluxes, and free energies constitute the basis for understanding and controlling metabolism. Recently, using high-resolution mass spectrometry and multi-isotope tracing, improved flux quantitation led to determination of metabolic fates, pathway and nutrient contributions, and Gibbs free energy of reaction (ΔG) in central carbon metabolism using a relationship between reaction reversibility and thermodynamic driving force. We applied this quantitative analysis scheme towards the rational design of a pathway to assimilate formate in E. coli. Several synthetic pathways utilizing formate have been constructed in E. coli, indicative of its appeal as a raw material; however, these pathways either use formate as an electron donor rather than a carbon source or lose carbons as carbon dioxide. We have designed a pathway capable of incorporating formate into central carbon metabolism. Of the six engineered strains theoretically capable of operating this engineered metabplic pathway, we determined, through media sampling and tracing of the metabolic fate of 13C-formate, that the JXG2 strain incorporated formate into central carbon metabolism. Formate was responsible for 0.4% of carbons in hexose phosphate. Thus, we have engineered an E. coli strain that can serve as a platform for one-carbon utilization and sustainable biotechnology. At this point, further pathway optimization is needed, as flux through the engineered pathway is low; this may involve conducting targeted strain evolution on formate or knockout of competing pathways. When further optimized, our engineered metabolic pathway will be able to efficiently convert one-carbon compounds to advanced bioproducts.