Microbes have been engineered to produce a variety of chemicals, including biofuels, commodity chemicals, and therapeutics. Production can be enhanced by connecting biosynthesis pathways to host metabolism and optimizing the pathway’s expression. However, another important step in engineering microbes to produce desired chemicals is to increase production of their chemical precursors. In this work, we evaluated what precursors are needed to synthesize a variety of chemicals, and then designed and engineered strains to produce one of these precursors—pyruvate. We systematically evaluated what potential non-native products could be produced by Escherichia coli (using both native and heterologous pathways) and then evaluated their distance (in terms of metabolic reactions steps) from central metabolic precursors. Using a genome-scale metabolic model of E. coli and a set of potential heterologous reactions (from the KEGG database), ~1,800 non-native products could potentially be produced in E. coli using heterologous enzymes, with ~300 having commercial applications. Subsequent analysis found that pyruvate was the closest central metabolic precursor to the most non-native commercial products. Since pyruvate has industrial applications and can be converted into valuable products, we sought to develop a strain of E. coli that could produce pyruvate at high yields. Guided by a genome-scale metabolic model of E. coli, we identified different strategies for enhancing production of pyruvate from glucose. We constructed a number of strains which achieved yields up to 0.92 g pyruvate per g substrate (~95% theoretical yield) under aerobic conditions. These results illustrate how computational models can be used to prioritize precursor-based strategies and identify genetic modifications to enhance precursor production. Current efforts are focused on engineering the pyruvate production strains to produce other native and non-native products, as well as developing other precursor over-production strains.

Development and analysis of precursor production strains for chemical production

251st National Meeting (2016)

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