An abundance of methane from natural gas reserves has raised interest in engineering synthetic methylotrophs that can utilize methane or methanol, the oxidized form of methane, for growth and chemical production. Synthetic methylotrophs are non-methylotrophic platform organisms that have been engineered to utilize reduced C1 compounds. Since native methylotrophs lack many of the well-developed genetic engineering tools that platform organisms possess, the development of robust synthetic methylotrophs is of considerable interest. Our lab previously developed a synthetic Escherichia coli methylotroph that consumes 1 mol of methanol with 1 mol of glucose concurrently, making this strain “methanol-dependent.” This allows the sugar feedstock cost to be reduced by supplying a portion of the carbon substrate as methanol, which is less expensive. Using adaptive laboratory evolution and process optimization, we report improvements to growth and chemical production. One such evolved strain exhibited robust methanol-dependent growth in glucose minimal medium, whereas negligible growth was observed using the ancestral strain. Furthermore, methanol-dependent acetone and 1-butanol production was achieved via incorporation of heterologous enzymes from Clostridium. As a result of dependence, high average carbon labeling (up to 33%) in acetone and 1-butanol was observed when using a 13C-methanol tracer during fermentation. Future work will involve scale-up to a recently developed cell-recycle bioreactor system for high-titer production at bench-scale. Additionally, this work has been extended to methane utilization via incorporation of methanotrophic enzymes that allow the biological conversion of methane to methanol in recombinant E. coli. By using a 13C-methane tracer, we demonstrate incorporation of methane-derived carbon into central metabolites during gas fermentations in engineered E. coli. This work greatly expands the knowledge of the synthetic methylotrophy field by highlighting the importance of using reduced C1 compounds, alone or together with sugar-based feedstocks, for the production of liquid fuels and chemicals.

Engineering Escherichia coli for methanol-dependent growth and production

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

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