The abundance of natural gas has made reduced one carbon compounds like methane and methanol attractive feedstocks for commodity chemical and fuel production, particularly in bioprocesses where they can replace more expensive sugar substrates. While many organisms natively consume one carbon substrates, these microbes (known as methylotrophs) lack many of the well-developed genetic engineering tools that platform organisms possess, which limits their production capacity. Engineering methylotrophy in industrially popular organisms is a promising alternative – in particular, progress has been made to engineer Escherichia coli to consume methanol. However, to date, no E. coli strain has been developed that can grow on methanol as a sole carbon substrate. We have identified amino acid biosynthesis as a crucial bottleneck to sole methylotrophy using 13C labelling – while most metabolites in glycolysis and the TCA cycle show substantial methanol incorporation, labelling in amino acids is significantly lower, and many amino acids lack methanol carbon entirely. Starting with an E. coli ΔfrmA variant expressing heterologous ribulose monophosphate (RuMP) pathway enzymes, we achieved greater methanol incorporation into amino acids using a variety of metabolic engineering strategies. Improvements were made by modulating regulators of amino acid biosynthesis, overexpressing enzymes for the biosynthesis of specific amino acids, and utilizing enzyme variants that do not possess traditional allosteric inhibition. The best variants from each method were then combined, yielding additive benefits to methanol incorporation. Starting with strains generated wth these combined strategies, adaptive laboratory evolution was performed to further improve methanol incorporation. Evolved isolates demonstrate methanol utilization for the production of all proteinogenic amino acids, providing a powerful platform for engineering sole methylotrophy.

Rewiring amino acid biosynthesis via regulator modulation and operon overexpression improves methanol utilization in a synthetic Escherichia coli methylotroph

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.