(268e) Genetic Refactoring of Escherichia coli with a Formaldehyde-Inducible Promoter for Synthetic Methylotrophy

The discovery of large natural gas reserves within the United States have led to increased interest in converting natural gas into liquid fuels for use in existing transportation infrastructure. Methanol production from methane has increased recently through chemical and bioconversion processes, and its increasing supply, declining price, and low contamination risk make it a promising substrate for the production of chemicals and biofuels. Initial attempts to generate a strain of the model organism Escherichia coli capable of efficiently utilizing methanol as a substrate have been met with various metabolic bottlenecks.

Formaldehyde is a cytotoxic compound and the product of the first step of methanol assimilation, catalyzed by methanol dehydrogenase (MDH). Improper pathway balancing and gene regulation can lead to formaldehyde accumulation and low levels of methanol assimilation in engineered methylotrophic E. coli strains. Utilizing an E. coli formaldehyde-inducible promoter to drive expression of key methanol assimilation genes, including 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI) in the ribulose monophosphate (RuMP) pathway, emulates native methylotrophic regulation and avoids the need to add costly inducers. It also reduces the metabolic burden that high expression promoters can place on the cell, and instead allows for expression levels driven directly in response to cell needs.

We genetically refactored methylotrophic E. coli strains by strategically placing formaldehyde-responsive promoters before key genes of interest. Refactored strains were evaluated by assessing MDH and HPS activities, transcriptional expression levels, biomass yields on methanol, and methanol consumption rates. A promoter engineering approach was used to elucidate transcription factor binding sites and generate customized promoters for placement. A formaldehyde-responsive promoter library was generated with error-prone PCR and evaluated using a GFP reporter and fluorescence-activated cell sorting (FACS). Utilizing key metabolic intermediates as inducers is a promising approach for automated pathway balancing and increased product yields.

This work was supported by the US DOE ARPA-E agency through contract no. DE-AR0000432.