(256f) Genetic Refactoring for the Implementation of Formaldehyde-Based Regulation in Escherichia coli for Synthetic Methylotrophy

Industrial microbial fermentation is frequently used to produce a wide array of desirable products, including amino acids, vitamins, recombinant proteins, pharmaceuticals, and alternative renewable fuels. Production of these compounds is most commonly accomplished utilizing glucose or other sugar substrates. Methanol is an attractive non-food feedstock option due to its high degree of reduction, increasing supply via chemical and bioconversion processes from natural gas methane, and low contamination risk. Attempts to generate a strain of the model organism Escherichia coli capable of efficiently utilizing methanol as a substrate have been met with various 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 easily lead to formaldehyde accumulation, limiting the efficient assimilation of methanol 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 mechanisms 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 dynamic regulation driven directly in response to cell needs.

We genetically refactored methylotrophic E. coli strains by combinatorially placing engineered formaldehyde-responsive promoters before key genes of interest. Refactored strains were evaluated with a growth-based selection, and high performing strains were isolated and analyzed for the ideal transcriptional balancing of key genes in methylotrophic E. coli.

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