Implementing dynamic formaldehyde regulation in E. coli for synthetic methylotrophy

Conference Dates

July 14-18, 2019


Genetic engineering of microbes frequently focuses on over-expression of heterologous enzymes to increase product titers and yields. However, recent trends suggest that more balanced approaches are capable of achieving desired cellular outcomes while minimizing the metabolic burden and cost associated with extremely high expression. These approaches include aligning core cell interests with the researcher through precise regulatory changes, and balancing expression levels directly in response to cell needs. Implementing dynamic regulation is particularly appropriate for engineering non-native substrate utilization, due to the complexity of entrenched regulatory networks necessary for existing substrate pathways. Here, we apply a dynamic regulation approach to the utilization of non-native substrate methanol by the model organism Escherichia coli. 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, and decreasing cost. As a model organism, E. coli has been engineered to produce a wide array of products which could in turn be produced from methanol. Attempts to generate a strain of E. 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. An E. coli formaldehyde-inducible promoter was used to drive expression of key methanol assimilation genes in the ribulose monophosphate (RuMP) pathway, emulating native methylotrophic regulation mechanisms and avoiding the need to add costly inducers. Additional regulatory targets were identified with RNA-seq and regulated with formaldehyde. Expressing accessory genes to aid with the regeneration of intermediates and placing key genes under formaldehyde control led to 34% higher average carbon labeling when grown with 13C-labeled methanol, and 10% higher methanol growth benefit. Strains were further improved with adaptive laboratory evolution, and high performing strains were isolated and analyzed. This work was supported by the US DOE ARPA-E agency through contract no. DE-AR0000432.

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