Converting Escherichia coli to a Synthetic Methylotroph Growing Solely on Methanol

Methanol, being electron rich and derivable from methane or CO2, is a potentially renewable one-carbon (C1) feedstock for microorganisms. Although the ribulose monophosphate (RuMP) cycle used by methylotrophs to assimilate methanol differs from the typical sugar metabolism by only three enzymes, turning a non-methylotrophic organism to a synthetic methylotroph that grows to a high cell density has been challenging. Here we reprogrammed E. coli using metabolic robustness criteria followed by laboratory evolution to establish a strain that can efficiently utilize methanol as the sole carbon source. This synthetic methylotroph alleviated a so far uncharacterized hurdle, DNA-protein crosslinking (DPC), by insertion sequence (IS)-mediated copy number variations (CNVs) and balanced the metabolic flux by mutations. Being capable of growing at a rate comparable with natural methylotrophs in a wide range of methanol concentrations, this synthetic methylotrophic strain illustrates genome editing and evolution for microbial tropism changes and expands the scope of biological C1 conversion. [Display omitted] •E. coli was engineered to grow on methanol alone by rational design and evolution•A doubling time of 8.5 h and maximum OD of 2 were achieved•Formaldehyde-induced DNA-protein crosslinking was identified and solved•Copy number variation of a region spanning 70kb facilitates carbon source shifts Chen et al. demonstrate genetic reprogramming of E. coli to efficiently grow with methanol as the sole carbon source.