Biological conversion of methanol by evolved Escherichia coli carrying a linear methanol assimilation pathway

Background: Methanol is regarded as a biorenewable platform feedstock because nearly all bioresources can be converted into methanol through syngas. Biological conversion of methanol using synthetic methylotrophs has thus gained worldwide attention. Results: Herein, to endow Escherichia coli with the ability to utilize methanol, an artificial linear methanol assimilation pathway was assembled in vivo for the first time. Distinct from native cyclic methanol utilization pathways, such as ribulose monophosphate cycle, the linear pathway requires no formaldehyde acceptor and only consists of two enzymatic reactions: oxidation of methanol into formaldehyde by methanol dehydrogenase and carboligation of formaldehyde into dihydroxyacetone by formolase. After pathway engineering, genome replication engineering assisted continuous evolution was applied to improve methanol utilization. C-13-methanol-labeling experiments showed that the engineered and evolved E. coli assimilated methanol into biomass. Conclusions: This study demonstrates the usability of the linear methanol assimilation pathway in bioconversion of C1 resources such as methanol and methane.