Methanol Assimilation with CO₂Reduction inButyribacterium methylotrophicumand Development of Genetic Toolkits for Its Engineering

CO₂-derived methanol is an attractive raw material for biobased production of value-added chemicals. Here, we investigated the native methylotrophButyribacterium methylotrophicum, which could synchronously assimilate methanol and CO₂to butyric acid anaerobically. Supplementation with an approximate amount of bicarbonate could improve methanol metabolism ofB. methylotrophicum, and 2.04 g/L butyric acid was finally obtained from 100 mM methanol and 20 mM bicarbonate. The genes involved in methanol metabolism were further identified through homologous alignment and transcriptome analysis. The methyltransferase cluster along with genes of the carbonyl branch of the Wood-Ljungdahl pathway (WLP) was found to be transcriptionally activated for the assimilation of methanol and CO₂. To engineerB. methylotrophicum, an efficient electrotransformation protocol and several functional promoters were subsequently developed. Following a systematic investigation of various parameters, the electrotransformation efficiency was increased to 3.2 × 10³transformants/μg DNA. The activities of four heterologous promoters includingP_thl,P_araE,P_ptb, andP_adcwere comparatively determined. With these genetic toolkits, transformants overexpressing genes associated with methyltransferase system or butyric acid synthesis were obtained, where methanol consumption was increased by 16.9 and 14%, and butyric acid production was increased by 13.8 and 28.6%, respectively, in methanol and CO₂medium. These results exhibit the great potential ofB. methylotrophicumas a chassis for C1 bioconversion.