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Synthetic methylotrophy has recently been intensively studied to achieve methanol-based biomanufacturing of fuels and chemicals. However, attempts to engineer platform microorganisms to utilize methanol mainly focus on enzyme and pathway engineering. Herein, we enhanced methanol bioconversion of synthetic methylotrophs by improving cellular tolerance to methanol. A previously engineered methanol-dependent  Corynebacterium glutamicum  is subjected to adaptive laboratory evolution with elevated methanol content. Unexpectedly, the evolved strain not only tolerates higher concentrations of methanol but also shows improved growth and methanol utilization. Transcriptome analysis suggests increased methanol concentrations rebalance methylotrophic metabolism by down-regulating glycolysis and up-regulating amino acid biosynthesis, oxidative phosphorylation, ribosome biosynthesis, and parts of TCA cycle. Mutations in the  O -acetyl- l -homoserine sulfhydrylase Cgl0653 catalyzing formation of  l -methionine analog from methanol and methanol-induced membrane-bound transporter Cgl0833 are proven crucial for methanol tolerance. This study demonstrates the importance of tolerance engineering in developing superior synthetic methylotrophs.

Adaptive laboratory evolution enhances methanol tolerance and conversion in engineered Corynebacterium glutamicum