Engineering Corynebacterium glutamicum for methanol-dependent growth and glutamate production.

Methanol is a promising feedstock for bioproduction of fuels and chemicals, thus massive efforts have been devoted to engineering non-native methylotrophic platform microorganisms to utilize methanol. Herein, we rationally designed and experimentally engineered the industrial workhorse Corynebacterium glutamicum to serve as a methanol-dependent synthetic methylotroph. The cell growth of the methanol-dependent strain relies on co-utilization of methanol and xylose, and most notably methanol is an indispensable carbon source. Due to the methanol-dependent characteristic, adaptive laboratory evolution was successfully applied to improving methanol utilization. The evolved mutant showed a 20-fold increase in cell growth on methanol-xylose minimal medium and utilized methanol and xylose with a high mole ratio of 3.83:1. 13C-labeling experiments demonstrated that the carbon derived from methanol was assimilated into intracellular building blocks, high-energy carriers, cofactors, and biomass (up to 63% 13C-labeling). By inhibiting cell wall biosynthesis, methanol-dependent glutamate production was also achieved, demonstrating the potential application in bioconversion of methanol into useful chemicals. Genetic mutations detected in the evolved strains indicate the importance of intracellular NAD+/NADH ratio, substrate uptake, and methanol tolerance on methanol utilization. This study reports significant improvement in the area of developing fully synthetic methylotrophs.