Sequencing of Corynebacterium glutamicum strains engineered for methanol-essential growth

Corynebacterium glutamicum was engineered for methanol-dependent growth with a sugar co-substrate by heterologous expression of genes for methanol dehydrogenase from Bacillus methanolicus and of ribulose monophosphate pathway genes for hexulose phosphate synthase and isomerase from Bacillus subtilis. Mutants carrying one of two independent metabolic cut-offs, i.e. either lacking ribose-5-phosphate isomerase or ribulose-5-phosphate epimerase were selected by adaptive laboratory evolution (ALE) for faster methanol-dependent growth. Whole genome sequencing of these strains was performed, resulting in identification of three mutations that caused improved methanol-dependent growth by 1) increased plasmid copy numbers, 2) enhanced riboflavin supply and 3) reduced formation of the methionine-analogue O-methyl-homoserine in the methanethiol pathway.