Artificial metabolic pathway for homoserine biosynthesis enables formaldehyde-dependent growth of Corynebacterium glutamicum

We designed an artificial homoserine biosynthetic pathway composed of 4-hydroxy-2-oxoheptanedioate aldolase (HpaI) from Escherichia coli B and leucine dehydrogenase (LeuDH) from Lysinibacillus sphaericus and implemented it in a homoserine auxotrophic mutant strain of Corynebacterium glutamicum. The engineered strain grew on glucose plus formaldehyde without homoserine addition. To enhance the metabolic flux of this artificial homoserine biosynthetic pathway, adaptive laboratory evolution of the engineered strain on glucose plus formaldehyde was conducted, and evolved strains exhibiting significant growth were obtained. A nonsense mutation in the leuDH gene was identified in one evolved mutant, suggesting that LeuDH is not necessary for the artificial homoserine biosynthesis pathway in the evolved strains. Whole genome resequencing revealed mutations in the upstream region of the NCgl0780 gene, which encodes an aminotransferase, in all evolved strains. Moreover, the expression of NCgl0780 was upregulated in the evolved strains compared to that in the parental strain, and the homoserine auxotrophic strain of C. glutamicum overexpressing both hpaI and NCgl0780 exhibited high growth on glucose plus formaldehyde in the absence of homoserine. These results support a role for HpaI and NCgl0780 in the observed growth phenotype of the evolved strains and suggest that NCgl0780 may substitute for or complement LeuDH in the evolved strains. Although homoserine formation through the proposed artificial metabolic pathway has not been directly demonstrated, this study provides genetic and physiological evidence supporting complementation of homoserine auxotrophy by a novel candidate pathway for formaldehyde-dependent homoserine biosynthesis composed of HpaI and NCgl0780 in C. glutamicum.