ObjectiveTo identify potential target genes involved in l-serine biosynthesis in Methylobacterium sp. MB200 and to evaluate the gnd genetically-engineered strains for l-serine production.ResultsFive genes that are not associated with the central metabolic pathway but with l-serine biosynthesis were identified from Methylobacterium sp. MB200 mutants. Gene gnd, encoding 6-phosphogluconate dehydrogenase (PGDH), was selected for further evaluation. The gnd deletion mutant showed a 600% increase in d-serine tolerance and an 80% decrease in PGDH activity compared to Methylobacterium sp. MB200. gnd over-expression did not affect d-serine tolerance, whereas it did increase enzyme-activity up to 136%. Additionally, analysis revealed that in Methylobacterium sp. MB200, l-serine inhibited PGDH activity. The deletion of gnd did not affect growth, whereas it did enhance the biosynthesis of l-serine, resulting in a 225% increase in production of l-serine compared to the wild-type.Conclusiongnd, one of the five genes identified here that is associated with l-serine synthesis, can be developed as a potential candidate for metabolic engineering to promote l-serine synthesis in Methylobacterium sp. MB200.