Ketogulonicigenium vulgare is used widely during the industrial production of 2-keto-L-gulonic acid (2-KLG), the precursor of vitamin C, in a coculture with Bacillus megaterium. We analyzed the sulfate and coenzyme A metabolic module in the genome-scale metabolic model (GSMM) iWZ663 and found that the poor growth of K. vulgare was due to a deficiency in key reductases in the sulfate metabolic pathway. To carefully investigate the metabolism of sulfate, we developed a chemically defined medium (CDM) to produce pure cultures of K. vulgare. The addition of glutathione and L-cysteine to a flask culture of K. vulgare increased the cell growth, 2-KLG titer, and the intracellular coenzyme A level by 38.7%, 45.5%, and 85.3%, respectively, with glutathione, and by 25.6%, 35.8%, and 44.7%, respectively, with L-cysteine. The addition of glutathione to a 7-L fermenter culture of K. vulgare and B. megaterium increased the 2-KLG productivity by 20.9%. This study shows that the analysis of a specific metabolic module in GSMM can provide a potential strategy for optimizing microbial physiological functions. (c) 2013 Elsevier B.V. All rights reserved.