A new exogenous glutamic acid-independent gamma-PGA producing strain was isolated and characterized as Bacillus subtilis C10. The factors influencing the endogenous glutamic acid supply and the biosynthesis of gamma-PGA in this strain were investigated. The results indicated that citric acid and oxalic acid showed the significant capability to support the overproduction of gamma-PGA. This stimulated increase of gamma-PGA biosynthesis by citric acid or oxalic acid was further proved in the 10 L fermentor. To understand the possible mechanism contributing to the improved gamma-PGA production, the activities of four key intracellular enzymes were measured, and the possible carbon fluxes were proposed. The result indicated that the enhanced level of pyruvate dehydrogenase (PDH) activity caused by oxalic acid was important for glutamic acid synthesized de novo from glucose. Moreover, isocitrate dehydrogenase (ICDH) and glutamate dehydrogenase (GDH) were the positive regulators of glutamic acid biosynthesis, while 2-oxoglutarate dehydrogenase complex (ODHC) was the negative one. (C) 2012 Elsevier Ltd. All rights reserved.