Poly-gamma-glutamate (gamma-PGA) has applications in food, medical, cosmetic, animal feed, and wastewater industries. Bacillus subtilis DB430, which possesses the gamma-PGA synthesis ywsC-ywtAB genes in its chromosome, cannot produce gamma-PGA. An efficient synthetic expression control sequence (SECS) was introduced into the upstream region of the ywtABC genes, and this resulted in gamma-PGA-producing B. subtilis mutant strains. Mutant B. subtilis PGA6-2 stably produces high levels of gamma-PGA in medium A without supplementation of extra glutamic acid or ammonium chloride. The mutant B. subtilis PGA 6-2 is not only a gamma-PGA producer, but it is also a candidate for the genetic and metabolic engineering of gamma-PGA production. (C) 2010 American Institute of Chemical Engineers Biotechnol. Prog., 26: 1001-1007, 2010