Poly(gamma-glutamic acid) (gamma-PGA) is a natural, biodegradable polyamide that is mostly produced by glutamate-dependent microorganisms such as Bacillus licheniforrnis, Bacillus amyloliquefaciens and Bacillus subtilis. In this study, a gamma-PGA-producing glutamate-independent strain, SK19.001, was screened and characterized as Bacillus methylotrophicus. The effects of various carbon and nitrogen sources on the gamma-PGA productivity of this strain were investigated. Many carbon sources, such as sodium glutamate, glucose, glycerol, soluble starch, alpha-lactose, maltose, sucrose, citric acid and fructose, proved suitable for SK19.001 to synthesize gamma-PGA with high molecular weight. Compared with the previously reported production from glutamate-independent strains, a high level of production of gamma-PGA (33.84-35.34 g/L) was obtained when SK19.001 was grown in a medium containing 30 g/L glycerol, 15 g/L sodium citrate and 50 g/L peptone. The gamma-PGA product showed an ultra-high molecular weight of over 10,000 kDa. The genome of B. methylotrophicus SK19.001 was sequenced and a comparative investigation revealed that the PgsB, PgsC, and PgsA protein sequences were almost identical between the glutamate-independent strains and less identical between the glutamate-independent and glutamate-dependent strains. This study is the first to demonstrate high levels of gamma-PGA production using B. methylotrophicus. The results suggest a promising new approach for gamma-PGA production. (C) 2015 Elsevier Ltd. All rights reserved.