A new glutamic acid independent poly-gamma-glutamic acid (gamma-PGA) producing strain, which was identified as Bacillus amyloliquefaciens LL3 by analysis of 16S rDNA and gyrase subunit A gene (gyrA), was isolated from fermented food. The product had a molecular weight of 470, 801 and L-glutamate monomer content of 98.47%. The pre-optimal medium, based on single-factor tests and orthogonal design, contained 50 g/L sucrose, 2 g/L (NH(4))(2)SO(4), 0.6 g/L MgSO(4), and provided well-balanced changes in processing parameters and a gamma-PGA yield of 4.36 g/L in 200 L system. The gamma-PGA synthetase genes pgsBCA were cloned from LL3, and successfully expressed by pTrcLpgs vector in Escherichia coil JM109, resulting the synthesis of gamma-PGA without glutamate. This study demonstrates the designedly improved yield of gamma-PGA in 200 L system and the first report of pgsBCA from glutamic acid independent strain, which will benefit the metabolized mechanism investigation and the wide-ranging application of gamma-PGA. (C) 2010 Elsevier Ltd. All rights reserved.