Enhanced poly(γ-glutamic acid) production by H₂O₂-induced reactive oxygen species in the fermentation of Bacillus subtilis NX-2

Effects of reactive oxygen species (ROS) on cell growth and poly(γ-glutamic acid) (γ-PGA) synthesis were studied by adding hydrogen peroxide to a medium of Bacillus subtilis NX-2. After optimizing the addition concentration and time of H₂O₂, a maximum concentration of 33.9 g/L γ-PGA was obtained by adding 100 µM H₂O₂ to the medium after 24 H. This concentration was 20.6% higher than that of the control. The addition of diphenyleneiodonium chloride (ROS inhibitor) can interdict the effect of H₂O₂-induced ROS. Transcriptional levels of the cofactors and relevant genes were also determined under ROS stress to illustrate the possible metabolic mechanism contributing to the improve γ-PGA production. The transcriptional levels of genes belonging to the tricarboxylic acid cycle and electron transfer chain system were significantly increased by ROS, which decreased the NADH/NAD⁺ ratio and increased the ATP levels, thereby providing more reducing power and energy for γ-PGA biosynthesis. The enhanced γ-PGA synthetic genes also directly promoted the formation of γ-PGA. This study was the first to use the ROS control strategy for γ-PGA fermentation and provided valuable information on the possible mechanism by which ROS regulated γ-PGA biosynthesis in B. subtilis NX-2.