Investigation of Glutamate Dependence Mechanism for Poly-γ-glutamic Acid Production in Bacillus subtilis on the Basis of Transcriptome Analysis

The development of commercial poly-gamma-glutamic acid (gamma-PGA) production by glutamate-dependent strains requires understanding the glutamate dependence mechanism in the strains. Here, we first systematically analyzed the response pattern of Bacillus subtilis to glutamate addition by comparative transcriptomics. Glutamate addition induced great changes in intracellular metabolite concentrations and significantly upregulated genes involved in the central metabolic pathways. Subsequent gene overexpression experiments revealed that only the enhancement of glutamate synthesis pathway successfully led to gamma-PGA accumulation without glutamate addition, indicating the key role of intracellular glutamate for gamma-PGA synthesis in glutamate-dependent strains. Finally, by a combination of metabolic engineering targets, the gamma-PGA titer reached 10.21 +/- 0.42 g/L without glutamate addition. Exogenous glutamate further enhanced the gamma-PGA yield (35.52 +/- 0.26 g/L) and productivity (0.74 g/(L h)) in shake-flask fermentation. This work provides insights into the glutamate dependence mechanism in B. subtilis and reveals potential molecular targets for increasing economical gamma-PGA production.