Efficient Biosynthesis of Low-Molecular-Weight Poly-γ-glutamic Acid by Stable Overexpression of PgdS Hydrolase in Bacillus amyloliquefaciens NB

Low-molecular-weight poly-gamma-glutamic acid (LMW-gamma-PGA) has attracted much attention owing to its great potential in food, agriculture, medicine, and cosmetics. Current methods of LMW-gamma-PGA production, including enzymatic hydrolysis, are associated with low operational stability. Here, an efficient method for stable biosynthesis of LMVV-gamma-PGA was conceived by overexpression of gamma-PGA hydrolase in Bacillus amyloliquefaciens NB. To establish stable expression of gamma-PGA hydrolase (PgdS) during fermentation, a novel plasmid pNX01 was constructed with a native replicon from endogenous plasmid p2Sip, showing a loss rate of 4% after 100 consecutive passages. Subsequently, this plasmid was applied in a screen of high activity PgdS hydrolase, leading to substantial improvements to gamma-PGA titer with concomitant decrease in the molecular weight. Finally, a satisfactory yield of 17.62 +/- 0.38 g/L LMW-gamma-PGA with a weight-average molecular weight of 20-30 kDa was achieved by direct fermentation of Jerusalem artichoke tuber extract. Our study presents a potential method for commercial production of LMW-gamma-PGA.