Low-molecular-weight poly-γ-glutamic acid (LMW-γ-PGA) has attracted much attention owing to its great potential in food, agriculture, medicine, and cosmetics. Current methods of LMW-γ-PGA production, including enzymatic hydrolysis, are associated with low operational stability. Here, an efficient method for stable biosynthesis of LMW-γ-PGA was conceived by overexpression of γ-PGA hydrolase in Bacillus amyloliquefaciens NB. To establish stable expression of γ-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 γ-PGA titer with concomitant decrease in the molecular weight. Finally, a satisfactory yield of 17.62 ± 0.38 g/L LMW-γ-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-γ-PGA.

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The Jerusalem artichoke is a perennial plant that belongs to the sunflower family. As a non-grain crop, Jerusalem artichoke possesses a number of desirable characteristics that make it a valuable feedstock for biorefinery, such as inulin content, rapid growth, strong adaptability, and high yields. This review provides a comprehensive introduction to renewable Jerusalem artichoke-based biomass resources and recent advances in bio-based product conversion. Furthermore, we discuss the latest in the development of inulinase-producing microorganisms and enhanced inulin hydrolysis capacity of microbes by genetic engineering, which lead to a more cost-effective Jerusalem artichoke biorefinery. The review is aimed at promoting Jerusalem artichoke industry and new prospects for higher value-added production.

Bacillus amyloliquefaciens NB, a glutamate-independent poly-γ-glutamic acid (γ-PGA)-producing strain, can directly utilize inulin-containing sustainable materials. However, low γ-PGA yield and lack of efficient genetic engineering approaches have hindered the industrial use of this strain. Here, we used the CRISPR-Cas9n technique to engineer B. amyloliquefaciens to enhance γ-PGA production. We engineered three modules involved in inulin hydrolysis, reducing sugars metabolism, and γ-PGA synthesis in B. amyloliquefaciens. Specifically, overexpresed the native inulin hydrolase CscA and two expressionoptimized levanase and endoinulinase, overexpressed of key genes related to reducing sugar metabolism to increased ATP production, and removed polysaccharide operon epsA-O and γ-PGA hydrolase cwlO. Finally, the highest production of γ-PGA (32.14 ± 0.38 g/L) was obtained in a 7.5 L fed-batch fermenter. Thus, we successfully constructed an ideal candidate strain for efficient γ-PGA production from inulin, which provides an important research basis for the development of more biobased products.