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

Abstract

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 H O , a maximum concentration of 33.9 g/L γ-PGA was obtained by adding 100 µM H O 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 H O -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.

Enhanced production of poly‐γ‐glutamic acid by improving ATP supply in metabolically engineered Bacillus licheniformis

“…Recently, many strategies have been attempted to improve the γ‐PGA yield, including the elimination of byproduct synthetic pathways, overexpression of glutamic acid synthesis pathways, and deletion of γ‐PGA hydrolase genes (Feng et al, , ; Scoffone et al, ). As the conversion of glutamic acid to γ‐PGA consumes ATP, a sufficient supply of ATP is crucial for a high‐level γ‐PGA synthesis (Su et al, ; Tang et al, ; Zhang et al, ). However, due to the unbalanced ATP production and consumption, γ‐PGA production microbes are often hindered by ATP deficiency.…”

Enhanced Synthesis of Poly Gamma Glutamic Acid by Increasing the Intracellular Reactive Oxygen Species in the Bacillus licheniformis Δ1-pyrroline-5-carboxylate Dehydrogenase Gene ycgN Deficient Strain

“…ROS has been reported to promote γ -PGA synthesis capability in B. subtilis NX-2 previously (9). Thus, the transiently increased ROS level observed in WXΔ ycgN was proposed to be the primary cause of γ -PGA enhancement.…”

“…γ -PGA is mainly produced by Bacillus species as an extracellular polymer (4, 6-8). Several strategies have been conducted to improve γ -PGA production via metabolic engineering of γ -PGA synthesis-related metabolic network (9). For instance, γ -PGA yield was enhanced by 63.2% in B. amyloliquefaciens LL3 via double-deletion of genes cwlO (encodes a cell wall lytic enzyme) and epsA-O cluster (responsible for extracellular polysaccharide synthesis), as well as introduction of the gene vgb (encodes Vitreoscilla hemoglobin) (10).…”

“…In budding yeast, P5C directly inhibits the mitochondrial respiration and induces a burst of superoxide anions from the mitochondria (24). Since addition of H 2 O 2 was proven as an efficient strategy for enhancement of γ -PGA yield by improving the intracellular ROS (9), we hypothesized that the manipulating of proline metabolism might also affect γ -PGA synthesis.…”

“…In addition, ROS could serve as the secondary messengers to activate the downstream defense against invaded microorganisms in plants (29, 30), and played an important role in plant secondary metabolism (31). Moreover, ROS could regulate the product synthesis in microorganism, and the yields of xanthan gum produced by Xanthomonas campestris (29), validamycin A produced by Streptomyces hygroscopicus 5008 (31), γ -PGA produced by Bacillus subtilis NX-2 (9) and fumonisin produced by Fusarium verticillioides (32, 33) were all improved obviously by the ROS induction.…”