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

Abstract

The development of commercial poly-γ-glutamic acid (γ-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 γ-PGA accumulation without glutamate addition, indicating the key role of intracellular glutamate for γ-PGA synthesis in glutamate-dependent strains. Finally, by a combination of metabolic engineering targets, the γ-PGA titer reached 10.21 ± 0.42 g/L without glutamate addition. Exogenous glutamate further enhanced the γ-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 γ-PGA production.

Study on Mechanism of γ-PGA and Nattokinase Produced by Bacillus Subtilis Natto Based on RNA-Seq Analysis

“…The genes encoding these proteins are also present in sodium. In B. subtilis natto, we found that the three ycg MNO genes are up-regulated in the co-production environment, which is consistent with the results in Bacillus subtilis (Sha Y et al, 2019). That may be the reason for the co-production of γ-PGA and NK simultaneously in soybean medium.…”

“…In this study, soybean was used as basic substrate for co-producing γ-PGA and nattokinase based on this phenomenon. Carbon sources and sodium glutamate which added to the substrates could improve γ-PGA and nattokinase production obviously (Zeng W et al, 2013;Sha Y et al, 2019). Wherein selecting sucrose as the carbon source based on the previous study that glucose, sucrose and fructose have similar effects on γ-PGA production (Shi F et al, 2006).…”

“…Combined with the fermentation curve, it can be seen that the NK activity stabilized after 24h, while the yield of γ-PGA reached the highest at 36h. Subsequently, a part of the γ-PGA is degraded due to the γ-PGA depolymerase, which is largely excreted by B. subtilis natto in the later stage of xation (Zeng W et al, 2013;Sha Y et al, 2019 (Fig. 6).…”

Bacillus velezensis 83 a bacterial strain from mango phyllosphere, useful for biological control and plant growth promotion

“…We know that the gene cluster ( pgdS , pgsEACB ) involved in the synthesis γ-PGA are present in both strains B. velezensis 83 and B. velezensis FZB42 and they are of high identity (98%). Nevertheless, is known that B. velezensis 83 is a glutamic acid independent γ-PGA producing strain (Cristiano-Fajardo et al 2019 ) as other few Bacillus strains ( B. subtilis C10, Bacillus licheniformis A13, B. licheniformis TISTR 1010, B. methylotrophicus , B. subtilis NX2, B. amyloliquefaciens LL3) which only need glucose and NH 4 Cl as carbon and nitrogen sources, respectively, for γ-PGA synthesis (Cao et al 2011 ; Hsueh et al 2017 ; Sha et al 2019 ). In contrast, lack of γ-PGA production by B. velezensis FZB42 could be explained due to a glutamate dependent mechanism.…”

Recent Advances in Microbial Synthesis of Poly-γ-Glutamic Acid: A Review

“…The microbial synthetic pathway of γ-PGA principally covers three distinct stages: racemization, polymerization and transfer, and catabolism ( Figure 2 ) [ 10 , 11 , 22 ]. Glycolysis, the Pentose Phosphate Pathway (PPP), Tricarboxylic Acid (TCA) cycle, amino acid metabolism, and glutamate synthesis all participate in γ-PGA biosynthesis [ 56 ].…”