γ-PGA Production Research Articles

The ClpY-ClpQ protease regulates multicellular development in Bacillus subtilis.

ATP-dependent proteases play essential roles in both protein quality control and the regulation of protein activities in bacteria. ClpYQ (also known as HslVU) is one of several highly conserved ATP-dependent proteases in bacteria. The regulation and biological function of ClpYQ have been well studied in Gram-negative bacteria, but are poorly understood in Gram-positive species. In this study, we showed that in the Gram-positive bacterium Bacillus subtilis, the ΔclpYQ deletion mutant formed early and robust biofilms, while swarming motility was severely impaired. Colonies of the ΔclpYQ mutant were also much less mucoid on agar plates, indicating the loss of the production of secreted γ-poly-dl-glutamic acid (γ-PGA). Global proteomic analysis using isobaric tags for relative and absolute quantification (iTRAQ) confirmed that a number of proteins involved in motility, chemotaxis and the production of γ-PGA were less abundant in the ΔclpYQ mutant. The results from both iTRAQ and Western immunoblotting showed that levels of the biofilm master repressor SinR were modestly reduced in the ΔclpYQ mutant, but probably significantly enough to alter biofilm regulation due to the ultrasensitivity of the expression of biofilm genes to SinR protein levels. Western immunoblotting also showed that the abundance of CodY, whose gene is clustered with clpYQ in the same operon, was not impacted on by ΔclpYQ. Lastly, our results suggested that, unlike in Escherichia coli, ClpYQ does not play an essential role in heat-shock response in both B. subtilis and Bacillus cereus. In conclusion, we propose that the ClpYQ protease is primarily involved in multicellular development in B. subtilis.

Purification of extra cellular poly-γ-glutamic acid as an antibacterial agent using anion exchange chromatography

BLAST analysis of the 16S rRNA gene sequence for the newly isolated bacterium, revealed significant identity (99.5%) with Bacillus sonorensis [Ijadi Bajestani, M., et al., International Journal of Biological Macromolecules, 2017. 96: p. 100–110]. According to the literature review for closely related species of Bacillus sonorensis, the production of poly-γ-glutamic acid (γ-PGA) as an extra cellular biopolymer was investigated for the isolated bacteria which is deposited in IBRC (Iranian Biological Resource Center) as Bacillus sp. Strain M2 (IBRC-M11173). To determine if γ-PGA production by Bacillus sp. Strain M2 is glutamate dependent, it was grown on PGA medium, consisted of sodium glutamate. The results proved that γ-PGA production is highly dependent on glutamate component. In the following, the bioproduct has undergone different purification processes mainly consisting of dialysis, deproteinization and anion exchange chromatography. Based on the high-performance liquid chromatography (HPLC) results for ion chromatography effluents, 59% of the initial PGA in main solution was eluted via NaCl elution. Gel permeation chromatography (GPC) characterization analysis was accomplished to determine the polydispersity and γ-PGA molecular weight. Two major average molecular weights were distinguished; the heavy weight fraction of 7.7×106g/mol with polydispersity index of 1.73 and the other one with an average molecular weight number of 1.7×104g/mol and polydispersity index of 4.4. The antibacterial activity of the extracellular γ-PGA, as an anionic biopolymer, toward Staphylococcus aureus and E. coli, was assayed using the clinical and laboratory standards institute (CLSI) guidelines. For Staphylococcus aureus the minimum inhibitory concentration (MIC) value was about 34g/L while for E. coli this value reaches 53g/L.

Enhanced Production of Poly-γ-glutamic acid by Overexpression of the Global Anaerobic Regulator Fnr in Bacillus licheniformis WX-02.

Poly-γ-glutamic acid is a multi-functional biopolymer with various applications. ATP supply plays an important role in poly-γ-glutamic acid (γ-PGA) synthesis. Global anaerobic regulator Fnr plays a key role in anaerobic adaptation and nitrate respiration, which might affect ATP generation during γ-PGA synthesis. In this study, we have improved γ-PGA production by overexpression of Fnr in Bacillus licheniformis WX-02. First, the gene fnr was knocked out in WX-02, and the γ-PGA yields have no significant differences between WX-02 and the fnr-deficient strain WXΔfnr in the medium without nitrate (BFC medium). However, the γ-PGA yield of 8.95g/L, which was produced by WXΔfnr in the medium with nitrate addition (BFCN medium), decreased by 74% compared to WX-02 (34.53g/L). Then, the fnr complementation strain WXΔfnr/pHY-fnr restored the γ-PGA synthesis capability, and γ-PGA yield was increased by 13% in the Fnr overexpression strain WX/pHY-fnr (39.96g/L) in BFCN medium, compared to WX/pHY300 (35.41g/L). Furthermore, the transcriptional levels of narK, narG, and hmp were increased by 5.41-, 4.93-, and 3.93-fold in WX/pHY-fnr, respectively, which led to the increases of nitrate consumption rate and ATP supply for γ-PGA synthesis. Collectively, Fnr affects γ-PGA synthesis mainly through manipulating the expression level of nitrate metabolism, and this study provides a novel strategy to improve γ-PGA production by overexpression of Fnr.

Poly-γ-glutamic Acid Synthesis, Gene Regulation, Phylogenetic Relationships, and Role in Fermentation.

Poly-γ-glutamic acid (γ-PGA) is a biodegradable biopolymer produced by several bacteria, including Bacillus subtilis and other Bacillus species; it has good biocompatibility, is non-toxic, and has various potential biological applications in the food, pharmaceutical, cosmetic, and other industries. In this review, we have described the mechanisms of γ-PGA synthesis and gene regulation, its role in fermentation, and the phylogenetic relationships among various pgsBCAE, a biosynthesis gene cluster of γ-PGA, and pgdS, a degradation gene of γ-PGA. We also discuss potential applications of γ-PGA and highlight the established genetic recombinant bacterial strains that produce high levels of γ-PGA, which can be useful for large-scale γ-PGA production.

Selection of an Effective Indicator for Rapid Detection of Microorganisms Producing γ-Polyglutamic Acid and Its Biosynthesis Under Submerged Fermentation Conditions Using Bacillus methylotrophicus.

γ-Polyglutamic acid (γ-PGA) is a biosynthetic outcome of glutamic acid polymerization by microbes. In the current study, we have isolated Bacillus methylotrophicus on solid differential media containing methylene blue. This is the first report mentioning the use of methylene blue to distinguish the monomeric and polymeric form of glutamic acid in the liquid medium using UV-Vis spectrophotometer. Our method can simplify the analytical process of γ-PGA confirmation using the aforementioned studies. This screening protocol is sensitive to the detection of γ-PGA quantities as low as 3μg/mL; thus, the potent producers can be effectively screened. Furthermore, we have carried out process optimization of the present strain for γ-PGA production wherein we could obtain 1.4-fold improvement in the yield with respect to utilization of carbon source and 2.6-fold increase with respect to nitrogen source under submerged fermentation at a shake flask level. We have shown an increase in γ-PGA titer from 1.5 to 36g/L using mannitol, monosodium glutamate, peptone, and tween 20.

Fishmeal Wastewater as A Low-Cost Nitrogen Source for γ-Polyglutamic Acid Production Using Bacillus subtilis

Fishmeal wastewater, a fishmeal processing waste, was used as culture medium to study the effect of Bacillus subtilis A3 on the production of γ-polyglutamic acid (γ-PGA). The results showed that the optimum concentration of chemical oxygen demand (CODCr) for fishmeal wastewater was 15 g/L. Moreover, addition of 30 g/L glucose and 25 g/L glutamic acid in the medium was beneficial to cell growth and production of γ-PGA. The study also showed that the high salinity of wastewater had little effect on cell growth and production of γ-PGA after dilution. Thus, the optimal medium consisted of COD 15, 30 g/L glucose, 25 g/L glutamic acid, in which the average yield of γ-PGA (25.07 ± 0.34 g/L) was obtained. The study suggested that fishmeal wastewater can be a replacement for nitrogen source for γ-PGA production, and hence it can be the cost-effective alternative in γ-PGA production. Meanwhile, the process can offset the disposal costs of the wastes.

Microbial production of poly-γ-glutamic acid.

Poly-γ-glutamic acid (γ-PGA) is a natural, biodegradable and water-soluble biopolymer of glutamic acid. This review is focused on nonrecombinant microbial production of γ-PGA via fermentation processes. In view of its commercial importance, the emphasis is on L-glutamic acid independent producers (i.e. microorganisms that do not require feeding with the relatively expensive amino acid L-glutamic acid to produce γ-PGA), but glutamic acid dependent production is discussed for comparison. Strategies for improving production, reducing costs and using renewable feedstocks are discussed.

Poly-γ-glutamic acid productivity of Bacillus subtilis BsE1 has positive function in motility and biocontrol against Fusarium graminearum.

In this study, we investigate the relationship between γ-PGA productivity and biocontrol capacity of Bacillus subtilis BsE1; one bacterial isolate displayed 62.14% biocontrol efficacy against Fusarium root rot. The γ-PGA yield assay, motility assay, wheat root colonization assay, and biological control assay were analysed in different γ-PGA yield mutants of BsE1. The pgsB (PGA-synthase-CapB gene) deleted mutant of BsE1 reduced γ-PGA yield and exhibited apparent decline of in vitro motile ability. Deletion of pgsB impaired colonizing capacity of BsE1 on wheat root in 30 days, also lowered biocontrol efficacies from 62.08% (wild type BsE1) to 14.22% in greenhouse experiment against Fusarium root rot. The knockout of pgdS and ggt (genes relate to two γ-PGA degrading enzymes) on BsE1, leads to a considerable improvement in polymer yield and biocontrol efficacy, which attains higher level compared with wild type BsE1. Compared with ΔpgsB mutant, defense genes related to reactive oxygen species (ROS) and phytoalexin expressed changes by notable levels on wheat roots treated with BsE1, demonstrating the functional role γ-PGA plays in biocontrol against Fusarium root rot. γ-PGA is not only important to the motile and plant root colonization ability of BsE1, but also essential to the biological control performed by BsE1 against Fusarium root rot. Our goal in this study is to reveals a new perspective of BCAs screening on bacterial isolates, without good performance during pre-assays of antagonism ability.

Development of Jerusalem artichoke resource for efficient one-step fermentation of poly-(γ-glutamic acid) using a novel strain Bacillus amyloliquefaciens NX-2S

This study aimed to develop non-food fermentation for the cost-effective production of poly-(γ-glutamic acid) (γ-PGA) using a novel strain of Bacillus amyloliquefaciens NX-2S. The new isolate assimilated inulin more efficiently than other carbohydrates from Jerusalem artichoke, without hydrolytic treatment. To investigate the effect of inulin on γ-PGA production, the transcript levels of γ-PGA synthetase genes (pgsB, pgsC, pgsA), regulatory genes (comA, degQ, degS), and the glutamic acid biosynthesis gene (glnA) were analyzed; inulin addition upregulated these key genes. Without exogenous glutamate, strain NX-2S could produce 6.85±0.22g/L of γ-PGA during fermentation. Exogenous glutamate greatly enhances the γ-PGA yield (39.4±0.38g/L) and productivity (0.43±0.05g/L/h) in batch fermentation. Our study revealed a potential method of non-food fermentation to produce high-value products.

A novel approach to improve poly-\u03b3-glutamic acid production by NADPH Regeneration in Bacillus licheniformis WX-02

Poly-γ-glutamic acid (γ-PGA) is an important biochemical product with a variety of applications. This work reports a novel approach to improve γ-PGA through over expression of key enzymes in cofactor NADPH generating process for NADPH pool. Six genes encoding the key enzymes in NADPH generation were over-expressed in the γ-PGA producing strain B. licheniformis WX-02. Among various recombinants, the strain over-expressing zwf gene (coding for glucose-6-phosphate dehydrogenase), WX-zwf, produced the highest γ-PGA concentration (9.13 g/L), 35% improvement compared to the control strain WX-pHY300. However, the growth rates and glucose uptake rates of the mutant WX-zwf were decreased. The transcriptional levels of the genes pgsB and pgsC responsible for γ-PGA biosynthesis were increased by 8.21- and 5.26-fold, respectively. The Zwf activity of the zwf over expression strain increased by 9.28-fold, which led to the improvement of the NADPH generation, and decrease of accumulation of by-products acetoin and 2,3-butanediol. Collectively, these results demonstrated that NADPH generation via over-expression of Zwf is as an effective strategy to improve the γ-PGA production in B. licheniformis.

Improved production of poly-γ-glutamic acid with low molecular weight under high ferric ion concentration stress in Bacillus licheniformis ATCC 9945a

This study found that high ferric ion (Fe3+) concentrations lead to high poly-γ-glutamic acid (γ-PGA) production and low-molecular-weight γ-PGA. In cultures containing 7.4 mmol/L FeCl3, the average molecular weight of γ-PGA reached 3.18 ± 0.3 × 105 Da, which is a 76% decrease from that in the culture without FeCl3 (1.31 ± 0.14 × 106 Da). Cultures containing increasing concentrations of FeCl3 (0–7.4 mmol/L) showed a corresponding increase in γ-PGA yield from 26.57 ± 1.64 to 34.93 ± 1.05 g/L. Transcription level analysis indicated that high FeCl3 concentrations could enhance the expression of γ-PGA synthetase genes (pgsA, pgsB, and pgsC), which may contribute to the improved production of γ-PGA. In addition, γ-PGA degradation genes ggt (encoding γ-glutamyltransferase) and cwlO were found to contribute to a lower γ-PGA molecular weight in cultures containing 7.4 mmol/L FeCl3. The result of γ-glutamyltransferase activity was in agreement with the results of the transcription level analysis. Overall, this study would provide a novel method for the regulation of synthesis of γ-PGA with low molecular weight.

Glutamate dehydrogenase (RocG) in Bacillus licheniformis WX-02: Enzymatic properties and specific functions in glutamic acid synthesis for poly-γ-glutamic acid production.

Poly-γ-glutamic acid (γ-PGA), a natural biopolymer, is widely used in cosmetics, medicine, food, water treatment, and agriculture owing to its features of moisture sequestration, cation chelation, non-toxicity and biodegradability. Intracellular glutamic acid, the substrate of γ-PGA, is a limiting factor for high yield in γ-PGA production. Bacillus subtilis and Bacillus licheniformis are both important γ-PGA producing strains, and B. subtilis synthesizes glutamic acid in vivo using the unique GOGAT/GS pathway. However, little is known about the glutamate synthesis pathway in B. licheniformis. The aim of this work was to characterize the glutamate dehydrogenase (RocG) in glutamic acid synthesis from B. licheniformis with both in vivo and in vitro experiments. By re-directing the carbon flux distribution, the rocG gene deletion mutant WX-02ΔrocG produced intracellular glutamic acid with a concentration of 90ng/log(CFU), which was only 23.7% that of the wild-type WX-02 (380ng/log(CFU)). Furthermore, the γ-PGA yield of mutant WX-02ΔrocG was 5.37g/L, a decrease of 45.3% compared to the wild type (9.82g/L). In vitro enzymatic assays of RocG showed that RocG has higher affinity for 2-oxoglutarate than glutamate, and the glutamate synthesis rate was far above degradation. This is probably the first study to reveal the glutamic acid synthesis pathway and the specific functions of RocG in B. licheniformis. The results indicate that γ-PGA production can be enhanced through improving intracellular glutamic acid synthesis.

Poly-γ-Glutamic Acids Contribute to Biofilm Formation and Plant Root Colonization in Selected Environmental Isolates of Bacillus subtilis.

Bacillus subtilis is long known to produce poly-γ-glutamic acids (γ-PGA) as one of the major secreted polymeric substances. In B. subtilis, the regulation of γ-PGA production and its physiological role are still unclear. B. subtilis is also capable of forming structurally complex multicellular communities, or biofilms, in which an extracellular matrix consisting of secreted proteins and polysaccharides holds individual cells together. Biofilms were shown to facilitate B. subtilis–plant interactions. In this study, we show that different environmental isolates of B. subtilis, all capable of forming biofilms, vary significantly in γ-PGA production. This is possibly due to differential regulation of γ-PGA biosynthesis genes. In many of those environmental isolates, γ-PGA seems to contribute to robustness and complex morphology of the colony biofilms, suggesting a role of γ-PGA in biofilm formation. Our evidence further shows that in selected B. subtilis strains, γ-PGA also plays a role in root colonization by the bacteria, pinpointing a possible function of γ-PGA in B. subtilis–plant interactions. Finally, we found that several pathways co-regulate both γ-PGA biosynthesis genes and genes for the biofilm matrix in B. subtilis, but in an opposing fashion. We discussed potential biological significance of that.

Cryoprotection of probiotic bacteria with poly-γ-glutamic acid produced by Bacillus subtilis and Bacillus licheniformis.

Poly-γ-glutamic acid (γ-PGA) is a naturally occurring biopolymer made up of repeating units of glutamic acid and can be potentially used for multiple applications. This study compared the production of γ-PGA by Bacillus subtilis and Bacillus licheniformis in GS and E media. The highest γ-PGA production was achieved using initial glycerol concentration of 40 and 80 g/l, ammonium chloride as the nitrogen source, 20 g/l glutamic acid at pH 6.5 for 72 h using E medium. On characterization, it was observed that glutamic acid was the sole component of the purified material. It contained a mixture of Na-γ-PGA and H+-γ-PGA. The survival of probiotics during freeze drying was improved by combining them with γ-PGA polymer. For Lactobacilli, 10% γ-PGA protected the cells significantly than 10% sucrose during freeze drying. γ-PGA protection was shown to improve the viability of probiotic bacteria in orange juice for 40 days. No considerable change was observed in the concentrations of citric acid, malic acid and ascorbic acid when probiotic bacteria and γ-PGA were introduced into orange juice and hence, it could be used as a non-dairy delivery platform for these bacteria.

Effects of MreB paralogs on poly-γ-glutamic acid synthesis and cell morphology in Bacillus amyloliquefaciens.

Actin-like MreB paralogs play important roles in cell shape maintenance, cell wall synthesis and the regulation of the D,L-endopeptidases, CwlO and LytE. The gram-positive bacteria, Bacillus amyloliquefaciens LL3, is a poly-γ-glutamic acid (γ-PGA) producing strain that contains three MreB paralogs: MreB, Mbl and MreBH. In B. amyloliquefaciens, CwlO and LytE can degrade γ-PGA. In this study, we aimed to test the hypothesis that modulating transcript levels of MreB paralogs would alter the synthesis and degradation of γ-PGA. The results showed that overexpression or inhibition of MreB, Mbl or MreBH had distinct effects on cell morphology and the molecular weight of the γ-PGA products. In fermentation medium, cells of mreB inhibition mutant were 50.2% longer than LL3, and the γ-PGA titer increased by 55.7%. However, changing the expression level of mbl showed only slight effects on the morphology, γ-PGA molecular weight and titer. In the mreBH inhibition mutant, γ-PGA production and its molecular weight increased by 56.7% and 19.4%, respectively. These results confirmed our hypothesis that suppressing the expression of MreB paralogs might reduce γ-PGA degradation, and that improving the cell size could strengthen γ-PGA synthesis. This is the first report of enhanced γ-PGA production via suppression of actin-like MreB paralogs.

Microbial synthesis of poly-γ-glutamic acid: current progress, challenges, and future perspectives.

Poly-γ-glutamic acid (γ-PGA) is a naturally occurring biopolymer made from repeating units of l-glutamic acid, d-glutamic acid, or both. Since some bacteria are capable of vigorous γ-PGA biosynthesis from renewable biomass, γ-PGA is considered a promising bio-based chemical and is already widely used in the food, medical, and wastewater industries due to its biodegradable, non-toxic, and non-immunogenic properties. In this review, we consider the properties, biosynthetic pathway, production strategies, and applications of γ-PGA. Microbial biosynthesis of γ-PGA and the molecular mechanisms regulating production are covered in particular detail. Genetic engineering and optimization of the growth medium, process control, and downstream processing have proved to be effective strategies for lowering the cost of production, as well as manipulating the molecular mass and conformational/enantiomeric properties that facilitate screening of competitive γ-PGA producers. Finally, future prospects of microbial γ-PGA production are discussed in light of recent progress, challenges, and trends in this field.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0537-7) contains supplementary material, which is available to authorized users.

Improvement of poly-γ-glutamic acid biosynthesis in a moving bed biofilm reactor by Bacillus subtilis NX-2

The production of poly-γ-glutamic acid (γ-PGA) by Bacillus subtilis NX-2 using a moving bed biofilm reactor (MBBR) system was tested for the first time in this study. Polypropylene TL-2 was chosen as a suitable carrier, and γ-PGA concentration of 42.7±0.86g/L and productivity of 0.59±0.06g/(Lh) were obtained in batch fermentation. After application of the strategy of dissolved oxygen (DO)-stat feeding, higher γ-PGA concentration and productivity were achieved than with glucose feedback feeding. Finally, the repeated fed-batch cultures implemented in the MBBR system showed high stability, and the maximal γ-PGA concentration and productivity of 74.2g/L and 1.24g/(Lh) were achieved, respectively. In addition, the promotion of oxygen transfer by an MBBR carrier was well explained by a computational fluid dynamics (CFD) simulation. These results suggest that an MBBR system could be applied to large-scale γ-PGA production.

Loss of GltB Inhibits Biofilm Formation and Biocontrol Efficiency of Bacillus subtilis Bs916 by Altering the Production of γ-Polyglutamate and Three Lipopeptides.

AimsThis study examined the contribution of GltB on biofilm formation and biocontrol efficiency of B. subtilis Bs916.Methods and ResultsThe gltB gene was identified through a biofilm phenotype screen and a bioinformatics analysis of serious biofilm formation defects, and then a gltB single knockout mutant was constructed using homologous recombination. This mutant demonstrated severe deficits in biofilm formation and colonisation along with significantly altered production ofγ-polyglutamate (γ-PGA) and three lipopeptide antibiotics (LPs) as measured by a transcriptional analysis of both the wild type B. subtilis Bs916 and the gltB mutant. Consequently, the mutant strain retained almost no antifungal activity against Rhizoctonia solani and exhibited decreased biocontrol efficiency against rice sheath blight. Very few gltB mutant cells colonised the rice stem, and they exhibited no significant nutrient chemotaxis compared to the wild type B. subtilis Bs916. The mechanism underlying these deficits in the gltB mutant appears to be decreased significantly in production of γ-PGA and a reduction in the production of both bacillomycin L and fengycin. Biofilm restoration of gltB mutant by additionγ-PGA in the EM medium demonstrated that biofilm formation was able to restore significantly at 20 g/L.ConclusionsGltB regulates biofilm formation by altering the production ofγ-PGA, the LPs bacillomycin L and fengcin and influences bacterial colonisation on the rice stem, which consequently leads to poor biocontrol efficiency against rice sheath blight.Significance and Impact of StudyThis is the first report of a key regulatory protein (GltB) that is involved in biofilm regulation and its regulation mechanism and biocontrol efficiency by B. subtilis.

Regulation of poly-γ-glutamic acid production in Bacillus subtilis GXA-28 by potassium

It is a simple and effective method to control the production of fermented products by metal ions. In this study, effect of KCl on γ-PGA production by B. subtilis GXA-28 was investigated. K+ was proved to be the true effector, and concentration of 201.2 mM was optimized for γ-PGA production. Addition of KCl to medium effectively reduced broth viscosity from 4289.5 mPa · s to 748.0 mPa · s by 82.6% and increased γ-PGA yield from 18.36 g/L to 25.62 g/L by 39.5%. Although high concentration KCl (>67.1 mM) suppressed cell growth, the maximum biomass with 201.2 mM had been brought forward to 16 h and had a marked effect in prolonging cell viability. Metabolic flux and key enzyme activities analysis indicated that enhanced GDH, GOGAT and GLR are important for γ-PGA production. Specifically, the expression of pgsB was improved 9.6-fold by KCl added, which might be another reason for enhanced γ-PGA production. In addition, that modulation of the D/L-glutamate ratio in γ-PGA and molecular weight could also be accomplished by variation of KCl concentrations. This work provided a simple control strategy for γ-PGA production, and it will be applied to γ-PGA production on a large scale in the future.

Optimized Production of GABA and γ-PGA in a Turmeric and Roasted Soybean Mixture Co-fermented by <i>Bacillus subtilis</i> and <i>Lactobacillus plantarum</i>

Optimized Production of GABA and γ-PGA in a Turmeric and Roasted Soybean Mixture Co-fermented by <i>Bacillus subtilis</i> and <i>Lactobacillus plantarum</i>