Explore the vast range of titles available.

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.

Poly-γ-glutamic acid (γ-PGA) has diverse applications from cosmetic to drug delivery. The production of γ-PGA primarily relies on microbial fermentation using Bacillus spp. supplemented with l -glutamate supplementation. However, the high cost of l -glutamate supplementation limits industrial production. This study aimed to achieve direct γ-PGA production from glucose using a Corynebacterium glutamicum-Bacillus licheniformis coculture system. To create such a coculture system, we utilized B. licheniformis ATCC 9945a, a natural l -glutamate-dependent γ-PGA producing strain, and C. glutamicum F343, which exhibited an excellent capacity to produce l -glutamate from glucose. B. licheniformis ATCC 9945a grew well and produced small amounts of γ-PGA in the medium of C. glutamicum F343. Subsequently, B. licheniformis ATCC 9945a was cultured using the supernatant collected from the C. glutamicum F343 fermentation broth to investigate its effect on the fermentation profile. It was found that B. licheniformis ATCC 9945a produced more γ-PGA in the supernatant compared to when exogenously supplemented with l -glutamate. Moreover , nine intracellular metabolites were discovered to be strongly connected to γ-PGA synthesis by UPLC-MS. Finally, the coculture of C. glutamicum F343 and B. licheniformis ATCC 9945a to produce γ-PGA was conducted. We successfully achieved direct γ-PGA production from glucose under optimal conditions, including an inoculation time of 4 h for B. licheniformis after C. glutamicum inoculation, a 75% inoculum ratio of C. glutamicum , and a total inoculum size of 10% culture volume. The coculture system produced 12.49 g/L of γ-PGA in a shake flask and 22.7 g/L in a 5-L fermentor. Key points • C. glutamicum F343 could produce L-glutamate from glucose as a precursor for PGA synthesis by B. licheniformis ATCC 9945a . • The C. glutamicum-B. licheniformis coculture system could produce γ-PGA up to 22.7 g/L . • Nine intracellular metabolites demonstrated a remarkable influence on γ-PGA synesis by UPLC-MS and metabolite profiling .

Poly-gamma-glutamic acid (γ-PGA) is mainly synthesized by glutamate-dependent strains in the manufacturing industry. Therefore, understanding glutamate-dependent mechanisms is imperative. In this study, we first systematically analyzed the response of Bacillus subtilis SCP017-03 to glutamate addition by comparing transcriptomics and proteomics. The introduction of glutamate substantially altered gene expression within the central metabolic pathway of cellular carbon. Most genes in the pentose phosphate pathway (PPP), tricarboxylic acid (TCA) cycle, and energy-consuming phase of the glycolysis pathway (EMP) were down-regulated, whereas those in the energy-producing phase of glycolysis and those responsible for γ-PGA synthesis were up-regulated. Based on these findings, the fermentation conditions were optimized, and γ-PGA production was improved by incorporating oxygen carriers. In a batch-fed fermentor with glucose, the γ-PGA production reached 95.2 g/L, demonstrating its industrial production potential. This study not only elucidated the glutamate dependence mechanism of Bacillus subtilis but also identified a promising metabolic target for further enhancing γ-PGA production.

Background Genome-scale metabolic models (GEMs) allow predicting metabolic phenotypes from limited data on uptake and secretion fluxes by defining the space of all the feasible solutions and excluding physio-chemically and biologically unfeasible behaviors. The integration of additional biological information in genome-scale models, e.g., transcriptomic or proteomic profiles, has the potential to improve phenotype prediction accuracy. This is particularly important for metabolic engineering applications where more accurate model predictions can translate to more reliable model-based strain design. Results Here we present a GEM with Enzymatic Constraints using Kinetic and Omics data (GECKO) model of Bacillus subtilis , which uses publicly available proteomic data and enzyme kinetic parameters for central carbon (CC) metabolic reactions to constrain the flux solution space. This model allows more accurate prediction of the flux distribution and growth rate of wild-type and single-gene/operon deletion strains compared to a standard genome-scale metabolic model. The flux prediction error decreased by 43% and 36% for wild-type and mutants respectively. The model additionally increased the number of correctly predicted essential genes in CC pathways by 2.5-fold and significantly decreased flux variability in more than 80% of the reactions with variable flux. Finally, the model was used to find new gene deletion targets to optimize the flux toward the biosynthesis of poly-γ-glutamic acid (γ-PGA) polymer in engineered B. subtilis . We implemented the single-reaction deletion targets identified by the model experimentally and showed that the new strains have a twofold higher γ-PGA concentration and production rate compared to the ancestral strain. Conclusions This work confirms that integration of enzyme constraints is a powerful tool to improve existing genome-scale models, and demonstrates the successful use of enzyme-constrained models in B. subtilis metabolic engineering. We expect that the new model can be used to guide future metabolic engineering efforts in the important industrial production host B. subtilis .

Extensive research has been conducted to mitigate the hazards of coal mine dust. Dust suppressants are crucial for enhancing the dust and fall efficiency of water media. Currently, environmentally-friendly, functional, polymeric, and microbial dust suppressant, which represent new types of suppressants, are primarily in the experimental and exploratory stages. Commercial models are not yet mature, and further validation through field tests and over time is required to assess the continuous effectiveness, environmental friendliness, safety, economic feasibility, and simplicity of the process of new dust suppressant. Consequently, this study utilized response surface methodology to optimize the conditions for microbial fermentation to synthesize the biobased dust suppressant γ-PGA. The fermentation extracts of Bacillus licheniformis were analyzed by thermogravimetric analysis. Infrared spectroscopy was employed to explore the functional group structure of the synthesized products; the wettability of γ-PGA was tested using an optical method for measuring contact angle/surface tension and transmission electron microscopy. The results indicated that screening, optimizing, and culturing Bacillus licheniformis could produce γ-PGA fermentation fluid with a maximum yield of 23.76 g/L. Infrared spectroscopy analysis showed that the purified product contained typical functional groups of γ-PGA. Changes in the contact angle of γ-PGA solution with coal dust over time demonstrated that brown coal was wetted extremely quickly, with the largest change in contact angle; within 5 min of dropping the fermentation liquid, the contact angle sharply decreased from 69° to 0°, completely wetting the brown coal. Transmission electron microscopy revealed that the coal pores became looser when wetted by water, and when the γ-PGA wetted the surface of the coal dust, the solution penetrated into the pores, forming a liquid film that enveloped the medium within the coal pores and suppressed the native dust. Due to the abundance of free carboxyl groups (a-COOH), amino groups (NH–), and carbonyl groups (CO) on the molecular chains of γ-PGA, along with numerous hydrogen bonds between the γ-PGA chains, γ-PGA has a strong ability to absorb and retain water, making its capacity to wet solids significantly stronger than that of plain water. This research is expected to lay the experimental foundation for the development of green and efficient dust suppression materials for mining applications. In mining applications, γ-PGA solutions demonstrate versatile dust suppression capabilities. For operational face dust control, γ-PGA can be applied through high-pressure spray systems directly onto coal mining surfaces. Its rapid wetting properties enable immediate capture of airborne particulates. In material transportation systems, γ-PGA combined with foaming agents generates dust-suppressive foam that adheres to conveyor belts or coal loads in mining vehicles. Pre-wetting treatments using γ-PGA solutions prior to coal crushing operations leverage the polymer’s exceptional water absorption and retention capacities. This pretreatment reduces coal brittleness, thereby minimizing the generation of new particulates during mechanical fragmentation processes. The technology exhibits potential for cross-industry adaptation. In construction demolition scenarios, γ-PGA formulations could integrate with dust suppression cannons to mitigate transient particulate emissions. Road dust management represents another promising application, where γ-PGA solutions may enhance the longevity of surface moisture retention when deployed via standard road sprinkler systems, thereby reducing maintenance frequency.

Background The diverse metabolic mechanisms underlying bacterial extracellular polymeric substances give rise to a wide array of components with distinct functionalities, including exopolysaccharides (EPS) and poly-γ-glutamic acid (γ-PGA). The coordinated synthesis of various types of extracellular polymeric substances necessitates comprehensive investigation from a global regulatory perspective. Results In this study, we examined the impact of multiple environmental stressors on Bacillus species, revealing that the EPS and γ-PGA produced respond to stress through metabolic and cellular process reorganization. The overexpression of global transcriptional regulators influenced the production of EPS and γ-PGA differently. Specifically, quorum sensing-related global regulators such as rsbRA , rapA , and the carbon utilization regulator ccpA -2 were found to enhance EPS synthesis. Conversely, positive global transcriptional regulators associated with γ-PGA synthesis included carbon and nitrogen utilization-related regulators ccpA -2, cggR , and nrgB . Notably, the global regulators nrgB and cggR increased γ-PGA production by 33.64% and 44.14%, respectively, while this enhancement was accompanied by a concomitant reduction in EPS production. In B. licheniformis , omics analyses have elucidated critical pathways and metabolites implicated in stress response mechanisms that induce alterations in amino acid metabolism, carbon source utilization, alongside the activation of global regulatory elements. These studies indicated that nrgB predominantly governs downstream genes associated with carbon metabolism, energy metabolism, signal transduction, and membrane transport processes. Conclusions This work combines stress induction strategies and global transcription machinery engineering for investigating the coordinated synthesis of various types of extracellular polymeric substances, which has not been explored before. The insights gained from our research contribute to a deeper understanding of the regulatory networks governing the competition between γ-PGA and EPS, thereby providing a theoretical basis for the engineered modification of Bacillus licheniformis aimed at optimizing the production of extracellular polymeric substances. Graphical abstract

Starch, a crucial raw material, has been extensively investigated for biotechnological applications. However, its application in γ-polyglutamic acid (γ-PGA) production remains unexplored. Based on γ-PGA output of Bacillus subtilis SCP010-1, a novel asynchronous saccharification and fermentation process for γ-PGA synthesis was implemented. The results revealed that a starch concentration of 20%, α-amylase dosage of 75 U/g, liquefaction temperature of 72℃, and γ-PGA yield of 36.31 g/L was achieved. At a glucoamylase dosage of 100 U/g, saccharification 38 h at 60℃, the yield of γ-PGA increased to 48.88 g/L. The contents of total sugar, glucose, maltose and oligosaccharide in saccharified liquid were determined. Through batch fermentation of saccharified liquid in fermentor, the γ-PGA output was elevated to 116.08 g/L. This study can offer a potential cost reduction of 40%, which can be a promising advancement in industrial γ-PGA production. Moreover, our approach can be applied in other starch-based fermentation industries.

Poly-γ-glutamic acid (γ-PGA) is an anionic polymer with wide-ranging applications in the areas of medicine, light chemical industry, wastewater treatment, and agriculture. However, the production cost of γ-PGA is high for the requirement of adding the expensive precursor L-glutamic acid during fermentation, which hinders its widespread application. In this study, in order to improve γ-PGA yield, central carbon metabolism was engineered to enhance the carbon flux of tricarboxylic acid (TCA) cycle and glutamic acid synthesis in a γ-PGA production strain Bacillus licheniformis WX-02. Firstly, pyruvate dehydrogenase (PdhABCD) and citrate synthase (CitA) were overexpressed to strengthen the flux of pyruvate into TCA cycle, resulting in 34.93% and 11.14% increase of γ-PGA yield in B. licheniformis WX-02, respectively. Secondly, the carbon flux to glyoxylate shunt was rewired via varying the expression of isocitrate lyase (AceA), and a 23.24% increase of γ-PGA yield was obtained in AceA down-regulated strain WXPbacAaceBA. Thirdly, deletion of pyruvate formate-lyase gene pflB led to a 30.70% increase of γ-PGA yield. Finally, combinatorial metabolic engineering was applied, and γ-PGA titer was enhanced to 12.02 g/L via overexpressing pdhABCD and citA, repressing aceA, and deleting pflB, with a 69.30% improvement compared to WX-02. Collectively, metabolic engineering of central carbon metabolism is an effective strategy for enhanced γ-PGA production in B. licheniformis, and this research provided a promising strain for industrial production of γ-PGA.

Bacillus subtilis naturally produces large amounts of 2,3-butanediol (2,3-BD) as a main by-product during poly-γ-glutamic acid (γ-PGA) production. 2,3-BD is a promising platform chemical in various industries, and co-production of the two chemicals has great economic benefits. Co-production of γ-PGA and 2,3-BD by a newly isolated B. subtilis CS13 was investigated here. The fermentation medium and culture parameters of the process were optimized using statistical methods. It was observed that sucrose, l-glutamic acid, ammonium citrate, and MgSO4·7H2O were favorable for γ-PGA and 2,3-BD co-production at culture pH of 6.5 and 37 °C. An optimal medium composed of 119.8 g/L sucrose, 48.8 g/L l-glutamic acid, 21.1 g/L ammonium citrate, and 3.2 g/L MgSO4·7H2O was obtained by response surface methodology (RSM). The results show that the titers of γ-PGA and 2,3-BD reached 27.8 ± 0.9 g/L at 24 h and 57.1 ± 1.3 g/L at 84 h with the optimized medium, respectively. γ-PGA and 2,3-BD production by B. subtilis CS13 was significantly enhanced in fed-batch fermentations. γ-PGA (36.5 ± 1.1 g/L, productivity of 1.22 ± 0.04 g/L/h) and 2,3-BD concentrations (119.6 ± 2.8 g/L, productivity of 2.49 ± 0.66 g/L/h) were obtained in the optimized medium with feeding sucrose. The co-production of 2,3-BD and γ-PGA provides a new perspective for industrial production of γ-PGA and 2,3-BD.Key points• A strategy for co-production of γ-PGA and 2,3-BD was developed.• The culture parameters for the co-production of γ-PGA and 2,3-BD were studied.• RSM was used to optimize the medium for γ-PGA and 2,3-BD co-production.• 36.5 g/L γ-PGA and 119.6 g/L 2,3-BD were obtained from the optimum medium in fed-batch fermentation.

Poly gamma glutamic acid (γ-PGA) is an anionic polyamide with numerous applications. Previous studies revealed that L-proline metabolism is implicated in a wide range of cellular processes by increasing intercellular reactive oxygen species (ROS) generation. However, the relationship between L-proline metabolism and γ-PGA synthesis has not yet been analyzed. In this study, our results confirmed that deletion of Δ1-pyrroline-5-carboxylate dehydrogenase gene ycgN in Bacillus licheniformis WX-02 increased γ-PGA yield to 13.91 g L−1, 85.22% higher than that of the wild type (7.51 g L−1). However, deletion of proline dehydrogenase gene ycgM had no effect on γ-PGA synthesis. Furthermore, a 2.92-fold higher P5C content (19.24 μmol gDCW−1) was detected in the ycgN deficient strain WXΔycgN, while the P5C levels of WXΔycgM and the double mutant strain WXΔycgMN showed no difference, compared to WX-02. Moreover, the ROS level of WXΔycgN was increased by 1.18-fold, and addition of n-acetylcysteine (antioxidant) decreased its ROS level, which further reduced γ-PGA synthesis capability of WXΔycgN. Collectively, our results demonstrated that proline catabolism played an important role in maintaining ROS homeostasis, and deletion of ycgN-enhanced P5C accumulation, which induced a transient ROS signal to promote γ-PGA synthesis in B. licheniformis.