Characterization, modeling, and anticancer activity of L.arginase production from marine Bacillus licheniformis OF2.

BACKGROUND: L-arginase, is a powerful anticancer that hydrolyzes L-arginine to L-ornithine and urea. This enzyme is widely distributed and expressed in organisms like plants, fungi, however very scarce from bacteria. Our study is based on isolating, purifying, and screening the marine bacteria that can produce arginase. RESULTS: The highest arginase producing bacteria will be identified by using microbiological and molecular biology methods as Bacillus licheniformis OF2. Characterization of arginase is the objective of this study. The activity of enzyme was screened, and estimated beside partial sequencing of arginase gene was analyzed. In silico homology modeling was applied to generate the protein's 3D structure, and COACH and COFACTOR were applied to determine the protein's binding sites and biological annotations based on the I-TASSER structure prediction. The purified enzyme was undergone an in vitro anticancer test. CONCLUSIONS: L-arginase demonstrated more strong anti-cancer cells with an IC50 of 21.4 ug/ml in a dose-dependent manner. L-arginase underwent another investigation for its impact on the caspase 7 and BCL2 family of proteins (BCL2, Bax, and Bax/Bcl2). Through cell arrest in the G1/S phase, L-arginase signals the apoptotic cascade, which is supported by a flow cytometry analysis of cell cycle phases.

A smart RBS library and its prediction model for robust and accurate fine-tuning of gene expression in Bacillus species.

Bacillus species, such as Bacillus subtilis and Bacillus licheniformis, are important industrial bacteria. However, there is a lack of standardized and predictable genetic tools for convenient and reproducible assembly of genetic modules in Bacillus species to realize their full potential. In this study, we constructed a Ribosome Binding Site (RBS) library in B. licheniformis, which provides incremental regulation of expression levels over a 104-fold range. Additionally, we developed a model to quantify the resulting translation rates. We successfully demonstrated the robust expression of various target genes using the RBS library and showed that the model accurately predicts the translation rates of arbitrary coding genes. Importantly, we also extended the use of the RBS library and prediction model to B. subtilis, B. thuringiensis, and B. amyloliquefacie. The versatility of the RBS library and its prediction model enables quantification of biological behavior, facilitating reliable forward engineering of gene expression.

A new ROS response factor YvmB protects Bacillus licheniformis against oxidative stress under adverse environment.

Bacillus spp., a class of aerobic bacteria, is widely used as a biocontrol microbe in the world. However, the reactive oxygen species (ROS) will accumulate once the aerobic bacteria are exposed to environmental stresses, which can decrease cell activity or lead to cell death. Hydroxyl radical (·OH), the strongest oxide in the ROS, can damage DNA directly, which is generated through Fenton Reaction by H2O2 and free iron. Here, we proved that the synthesis of pulcherriminic acid (PA), an iron chelator produced by Bacillus spp., could reduce DNA damage to protect cells from oxidative stress by sequestrating excess free iron, which enhanced the cell survival rates in stressful conditions (salt, antibiotic, and high temperature). It was worth noting that the synthesis of PA was found to be increased under oxidative stress. Thus, we demonstrated that the YvmB, a direct negative regulator of PA synthesis cluster yvmC-cypX, could be oxidized at cysteine residue (C57) to form a dimer losing the DNA-binding activity, which led to an improvement in PA production. Collectively, our findings highlight that YvmB senses ROS to regulate PA synthesis is one of the evolved proactive defense systems in bacteria against adverse environments.IMPORTANCEUnder environment stress, the electron transfer chain will be perturbed resulting in the accumulation of H2O2 and rapidly transform to ·OH through Fenton Reaction. How do bacteria deal with oxidative stress? At present, several iron chelators have been reported to decrease the ·OH generation by sequestrating iron, while how bacteria control the synthesis of iron chelators to resist oxidative stress is still unclear. Our study found that the synthesis of iron chelator PA is induced by reactive oxygen species (ROS), which means that the synthesis of iron chelator is a proactive defense mechanism against environment stress. Importantly, YvmB is the first response factor found to protect cells by reducing the ROS generation, which present a new perspective in antioxidation studies.

Optimization of fermentation parameters to improve the biosynthesis of selenium nanoparticles by Bacillus licheniformis F1 and its comprehensive application.

BACKGROUND: Selenium nanoparticles (SeNPs) are increasingly gaining attention due to its characteristics of low toxicity, high activity, and stability. Additionally, Bacillus licheniformis, as a probiotic, has achieved remarkable research outcomes in diverse fields such as medicine, feed processing, and pesticides, attracting widespread attention. Consequently, evaluating the activity of probiotics and SeNPs is paramount. The utilization of probiotics to synthesize SeNPs, achieving large-scale industrialization, is a current hotspot in the field of SeNPs synthesis and is currently the most promising synthetic method. To minimize production costs and maximize yield of SeNPs, this study selected agricultural by-products that are nutrient-rich, cost-effective, and readily available as culture medium components. This approach not only fulfills industrial production requirements but also mitigates the impact on downstream processes. RESULTS: The experimental findings revealed that SeNPs synthesized by B. licheniformis F1 exhibited a spherical morphology with diameters ranging from 110 to 170 nm and demonstrating high stability. Both the secondary metabolites of B. licheniformis F1 and the synthesized SeNPs possessed significant free radical scavenging ability. To provide a more robust foundation for acquiring large quantities of SeNPs via fermentation with B. licheniformis F1, key factors were identified through single-factor experiments and response surface methodology (RSM) include a 2% seed liquid inoculum, a temperature of 37 ℃, and agitation at 180 rpm. Additionally, critical factors during the optimization process were corn powder (11.18 g/L), soybean meal (10.34 g/L), and NaCl (10.68 g/L). Upon validating the optimized conditions and culture medium, B. licheniformis F1 can synthesize nearly 100.00% SeNPs from 5 mmol/L sodium selenite. Subsequently, pilot-scale verification in a 5 L fermentor using the optimized medium resulted in a shortened fermentation time, significantly reducing production costs. CONCLUSION: In this study, the efficient production of SeNPs by the probiotic B. licheniformis F1 was successfully achieved, leading to a significant reduction in fermentation costs. The exploration of the practical applications of this strain holds significant potential and provides valuable guidance for facilitating the industrial-scale implementation of microbial synthesis of SeNPs.

Biosynthesis of silver nanoparticle using Bacillus licheniformis culture-supernatant for combating pathogenic biofilms.

Bacterial biofilms pose a significant threat to healthcare due to their recalcitrance to antibiotics and disinfectants. This study explores the anti-biofilm potential of Bacillus licheniformis cell-free culture supernatant (CFS) and its derived silver nanoparticles (bSNPs) against Staphylococcus aureus and Pseudomonas aeruginosa. The CFS exhibited potent anti-biofilm activity against both bacterial species, even at low concentrations, while devoid of significant bactericidal effects, mitigating resistance risks. Characterization studies revealed the non-proteinaceous nature and thermal stability of the CFS's anti-biofilm agent, suggesting a robust and heat-resistant structure. Green synthesis of bSNPs from CFS resulted in nanoparticles with significant anti-biofilm properties, particularly against P. aeruginosa, indicating differences in susceptibility between the bacterial species. Epifluorescence microscopy confirmed bSNPs' ability to inhibit and partially disrupt biofilm formation without inducing cellular lysis. The study highlights the potential of B. licheniformis CFS and bSNPs as promising biofilm control agents, offering insights into their mechanisms of action and broad-spectrum efficacy. Further research elucidating the underlying molecular mechanisms and identifying specific bioactive compounds is warranted for the translation of these findings into clinically relevant applications for combating biofilm-associated infections.

Enhanced sporulation of B. licheniformis BF-002 through automatic co-feeding of carbon and nitrogen sources.

Bacillus licheniformis formulations are effective for environmental remediation, gut microbiota modulation, and soil improvement. An adequate spore quantity is crucial for the activity of B. licheniformis formulations. This study investigated the synergistic effects of carbon/nitrogen source consumption and concentration on B. licheniformis BF-002 cultivation, with the aim of developing an automatic co-feeding strategy to enhance spore production. Initial glucose (10 g/L) and amino nitrogen (1.5 g/L) concentrations promote cell growth, followed by reduced glucose (2.0 g/L) and amino nitrogen (0.5 g/L) concentrations for sustained spore generation. The spore quantity reached 2.59 × 1010 CFU/mL. An automatic co-feeding strategy was developed and implemented in 5 and 50 L cultivations, resulting in spore quantities of 2.35 × 1010 and 2.86 × 1010 CFU/mL, respectively, improving by 6.81% and 30.00% compared to that with a fixed glucose concentration (10.0 g/L). The culture broth obtained at both the 5 and 50 L scales was spray-dried, resulting in bacterial powder with cell viability rates of 85.94% and 82.68%, respectively. Even after exposure to harsh conditions involving high temperature and humidity, cell viability remained at 72.80% and 69.89%, respectively. Employing the automatic co-feeding strategy increased the transcription levels of the spore formation-related genes spo0A, spoIIGA, bofA, and spoIV by 7.42%, 8.46%, 8.87%, and 9.79%, respectively. The proposed strategy effectively promoted Bacillus growth and spore formation, thereby enhancing the quality of B. licheniformis formulations.

Genome mining of Bacillus licheniformis MCC2514 for the identification of lasso peptide biosynthetic gene cluster and its characterization.

The probiotic strain Bacillus licheniformis MCC2514 has been shown to produce a strong antibacterial peptide and the whole genome sequence of this strain is also reported in our previous study. The present study is focused on the genome level investigation of this peptide antibiotic and its characterization. Genome mining of the culture revealed the presence of three putative bacteriocin clusters, viz. lichenicidin, sonorensin and lasso peptide. Hence, the mode of action of the peptide was investigated by reporter assay, scanning electron microscopy, and Fourier Transform Infrared spectroscopy. Additionally, the peptide treated groups of Kocuria rhizophila showed a reduction in the fold expression for transcription-related genes. The gene expression studies, quantitative ß-galactosidase induction assay using the RNA stress reporter strain, yvgS along with the homology studies concluded that lasso peptide is responsible for the antibacterial activity of the peptide which acts as an inhibitor of RNA biosynthesis. Gene expression analysis showed a considerable increase in fold expression of lasso peptide genes at various fermentation hours. Also, the peptide was isolated, and its time-kill kinetics and minimum inhibitory concentration against the indicator pathogen K. rhizophila were examined. The peptide was also purified and the molecular weight was determined to be ~ 2 kDa. Our study suggests that this bacteriocin can function as an effective antibacterial agent in food products as well as in therapeutics as it contains lasso peptide, which inhibits the RNA biosynthesis.

Transcriptome analysis reveals the underlying mechanism for over-accumulation of alkaline protease in Bacillus licheniformis.

AIMS: Bacillus licheniformis AQ is an industrial strain with high production of alkaline protease (AprE), which has great industrial application value. However, how to regulate the production of AprE in the process of industrial fermentation is still not completely clear. Therefore, it is important to understand the metabolic process of AprE production in the industrial fermentation medium. METHODS AND RESULTS: In this study, transcriptome sequencing of the whole fermentation course was performed to explore the synthesis and regulation mechanism of AprE in B. licheniformis AQ. During the fermentation process, the AprE got continuously accumulated, reaching a peak of 42 020 U/mL at the fermentation endpoint (48 h). Meanwhile, the highly expressed genes were observed. Compared with the fermentation endpoint, there were 61 genes in the intersection of differentially expressed genes, functioning as catabolic processes, peptidases and inhibitors, chaperones, and folding catalysts. Furthermore, the protein-protein interactions network of AprE was constructed. CONCLUSION: This study provides important transcriptome information for B. licheniformis AQ and potential molecular targets for further improving the production of AprE.

Impact of Bacillus licheniformis from yaks following antibiotic therapy in mouse model.

Gut microorganism (GM) is an integral component of the host microbiome and health system. Abuse of antibiotics disrupts the equilibrium of the microbiome, affecting environmental pathogens and host-associated bacteria alike. However, relatively little research on Bacillus licheniformis alleviates the adverse effects of antibiotics. To test the effect of B. licheniformis as a probiotic supplement against the effects of antibiotics, cefalexin was applied, and the recovery from cefalexin-induced jejunal community disorder and intestinal barrier damage was investigated by pathology, real-time PCR (RT-PCR), and high-throughput sequencing (HTS). The result showed that A group (antibiotic treatment) significantly reduced body weight and decreased the length of jejunal intestinal villi and the villi to crypt (V/C) value, which also caused structural damage to the jejunal mucosa. Meanwhile, antibiotic treatment suppressed the mRNA expression of tight junction proteins ZO-1, claudin, occludin, and Ki67 and elevated MUC2 expression more than the other Groups (P < 0.05 and P < 0.01). However, T group (B. licheniformis supplements after antibiotic treatment) restored the expression of the above genes, and there was no statistically significant difference compared to the control group (P > 0.05). Moreover, the antibiotic treatment increased the relative abundance of 4 bacterial phyla affiliated with 16 bacterial genera in the jejunum community, including the dominant Firmicutes, Proteobacteria, and Cyanobacteria in the jejunum. B. licheniformis supplements after antibiotic treatment reduced the relative abundance of Bacteroidetes and Proteobacteria and increased the relative abundance of Firmicutes, Epsilonbacteraeota, Lactobacillus, and Candidatus Stoquefichus. This study uses mimic real-world exposure scenarios by considering the concentration and duration of exposure relevant to environmental antibiotic contamination levels. We described the post-antibiotic treatment with B. licheniformis could restore intestinal microbiome disorders and repair the intestinal barrier. KEY POINTS: • B. licheniformis post-antibiotics restore gut balance, repair barrier, and aid health • Antibiotics harm the gut barrier, alter structure, and raise disease risk • Long-term antibiotics affect the gut and increase disease susceptibility.

Engineering the Tat-secretion pathway of Bacillus licheniformis for the secretion of cytoplasmic enzyme arginase.

The industrial bacterium Bacillus licheniformis has long been used as a microbial factory for the production of enzymes due to its ability to secrete copious amounts of native extracellular proteins and its generally regarded as safe (GRAS) status. However, most attempts to use B. licheniformis to produce heterologous and cytoplasmic enzymes primarily via the general secretory (Sec) pathway have had limited success. The twin-arginine transport (Tat) pathway offers a promising alternative for the extracellular export of Sec-incompatible proteins because it transports full, correctly folded proteins. However, compared to the Sec pathway, the yields of the Tat pathway have historically been too low for commercial use. To improve the export efficiency of the Tat pathway, we identified the optimal Tat-dependent signal peptides and increased the abundance of the Tat translocases, the signal peptidase (SPase), and the intracellular chaperones. These strategic modifications significantly improved the Tat-dependent secretion of the cytoplasmic enzyme arginase into the culture medium using B. licheniformis. The extracellular enzymatic activity of arginase showed a 5.2-fold increase after these modifications. Moreover, compared to the start strain B. licheniformis 0F3, the production of extracellular GFP was improved by 3.8 times using the strategic modified strain B. licheniformis 0F13, and the extracellular enzymatic activity of SOX had a 1.3-fold increase using the strain B. licheniformis 0F14. This Tat-based production chassis has the potential for enhanced production of Sec-incompatible enzymes, therefore expanding the capability of B. licheniformis as an efficient cellular factory for the production of high-value proteins. KEY POINTS: • Systematic genetic modification of Tat-pathway in B. licheniformis. • Significant enhancement of the secretion capacity of Tat pathway for delivery the cytoplasmic enzyme arginase. • A new platform for efficient extracellular production of Sec-incompatible enzymes.

Poly-γ-glutamic acid overproduction of Bacillus licheniformis ATCC 9945a by developing a novel optimum culture medium and glutamate pulse feeding using different experimental design approaches.

The commercial production of multifunctional, biocompatible, and biodegradable biopolymers such as poly-γ-glutamic acid via microbial fermentation requires the development of simple and cheap methods for mass production. This study optimized the poly-γ-glutamic acid production of Bacillus licheniformis ATCC 9945a in several steps. At first, the most critical components of the culture medium, including l-glutamic acid, citric acid, and glycerol, were selected by screening nine factors through the Plackett-Burman experimental design and then were optimized using the response surface method and the central composite design algorithm. Under optimal conditions, the production of poly-γ-glutamic acid increased by more than 4.2 times from 11.2 to 47.2 g/L. This is one of the highest production rates of this strain in submerged batch fermentation reported so far using the optimized medium compared to the conventional base medium. A novel and efficient sudden pulse feeding strategy (achieved by a novel one-factorial statistical technique) of l-glutamic acid to the optimized medium increased biopolymer production from 47.2 to 66.1 g/L, the highest value reported in published literature with this strain. This simple, reproducible, and cheap fermentation process can considerably enhance the commercial applications of the poly-γ-glutamic acid synthesized by B. licheniformis ATCC 9945a.

Secretory expression of amylosucrase in Bacillus licheniformis through twin-arginine translocation pathway.

Amylosucrase (EC 2.4.1.4) is a versatile enzyme with significant potential in biotechnology and food production. To facilitate its efficient preparation, a novel expression strategy was implemented in Bacillus licheniformis for the secretory expression of Neisseria polysaccharea amylosucrase (NpAS). The host strain B. licheniformis CBBD302 underwent genetic modification through the deletion of sacB, a gene responsible for encoding levansucrase that synthesizes extracellular levan from sucrose, resulting in a levan-deficient strain, B. licheniformis CBBD302B. Neisseria polysaccharea amylosucrase was successfully expressed in B. licheniformis CBBD302B using the highly efficient Sec-type signal peptide SamyL, but its extracellular translocation was unsuccessful. Consequently, the expression of NpAS via the twin-arginine translocation (TAT) pathway was investigated using the signal peptide SglmU. The study revealed that NpAS could be effectively translocated extracellularly through the TAT pathway, with the signal peptide SglmU facilitating the process. Remarkably, 62.81% of the total expressed activity was detected in the medium. This study marks the first successful secretory expression of NpAS in Bacillus species host cells, establishing a foundation for its future efficient production. ONE-SENTENCE SUMMARY: Amylosucrase was secreted in Bacillus licheniformis via the twin-arginine translocation pathway.

Improved production of RNA-inhibiting antimicrobial peptide by Bacillus licheniformis MCC 2514 facilitated by a genetic algorithm optimized medium.

One of the significant challenges during the purification and characterization of antimicrobial peptides (AMPs) from Bacillus sp. is the interference of unutilized peptides from complex medium components during analytical procedures. In this study, a semi-synthetic medium was devised to overcome this challenge. Using a genetic algorithm, the production medium of AMP is optimized. The parent organism, Bacillus licheniformis MCC2514, produces AMP in very small quantities. This AMP is known to inhibit RNA biosynthesis. The findings revealed that lactose, NH4Cl and NaNO3 were crucial medium constituents for enhanced AMP synthesis. The potency of the AMP produced was studied using bacterium, Kocuria rhizophila ATCC 9341. The AMP produced from the optimized medium was eightfold higher than that produced from the unoptimized medium. Furthermore, activity was increased by 1.5-fold when cultivation conditions were standardized using the optimized medium. Later, AMP was produced in a 5 L bioreactor under controlled conditions, which led to similar results as those of shake-flask production. The mode of action of optimally produced AMP was confirmed to be inhibition of RNA biosynthesis. Here, we demonstrate that improved production of AMP is possible with the developed semi-synthetic medium recipe and could help further AMP production in an industrial setup.

Probiotic Bacillus licheniformis ZW3 Alleviates DSS-Induced Colitis and Enhances Gut Homeostasis.

Despite Bacillus species having been extensively utilized in the food industry and biocontrol as part of probiotic preparations, limited knowledge exists regarding their impact on intestinal disorders. In this study, we investigated the effect of Bacillus licheniformis ZW3 (ZW3), a potential probiotic isolated from camel feces, on dextran sulfate sodium (DSS)-induced colitis. The results showed ZW3 partially mitigated body weight loss, disease activity index (DAI), colon shortening, and suppressed immune response in colitis mice, as evidenced by the reduction in the levels of the inflammatory markers IL-1ß, TNF-α, and IL-6 (p < 0.05). ZW3 was found to ameliorate DSS-induced dysfunction of the colonic barrier by enhancing mucin 2 (MUC2), zonula occluden-1 (ZO-1), and occludin. Furthermore, enriched beneficial bacteria Lachnospiraceae_NK4A136_group and decreased harmful bacteria Escherichia-Shigella revealed that ZW3 improved the imbalanced gut microbiota. Abnormally elevated uric acid levels in colitis were further normalized upon ZW3 supplementation. Overall, this study emphasized the protective effects of ZW3 in colitis mice as well as some potential applications in the management of inflammation-related diseases.

High-level production of chitinase by multi-strategy combination optimization in Bacillus licheniformis.

In view of the extensive potential applications of chitinase (ChiA) in various fields such as agriculture, environmental protection, medicine, and biotechnology, the development of a high-yielding strain capable of producing chitinase with enhanced activity holds significant importance. The objective of this study was to utilize the extracellular chitinase from Bacillus thuringiensis as the target, and Bacillus licheniformis as the expression host to achieve heterologous expression of ChiA with enhanced activity. Initially, through structural analysis and molecular dynamics simulation, we identified key amino acids to improve the enzymatic performance of chitinase, and the specific activity of chitinase mutant D116N/E118N was 48% higher than that of the natural enzyme, with concomitant enhancements in thermostability and pH stability. Subsequently, the expression elements of ChiA(D116N/E118N) were screened and modified in Bacillus licheniformis, resulting in extracellular ChiA activity reached 89.31 U/mL. Further efforts involved the successful knockout of extracellular protease genes aprE, bprA and epr, along with the gene clusters involved in the synthesis of by-products such as bacitracin and lichenin from Bacillus licheniformis. This led to the development of a recombinant strain, DW2△abelA, which exhibited a remarkable improvement in chitinase activity, reaching 145.56 U/mL. To further improve chitinase activity, a chitinase expression frame was integrated into the genome of DW2△abelA, resulting in a significant increas to 180.26 U/mL. Optimization of fermentation conditions and medium components further boosted shake flask enzyme activity shake flask enzyme activity, achieving 200.28 U/mL, while scale-up fermentation experiments yielded an impressive enzyme activity of 338.79 U/mL. Through host genetic modification, expression optimization and fermentation optimization, a high-yielding ChiA strain was successfully constructed, which will provide a solid foundation for the extracellular production of ChiA.

A novel toolbox for precise regulation of gene expression and metabolic engineering in Bacillus licheniformis.

Despite industrial bio-manufacturing progress using Bacillus licheniformis, the absence of a well-characterized toolbox allowing precise regulation of multiple genes limits its expansion for basic research and application. Here, a novel gene expression toolbox (GET) was developed for precise regulation of gene expression and high-level production of 2-phenylethanol. Firstly, we established a novel promoter core region mosaic combination model to combine, characterize and analyze different core regions. Characterization and orthogonal design of promoter ribbons allowed convenient construction of an adaptable and robust GET, gene gfp expression intensity was 0.64%-16755.77%, with a dynamic range of 2.61 × 104 times, which is the largest regulatory range of GET in Bacillus based on modification of promoter P43. Then we verified the protein and species universality of GET using different proteins expressed in B. licheniformis and Bacillus subtilis. Finally, the GET for 2-phenylethanol metabolic breeding, resulting in a plasmid-free strain producing 6.95 g/L 2-phenylethanol with a yield and productivity of 0.15 g/g glucose and 0.14 g/L/h, respectively, the highest de novo synthesis yield of 2-phenylethanol reported. Taken together, this is the first report elucidating the impact of mosaic combination and tandem of multiple core regions to initiate transcription and improve the output of proteins and metabolites, which provides strong support for gene regulation and diversified product production in Bacillus.

Systematic Adaptation of Bacillus licheniformis to 2-Phenylethanol Stress.

The compound 2-phenylethanol (2-PE) is a bulk flavor and fragrance with a rose-like aroma that can be produced by microbial cell factories, but its cellular toxicity inhibits cellular growth and limits strain performance. Specifically, the microbe Bacillus licheniformis has shown a strong tolerance to 2-PE. Understanding these tolerance mechanisms is crucial for achieving the hyperproduction of 2-PE. In this report, the mechanisms of B. licheniformis DW2 resistance to 2-PE were studied by multi-omics technology coupled with physiological and molecular biological approaches. 2-PE induced reactive oxygen species formation and affected nucleic acid, ribosome, and cell wall synthesis. To manage 2-PE stress, the antioxidant and global stress response systems were activated; the repair system of proteins and homeostasis of the ion and osmotic were initiated. Furthermore, the tricarboxylic acid cycle and NADPH synthesis pathways were upregulated; correspondingly, scanning electron microscopy revealed that cell morphology was changed. These results provide deeper insights into the adaptive mechanisms of B. licheniformis to 2-PE and highlight the potential targets for genetic manipulation to enhance 2-PE resistance. IMPORTANCE The ability to tolerate organic solvents is essential for bacteria producing these chemicals with high titer, yield, and productivity. As exemplified by 2-PE, bioproduction of 2-PE represents a promising alternative to chemical synthesis and plant extraction approaches, but its toxicity hinders successful large-scale microbial production. Here, a multi-omics approach is employed to systematically study the mechanisms of B. licheniformis DW2 resistance to 2-PE. As a 2-PE-tolerant strain, B. licheniformis displays multifactorial mechanisms of 2-PE tolerance, including activating global stress response and repair systems, increasing NADPH supply, changing cell morphology and membrane composition, and remodeling metabolic pathways. The current work yields novel insights into the mechanisms of B. licheniformis resistance to 2-PE. This knowledge can also be used as a clue for improving bacterial performances to achieve industrial-scale production of 2-PE and potentially applied to the production of other relevant organic solvents, such as tyrosol and hydroxytyrosol.

Extracellular nanovesicles produced by Bacillus licheniformis: A potential anticancer agent for breast and lung cancer.

Cancer is a major public burden and leading cause of death worldwide; furthermore, it is a significant barrier to increasing life expectancy in most countries of the world. Among various types of cancers, breast and lung cancers lead to significant mortality in both males and females annually. Bacteria-derived products have been explored for their use in cancer therapy. Although bacteria contain significant amounts of anticancer substances, attenuated bacteria may still pose a potential risk for infection owing to the variety of immunomodulatory molecules present in the parental bacteria; therefore, non-cellular bacterial extracellular vesicles (BEVs), which are naturally non-replicating, safer, and are considered to be potential anticancer agents, are preferred for cancer therapy. Gram-positive bacteria actively secrete cytoplasmic membrane vesicles that are spherical and vary between 10 and 400 nm in size. However, no studies have considered cytoplasmic membrane vesicles derived from Bacillus licheniformisin cancer treatment. In this study, we investigated the potential use of B. licheniformis extracellular nanovesicles (BENVs) as therapeutic agents to treat cancer. Purified BENVs from the culture supernatant of B. licheniformis using ultracentrifugation and ExoQuick were characterized using a series of analytical techniques. Human breast cancer cells (MDA-MB-231) and lung cancer cells (A549) were treated with different concentrations of purified BENVs, which inhibited the cell viability and proliferation, and increased cytotoxicity in a dose-dependent manner. To elucidate the mechanism underlying the anticancer activity of BENVs, the oxidative stress markers such as reactive oxygen species (ROS) and glutathione (GSH) levels were measured. The ROS levels were significantly higher in BENV-treated cells, whereas the GSH levels were markedly reduced. Cells treated with BENVs, doxorubicin (DOX), or a combination of BENVs and DOX showed significantly increased expression of p53, p21, caspase-9/3, and Bax, and concomitantly decreased expression of Bcl-2. The combination of BENVs and doxorubicin enhanced mitochondrial dysfunction, DNA damage, and apoptosis. To our knowledge, this is the first study to determine the anticancer properties of BENVs derived from industrially significant probacteria on breast and lung cancer cells.

Bacillus changyiensis sp. nov., isolated from coastal sediment.

Two Gram-stain-positive, facultatively anaerobic, motile, endospore-forming, rod-shaped bacteria, designated CLL-3-40T and CLL-7-23, were isolated from coastal sediment sampled in Changyi, Shandong Province, PR China. Phylogenetic analysis based on 16S rRNA gene sequences indicated that these strains were related to the genus Bacillus and close to six type strains of species within the Bacillus licheniformis group. In phenotypic characterization tests, strain CLL-3-40T could grow at 15-50 °C (optimum, 37 °C) and in media with pH 5-9 (optimum pH 7.0), and tolerate up to 12 % (w/v) NaCl. The fermentation broth supernatant extracted by ethyl acetate of strain CLL-3-40T could inhibit aquaculture pathogenic vibrios. The predominant cellular fatty acids of strain CLL-3-40T were anteiso-C15 : 0 (30.7 %) and iso-C15 : 0 (31.5 %); the peptidoglycan from cell-wall contained meso-diaminopimelic acid; the predominant quinone was menaquinone 7; and the major polar lipids were diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, an unidentified glycolipid and two unidentified phospholipids. The digital DNA-DNA hybridization values and average nucleotide identities among strains CLL-3-40T and CLL-7-23 and their close type strains were less than 21.9 and 48.4 %, respectively, thereby indicating that strain CLL-3-40T should represent a novel species of the genus Bacillus. The genomic DNA G+C contents were 38.4 mol% in strain CLL-3-40T and 38.3 mol% in strain CLL-7-23. The 12 biosynthetic gene clusters of strain CLL-3-40T were predicted based on results from the online server antiSMASH. Based upon the consensus of phenotypic and genotypic results, strain CLL-3-40T should be classified as representing a novel species of the genus Bacillus, for which the name Bacillus changyiensis sp. nov. is proposed. The type strain is CLL-3-40T (= MCCC 1A14857T=JCM 35755T).

Effects of Fe2+ addition to sugarcane molasses on poly-γ-glutamic acid production in Bacillus licheniformis CGMCC NO. 23967.

BACKGROUND: Poly-γ-glutamic acid (γ-PGA) is biodegradable, water-soluble, environment-friendly, and edible. Consequently, it has a variety of industrial applications. It is crucial to control production cost and increase output for industrial production γ-PGA. RESULTS: Here γ-PGA production from sugarcane molasses by Bacillus licheniformis CGMCC NO. 23967 was studied in shake-flasks and bioreactors, the results indicate that the yield of γ-PGA could reach 40.668 g/L in a 5L stirred tank fermenter. Further study found that γ-PGA production reached 70.436 g/L, γ-PGA production and cell growth increased by 73.20% and 55.44%, respectively, after FeSO4·7H2O was added. Therefore, we investigated the metabolomic and transcriptomic changes following FeSO4·7H2O addition. This addition resulted in increased abundance of intracellular metabolites, including amino acids, organic acids, and key TCA cycle intermediates, as well as upregulation of the glycolysis pathway and TCA cycle. CONCLUSIONS: These results compare favorably with those obtained from glucose and other forms of biomass feedstock, confirming that sugarcane molasses can be used as an economical substrate without any pretreatment. The addition of FeSO4·7H2O to sugarcane molasses may increase the efficiency of γ-PGA production in intracellular.