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1.
Arch Microbiol ; 206(4): 143, 2024 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-38443732

RESUMO

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.


Assuntos
Bacillus licheniformis , Bacteriocinas , Bacillus licheniformis/genética , Família Multigênica , Antibacterianos/farmacologia , Bacteriocinas/genética , Bacteriocinas/farmacologia , Peptídeos , RNA
2.
BMC Genom Data ; 25(1): 3, 2024 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-38166625

RESUMO

OBJECTIVES: The data presented in this study were collected with the aim of obtaining the complete genomes of specific strains of Bacillus bacteria, namely, Bacillus licheniformis T5. This strain was chosen based on its enzymatic activities, particularly amylolytic activity. In this study, nanopore sequencing technology was employed to obtain the genome sequences of this strain. It is important to note that these data represent a focused objective within a larger research context, which involves exploring the biochemical features of promising Bacilli strains and investigating the relationship between enzymatic activity, phenotypic features, and the microorganism's genome. DATA DESCRIPTION: In this study, the whole-genome sequence was obtained from one Bacillus strain, Bacillus licheniformis T5, isolated from soil samples in Kazakhstan. Sample preparation and genomic DNA library construction were performed according to the Ligation sequencing gDNA kit (SQK-LSK109) protocol and NEBNext module. The prepared library was sequenced on a MinION instrument (Oxford Nanopore Technologies nanopore sequencer with a maximum throughput of up to 30 billion nucleotides per run and no limit on read length), using a flow cell for nanopore sequencing FLO-MIN106D. The genome de novo assembly was performed using the long sequencing reads generated by MinION Oxford Nanopore platform. Finally, one circular contig was obtained harboring a length of 4,247,430 bp with 46.16% G + C content and the mean contig 428X coverage. B. licheniformis T5 genome assembly annotation revealed 5391 protein-coding sequences, 81 tRNAs, 51 repeat regions, 24 rRNAs, 3 virulence factors and 53 antibiotic resistance genes. This sequence encompasses the complete genetic information of the strain, including genes, regulatory elements, and noncoding regions. The data reveal important insights into the genetic characteristics, phenotypic traits, and enzymatic activity of this Bacillus strain. The findings of this study have particular value to researchers interested in microbial biology, biotechnology, and antimicrobial studies. The genomic sequence offers a foundation for understanding the genetic basis of traits such as endospore formation, alkaline tolerance, temperature range for growth, nutrient utilization, and enzymatic activities. These insights can contribute to the development of novel biotechnological applications, such as the production of enzymes for industrial purposes. Overall, this study provides valuable insights into the genetic characteristics, phenotypic traits, and enzymatic activities of the Bacillus licheniformis T5 strain. The acquired genomic sequences contribute to a better understanding of this strain and have implications for various research fields, such as microbiology, biotechnology, and antimicrobial studies.


Assuntos
Anti-Infecciosos , Bacillus licheniformis , Análise de Sequência de DNA/métodos , Bacillus licheniformis/genética , Cazaquistão , Genoma
3.
Int J Mol Sci ; 25(1)2024 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-38203732

RESUMO

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.


Assuntos
Bacillus licheniformis , Bacillus , Colite , Probióticos , Animais , Camundongos , Colite/induzido quimicamente , Colite/terapia , Camelus , Homeostase , Probióticos/farmacologia , Probióticos/uso terapêutico
4.
Bioorg Chem ; 143: 107068, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38181659

RESUMO

α-Amylase is a secretory enzyme commonly found in nature. The α-Amylase enzyme catalyzes the hydrolysis of α-D-(1,4)-glucosidic bonds in starch, glycogen, and polysaccharides. The chemical characterization of the composite carrier and the immobilized enzyme was performed, and the accuracy of the immobilization was confirmed by FTIR, SEM, and EDS analyses. The X-ray diffraction (XRD) analysis indicates that the magnetic nanoparticle retained its magnetic properties following the modification process. Based on the Thermogravimetric Analysis (TGA) outcomes, it was evident that the structural integrity of the FPT nanocomposite remained unchanged at 200°C. The optimal pH was determined to be 5.5, and no alteration was observed following the immobilization process. Purified α-amylases usually lose their activity rapidly above 50°C. In this study, Bacillus licheniformis α-Amylase enzyme was covalently immobilized on the newly synthesized magnetic composite carrier having more azole functional group. For novelty-designed immobilized enzymes, while there is no change in the pH and optimum operating temperature of the enzyme with immobilization, two essential advantages are provided to reduce enzyme costs: the storage stability and reusability are increased. Furthermore, our immobilization technique enhanced enzyme stability when comparing our immobilized enzyme with the reference enzyme in industrial applications. The activity of the immobilized enzyme was higher in presence of 1-3% H2O2.


Assuntos
Bacillus licheniformis , Compostos de Epóxi , Nanopartículas de Magnetita , Metacrilatos , Triazóis , Enzimas Imobilizadas/química , Bacillus licheniformis/metabolismo , Peróxido de Hidrogênio , Nanopartículas de Magnetita/química , Concentração de Íons de Hidrogênio , Estabilidade Enzimática , alfa-Amilases/metabolismo , Temperatura
5.
Appl Microbiol Biotechnol ; 108(1): 139, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38229401

RESUMO

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.


Assuntos
Bacillus licheniformis , Enteropatias , Probióticos , Animais , Camundongos , Bovinos , Antibacterianos/farmacologia , Suplementos Nutricionais , Probióticos/farmacologia , Enteropatias/microbiologia , Firmicutes/genética , Cefalexina
6.
Appl Microbiol Biotechnol ; 108(1): 89, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38194145

RESUMO

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.


Assuntos
Arginase , Bacillus licheniformis , Via Secretória/genética , Bacillus licheniformis/genética , Citoplasma , Citosol
7.
Appl Microbiol Biotechnol ; 108(1): 149, 2024 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-38240797

RESUMO

In this study, we successfully applied the strategy of combining tandem promoters and tandem signal peptides with overexpressing signal peptidase to efficiently express and produce γ-glutamyl peptidase (GGT) enzymes (BsGGT, BaGGT, and BlGGT) from Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus licheniformis in Bacillus subtilis ATCC6051Δ5. In order to avoid the problem of instability caused by duplicated strong promoters, we assembled tandem promoters of different homologous genes from different species. To achieve resistance marker-free enzyme in the food industry, we first removed the replication origin and corresponding resistance marker of Escherichia coli from the expression vector. The plasmid was then transformed into the B. subtilis host, and the Kan resistance gene in the expression plasmid was directly edited and silenced using the CRISPR/Cas9n-AID base editing system. As a result, a recombinant protein expression carrier without resistance markers was constructed, and the enzyme activity of the BlGGT strain during shake flask fermentation can reach 53.65 U/mL. The recombinant BlGGT was immobilized with epoxy resin and maintained 82.8% enzyme activity after repeated use for 10 times and 87.36% enzyme activity after storage at 4 °C for 2 months. The immobilized BlGGT enzyme was used for the continuous synthesis of theanine with a conversion rate of 65.38%. These results indicated that our approach was a promising solution for improving enzyme production efficiency and achieving safe production of enzyme preparations in the food industry. KEY POINTS: • Efficient expression of recombinant proteins by a combination of dual promoter and dual signal peptide. • Construction of small vectors without resistance markers in B. subtilis using CRISPR/Cas9n-AID editing system. • The process of immobilizing BlGGT with epoxy resin was optimized.


Assuntos
Bacillus licheniformis , Bacillus subtilis , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , gama-Glutamiltransferase/genética , gama-Glutamiltransferase/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Resinas Epóxi , Bacillus licheniformis/genética , Proteínas Recombinantes/genética , Enzimas Imobilizadas/metabolismo
8.
Curr Microbiol ; 81(2): 62, 2024 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-38216774

RESUMO

Microbial contamination of coffee beans arises from various factors such as harvesting, handling, and storage practices, during which ochratoxin A (OTA)-producing fungi develop and proliferate. The presence of elevated concentrations of OTA poses a serious health risk to coffee consumers. Therefore, the implementation of a post-harvest treatment involving the use of bacteria known to antagonize OTA-producing fungi constitutes a safe alternative for reducing or eliminating the toxin's concentration in coffee beans. In this study, coffee beans (Coffea arabica L.) were inoculated with Bacillus licheniformis M2-7, after which we monitored fungal growth, in vitro antagonism, and OTA concentration. Our findings demonstrated that coffee beans inoculated with this bacterial strain exhibited a significant decrease in fungal populations belonging to the genera Aspergillus and Penicillium, which are known to produce OTA. Moreover, strain M2-7 decreased the growth rates of these fungi from 67.8% to 95.5% (P < 0.05). Similarly, inoculation with B. licheniformis strain M2-7 effectively reduced the OTA concentration from 24.35 ± 1.61 to 5.52 ± 1.69 µg/kg (P < 0.05) in stored coffee beans. These findings suggest that B. licheniformis M2-7 holds promise as a potential post-harvest treatment for coffee beans in storage, as it effectively inhibits the proliferation of OTA-producing fungi and lowers the toxin's concentration.


Assuntos
Bacillus licheniformis , Coffea , Ocratoxinas , Contaminação de Alimentos/análise , Coffea/microbiologia
9.
Artigo em Inglês | MEDLINE | ID: mdl-38253396

RESUMO

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.


Assuntos
Bacillus licheniformis , Glucosiltransferases , Neisseria , Bacillus licheniformis/metabolismo , Sinais Direcionadores de Proteínas/genética , Frutanos , Arginina , Proteínas de Bactérias/genética
10.
BMC Biotechnol ; 24(1): 6, 2024 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-38273334

RESUMO

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.


Assuntos
Arginase , Bacillus licheniformis , Arginase/genética , Arginase/metabolismo , Bacillus licheniformis/genética , Bacillus licheniformis/metabolismo , Proteína X Associada a bcl-2/genética , Arginina/metabolismo , Ornitina/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2
11.
Appl Environ Microbiol ; 90(2): e0146823, 2024 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-38193675

RESUMO

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.


Assuntos
Bacillus licheniformis , Bacillus , Espécies Reativas de Oxigênio/metabolismo , Bacillus licheniformis/metabolismo , Peróxido de Hidrogênio , Estresse Oxidativo , Ferro/metabolismo , Quelantes de Ferro , Bacillus/metabolismo , DNA/metabolismo
12.
Int J Biol Macromol ; 258(Pt 1): 128822, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38114007

RESUMO

Bletilla striata polysaccharide (BP) is one of the main active ingredients in Orchidaceae plant Bletilla striata. BP has a high molecular weight, high viscosity, and complex diffusion, which is not conducive to the absorption and utilization of the human body. For the first time, we produced fermented Bletilla striata polysaccharide (FBP) with a low polymerization degree using Bacillus licheniformis BJ2022 one-step fermentation. FBP was a neutral polysaccharide with the molecular weight of 6790 Da. It was composed of glucose and mannose at a molar ratio of 1:2.7. The glycosidic bonds of FBP were composed of ß-1,4-linked mannose, ß-1,4-linked glucose and ß-1,6-linked mannose according to methylation and NMR analysis. Compared with BP, FBP has a lower viscosity and higher solubility. The scanning electron microscopy results showed that the surface of FBP was porous and honeycomb-like. The rheology properties of FBP solution were close to non-Newtonian fluid. Using in vitro fermentation, we proved that FBP could regulate human gut microbiota and significantly increase the content of Bifidobacterium and Bacteroides. Our results suggested that Bacillus licheniformis fermentation significantly improved the physical and prebiotic properties of FBP. This study provides a new strategy for developing and utilizing Bletilla striata resources in China.


Assuntos
Bacillus licheniformis , Orchidaceae , Humanos , Manose , Fermentação , Polissacarídeos/química , Orchidaceae/química , Glucose
13.
J Appl Microbiol ; 135(2)2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38159929

RESUMO

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.


Assuntos
Bacillus licheniformis , Bacillus licheniformis/genética , Endopeptidases/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Perfilação da Expressão Gênica , Fermentação , Transcriptoma
14.
Sci Rep ; 13(1): 22785, 2023 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-38129471

RESUMO

Bacillus strains from the Moroccan Coordinated Collections of Microorganisms (CCMM) were characterised and tested for fibrolytic function and safety properties that would be beneficial for maintaining intestinal homeostasis, and recommend beneficial microbes in the field of health promotion research. Forty strains were investigated for their fibrolytic activities towards complex purified polysaccharides and natural fibres representative of dietary fibres (DFs) entering the colon for digestion. We demonstrated hemicellulolytic activities for nine strains of Bacillus aerius, re-identified as Bacillus paralicheniformis and Bacillus licheniformis, using xylan, xyloglucan or lichenan as purified polysaccharides, and orange, apple and carrot natural fibres, with strain- and substrate-dependent production of glycoside hydrolases (GHs). Our combined methods, based on enzymatic assays, secretome, and genome analyses, highlighted the hemicellulolytic activities of B. paralicheniformis and the secretion of specific glycoside hydrolases, in particular xylanases, compared to B. licheniformis. Genomic features of these strains revealed a complete set of GH genes dedicated to the degradation of various polysaccharides from DFs, including cellulose, hemicellulose and pectin, which may confer on the strains the ability to digest a variety of DFs. Preliminary experiments on the safety and immunomodulatory properties of B. paralicheniformis fibrolytic strains were evaluated in light of applications as beneficial microbes' candidates for health improvement. B. paralicheniformis CCMM B969 was therefore proposed as a new fibrolytic beneficial microbe candidate.


Assuntos
Bacillus licheniformis , Bacillus , Bacillus/genética , Bacillus licheniformis/genética , Polissacarídeos/metabolismo , Glicosídeo Hidrolases/metabolismo
16.
Nucleic Acids Res ; 51(21): 11952-11966, 2023 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-37850640

RESUMO

Synthetic regulation of metabolic fluxes has emerged as a common strategy to improve the performance of microbial cell factories. The present regulatory toolboxes predominantly rely on the control and manipulation of carbon pathways. Nitrogen is an essential nutrient that plays a vital role in growth and metabolism. However, the availability of broadly applicable tools based on nitrogen pathways for metabolic regulation remains limited. In this work, we present a novel regulatory system that harnesses signals associated with nitrogen metabolism to redirect excess carbon flux in Bacillus licheniformis. By engineering the native transcription factor GlnR and incorporating a sorbitol-responsive element, we achieved a remarkable 99% inhibition of the expression of the green fluorescent protein reporter gene. Leveraging this system, we identified the optimal redirection point for the overflow carbon flux, resulting in a substantial 79.5% reduction in acetoin accumulation and a 2.6-fold increase in acetate production. This work highlight the significance of nitrogen metabolism in synthetic biology and its valuable contribution to metabolic engineering. Furthermore, our work paves the way for multidimensional metabolic regulation in future synthetic biology endeavors.


Assuntos
Bacillus licheniformis , Engenharia Metabólica , Sorbitol , Bacillus licheniformis/genética , Bacillus licheniformis/metabolismo , Carbono/metabolismo , Engenharia Metabólica/métodos , Nitrogênio/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Sorbitol/metabolismo
17.
Microb Pathog ; 185: 106396, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37863272

RESUMO

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.


Assuntos
Antineoplásicos , Bacillus licheniformis , Neoplasias Pulmonares , Masculino , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Espécies Reativas de Oxigênio/metabolismo , Bacillus licheniformis/metabolismo , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Apoptose
18.
Int J Mol Sci ; 24(18)2023 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-37762368

RESUMO

2,3-Butanediol (2,3-BD) is an alcohol highly demanded in the chemical, pharmaceutical, and food industries. Its microbial production, safe non-pathogenic producer strains, and suitable substrates have been avidly sought in recent years. The present study investigated 2,3-BD synthesis by the GRAS Bacillus licheniformis 24 using chicory inulin as a cheap and renewable substrate. The process appears to be pH-dependent. At pH 5.25, the synthesis of 2,3-BD was barely detectable due to the lack of inulin hydrolysis. At pH 6.25, 2,3-BD concentration reached 67.5 g/L with rapid hydrolysis of the substrate but was accompanied by exopolysaccharide (EPS) synthesis. Since inulin conversion by bacteria is a complex process and begins with its hydrolysis, the question of the acting enzymes arose. Genome mining revealed that several glycoside hydrolase (GH) enzymes from different CAZy families are involved. Five genes encoding such enzymes in B. licheniformis 24 were amplified and sequenced: sacA, sacB, sacC, levB, and fruA. Real-time RT-PCR experiments showed that the process of inulin hydrolysis is regulated at the level of gene expression, as four genes were significantly overexpressed at pH 6.25. In contrast, the expression of levB remained at the same level at the different pH values at all-time points. It was concluded that the sacC and sacA/fruA genes are crucial for inulin hydrolysis. They encode exoinulinase (EC 3.2.1.80) and sucrases (EC 3.2.1.26), respectively. The striking overexpression of sacB under these conditions led to increased synthesis of EPS; therefore, the simultaneous production of 2,3-BD and EPS cannot be avoided.


Assuntos
Bacillus licheniformis , Bacillus , Humanos , Bacillus licheniformis/genética , Bacillus licheniformis/metabolismo , Inulina/metabolismo , Bacillus/metabolismo , Concentração de Íons de Hidrogênio , Expressão Gênica , Fermentação
19.
Ultrason Sonochem ; 100: 106611, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37757602

RESUMO

The effect of low-intensity fixed-frequency continuous ultrasound (LIFFCU) on the growth of Bacillus licheniformis YYC4 was investigated. The changes in morphology and activity of the organism, contributing to the growth were also explored. Compared with the control, a significant increase (48.95%) in the biomass of B. licheniformis YYC4 (at the logarithmic metaphase) was observed following the LIFFCU (28 kHz, 1.5 h and 120 W (equivalent to power density of 40 W/L)) treatment. SEM images showed that ultrasonication caused sonoporation, resulting in increased membrane permeability, evidenced by increase in cellular membrane potential, electrical conductivity of the culture, extracellular protein and nucleic acid, and intracellular Ca2+ content. Furthermore, LIFFCU action remarkably increased the extracellular protease activity, volatile components of the culture medium, microbial metabolic activity, and spore germination of the strain. Therefore, LIFFCU could be used to efficiently promote the growth of targeted microorganisms.


Assuntos
Bacillus licheniformis , Esporos Bacterianos/metabolismo , Proteínas de Bactérias
20.
J Agric Food Chem ; 71(39): 14379-14389, 2023 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-37737871

RESUMO

Bacillus licheniformis has been widely utilized in the food industry as well as various agricultural industries. In particular, it is a main microorganism of fermented soybeans. In this study, the changes of the metabolome and transcriptome of B. licheniformis KACC15844, which had been isolated from fermented soybeans, were investigated depending on alkaline pH (BP) and a high salt concentration (BS) using an integrated-omics technology, focusing on leucine metabolism. Overall, carbohydrate (glycolysis, sugar transport, and overflow) and amino acid (proline, glycine betaine, and serine) metabolisms were strongly associated with BS, while fatty acid metabolism, malate utilization, and branched-chain amino acid-derived volatiles were closely related to BP, in both gene and metabolic expressions. In particular, in leucine metabolism, the formation of 3-methylbutanoic acid, which has strong cheesy odor notes, was markedly increased in BP compared to the other samples. This study provided information on how specific culture conditions can affect gene expressions and metabolite formations in B. licheniformis using an integrated-omics approach.


Assuntos
Bacillus licheniformis , Alimentos Fermentados , Bacillus licheniformis/genética , Transcriptoma , /metabolismo , Pressão Osmótica , Leucina/metabolismo , Concentração de Íons de Hidrogênio
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