Bacillus subtilis is widely used for industrial enzyme production due to its food safety and good capability of protein synthesis and secretion. However, the production of heterologous proteins is often inefficient, partly due to poor compatibility and versatility of genetic elements in B. subtilis. Recent study shows that transcription and translation is uncoupled in B. subtilis, which is quite different from general knowledge about the transcription-translation coupling mechanism in bacteria. The uncoupling mechanism in B. subtilis shows that the transcription rate is much faster than translation rate. Therefore, the translation regulation will play an important role in highly-effective synthesis of heterologous protein. To better understanding the different regulation strategies at the translation level in B. subtilis, this review will summarize the translation process in B. subtilis cell and its regulatory mechanisms as well as the differences in comparison to other bacteria. Besides, the genetic engineering strategies for engineering the translation regulatory elements are also summarized.

Human intestinal epithelial cells (IECs) play an important role in maintaining gut homeostasis by producing antimicrobial peptides (AMPs). Bacillus subtilis, a commensal bacterium, is considered a probiotic. Although its protective effects on intestinal health are widely reported, the key component of B. subtilis responsible for its beneficial effects remains elusive. In this study, we tried to identify the key molecules responsible for B. subtilis-induced AMPs and their molecular mechanisms in a human IEC line, Caco-2. B. subtilis increased human beta defensin (HBD)-2 mRNA expression in a dose- and time-dependent manner. Among the B. subtilis microbe-associated molecular patterns, lipoprotein (LPP) substantially increased the mRNA expression and protein production of HBD-2, whereas lipoteichoic acid and peptidoglycan did not show such effects. Those results were confirmed in primary human IECs. In addition, both LPP recognition and HBD-2 secretion mainly took place on the apical side of fully differentiated and polarized Caco-2 cells through Toll-like receptor 2-mediated JNK/p38 MAP kinase/AP-1 and NF-κB pathways. HBD-2 efficiently inhibited the growth of the intestinal pathogens Staphylococcus aureus and Bacillus cereus. Furthermore, LPPs pre-incubated with lipase or proteinase K decreased LPP-induced HBD-2 expression, suggesting that the lipid and protein moieties of LPP are crucial for HBD-2 expression. Q Exactive Plus mass spectrometry identified 35 B. subtilis LPP candidates within the LPP preparation, and most of them were ABC transporters. Taken together, these results suggest that B. subtilis promotes HBD-2 secretion in human IECs mainly with its LPPs, which might enhance the protection from intestinal pathogens.

The high-value utilization of okara is vital for promoting sustainable practices in the soybean industry and addressing global food security challenges. This study investigated the effects of Bacillus subtilis BSNK-5 fermentation on okara's nutritional, structural, and functional properties. BSNK-5 fermentation effectively degraded insoluble dietary fiber, protein, and fat into soluble fiber, peptides, amino acids, and fatty acids, while reducing trypsin inhibitors and antigenic proteins. It also increased phenolic compounds, promoted the conversion of glycosides to aglycones, and enhanced digestibility and nutritional quality. After fermentation with BSNK-5, the surface wrinkles of the okara were reduced, and the particles became smaller. Additionally, BSNK-5 fermentation improved water-holding, oil-holding, swelling capacities, antioxidant activity, cholesterol binding, glucose absorption, and antihypertensive properties. These results highlight the potential of BSNK-5 fermentation to enhance okara's nutritional and functional value, providing valuable raw materials for the food industry.

Bacillus species have attracted significant attention in recent years due to their potential for producing various bioactive compounds with diverse functional properties. This review highlights bioactive substances from food-grade Bacillus strains and their applications in functional foods and nutraceuticals. The metabolic capacities of Bacillus species have allowed them to generate a wide range of bioactive substances, including vitamins, enzymes, anti-microbial peptides, and other non-ribosomal peptides. These substances have a variety of positive effects, including potential cholesterol-lowering and immune-modulatory qualities in addition to anti-oxidant and anti-bacterial actions. The uses and mechanisms of action of these bioactive chemicals can be used to improve the functional qualities and nutritional profile of food products. Examples include the use of anti-microbial peptides to increase safety and shelf life, as well as the use of Bacillus-derived enzymes in food processing to improve digestibility and sensory qualities. The exploitation of bioactive compounds from food-grade Bacillus strains presents a promising frontier in the development of functional foods and nutraceuticals with enhanced health benefits. Due to their wide range of activity and applications, they are considered as important resources for the development of novel medications, agricultural biocontrol agents, and industrial enzymes. Ongoing research into the biosynthetic pathways, functional properties, and applications of these compounds is essential to fully realize their potential in the food industry. This review underscores the significance of various bioactive compounds generated from Bacillus in tackling global issues like environmental sustainability, sustainable agriculture, and antibiotic resistance. Future developments in microbiology and biotechnology will enable us to fully utilize the potential of these amazing microbes, resulting in novel approaches to industry, agriculture, and health. © 2024 Society of Chemical Industry.

Bacillus subtilis is a beneficial rhizobacterium extensively used in agriculture and industry due to its abilities in promoting plant growth and decomposing organic matter. To enhance its application potential, precise genetic characterization of native strains, particularly those associated with crop rhizospheres, is crucial.

This study focused on B. subtilis isolates obtained from the cassava rhizosphere. Genetic diversity among the isolates was assessed using the Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR method. Genomic DNA was extracted and amplified, with ERIC-PCR effectively differentiating strains based on unique banding patterns. Whole-genome sequencing and database comparisons further validated the strain identities and revealed significant genetic variation. While a few isolates shared high similarity (≥ 99.5%), the majority exhibited lower similarity levels (< 70%), indicating considerable genomic diversity. Several genes-spoVAF, spoVAEA, spoVAEB, spoVAD, hisIE, hisF, hisA, rsbRB, thiW, ispA, and thiX-were identified as potential markers for strain differentiation and functional characterization.

ERIC-PCR proved to be a reliable and efficient method for discriminating B. subtilis strains from the cassava rhizosphere. The observed genetic diversity suggests a rich reservoir of functional traits among native strains, offering new opportunities for targeted applications in plant-microbe interactions, such as biofertilization and biocontrol. These findings provide a foundation for the strategic use and further study of B. subtilis in sustainable agriculture.

Not applicable.

Bacillus subtilis exhibits strong adaptability and biotransformation potential in the fermentation of okara, but the effects of fermentation on their dietary fiber remain unclear. This study explored the impact of Bacillus subtilis BSNK-5 fermentation on converting insoluble dietary fiber (IDF) to soluble dietary fiber (SDF) in okara, focusing on structural and functional changes. After 72 h of fermentation, SDF increased 7.51-fold. The surface folds of fermented IDF were reduced. Meanwhile, SDF displayed a more porous structure with significant changes in its crystalline structure. FTIR analysis showed that surface disruption exposed both hydrophilic and hydrophobic groups. Thermal analysis showed that the peak of maximum degradation moved to a lower temperature. Both fermented SDF and IDF exhibited antioxidant activity, effective lipid- and glucose-lowering effects. These findings suggest that BSNK-5 effectively transforms IDF into SDF, with fermented dietary fiber showing great potential as a functional ingredient in the food industry.

Flaxseed meal (FSM) is a by-product of flaxseed product production that is wasted unreasonably at present. In this study, we used Bacillus subtilis K6, a dominant microbial strain, for solid-state fermentation (SSF) of FSM following preliminary screening to improve FSM utilization efficiency and enhance the soluble dietary fiber (SDF) content while modifying its functional properties. FSM's microstructure was characterized before and after fermentation, and the functional properties of the dietary fiber (DF) in the FSM were assessed. Single-factor experiments combined with response surface methodology were conducted to optimize SSF parameters using SDF yield as the response variable. The optimal conditions were determined as follows: 45 h fermentation time, 40.5 °C temperature, and 1:0.65 material-to-liquid ratio. Under these conditions, the SDF yield reached 33.45 ± 0.24%, an SDF yield increase of 36.92%. Scanning electron microscopy and confocal laser scanning microscopy demonstrated FSM's structural disruption during fermentation. Furthermore, SDF and insoluble DF showed improved water-holding, oil-holding, and swelling capacities following fermentation. These results indicate that SSF effectively enhances the SDF content in FSM and optimizes its functional properties, thereby providing a theoretical foundation for the valorization of flaxseed by-products.

Microbial cell factories for the production of high-quality commercial-grade enzymes have accelerated the development of advanced bio-manufacturing approaches, which in turn are environmentally friendly and sustainable. Myxobacteria, a term commonly used to refer to a group within the Myxococcota phylum, are of great interest for their biotechnological applications due to their ability to synthesize a wide range of natural products and lytic enzymes. These traits are essential for the development of robust expression systems. However, myxobacteria have remained an underexploited resource with industrial relevance. Nevertheless, a growing number of food and industrial enzymes have been identified, highlighting myxobacteria as suitable platforms for exploring enzymes with commercial applications, including biomass conversion. Yet, the discovered lytic enzymes are just the tip of the iceberg given their large genomes and diversity across myxobacteria taxa. Despite holding much promise, challenges in genetic engineering, slow growth, and limitations in metabolic remodeling and expression strategies have limited the construction of myxobacterial cell factories. In this review, we highlight recent advances in the discovery of new myxobacterial enzymes and biomass conversion resources, focusing on their potential applications in agriculture and industry. We describe how myxobacteria and their enzymes can be identified through bioprospecting and computational approaches and summarize current biotechnological applications and synthetic biology strategies for bio-manufacturing. Finally, we discuss the promising potential of myxobacteria as industrial cell factories and address open research questions and future directions.

In recent years, there has been a notable increase interest in engineered living materials (ELMs) owing to their considerable potential. One key area of research within this field is the utilisation of various species of bacteria to create innovative living materials. In order to accelerate the advancement of bacterial-based living materials, a systematic summary of bacterial species and their design strategies is essential. Yet, up to this point, no applicable reviews have been documented. This review offers a concise introduction to living materials derived from bacteria, delves into the strategies and applications of each bacterial species in this realm, and provides perspectives and future outlooks in this field.

Hyaluronic acid (HA) is a linear, unbranched polysaccharide composed of repeating disaccharide units of N-acetyl-d-glucosamine and D-glucuronic acid. It plays a crucial role in promoting soft tissue growth, elasticity, and scar reduction. The growing demand for HA in pharmaceutical and cosmetic applications has provoked extensive research into diverse production strategies. Current efforts focus on bacterial and yeast fermentation. However, the extraction process presents a significant challenge due to the complex nature of source materials like fermentation broth, which contains numerous components and solutes. Achieving high extraction yields and purity requires careful consideration of extraction techniques. This study provides a comprehensive overview of the primary methodologies employed for HA production, elaborating on the advantages and disadvantages of each approach. Additionally, it highlights recent advancements in HA extraction and purification, with a particular emphasis on bacterial sources and the applications of HA. This review critically evaluates current HA production strategies, identifies key challenges hindering scalability and efficiency, and discusses innovative solutions under development to overcome these limitations.

Antimicrobial peptides (AMPs) are critical effectors of innate immunity, presenting a compelling alternative to conventional antibiotics amidst escalating antimicrobial resistance. Their broad-spectrum efficacy and inherent low resistance development are countered by production challenges, including limited yields and proteolytic degradation, which restrict their clinical translation. While chemical synthesis offers precise structural control, it is often prohibitively expensive and complex for large-scale production. Heterologous expression systems provide a scalable, cost-effective platform, but necessitate optimization. This review comprehensively examines established and emerging AMP production strategies, encompassing fusion protein technologies, molecular engineering approaches, rational peptide design, and post-translational modifications, with an emphasis on maximizing yield, bioactivity, stability, and safety. Furthermore, we underscore the transformative role of artificial intelligence, particularly machine learning algorithms, in accelerating AMP discovery and optimization, thereby propelling their expanded therapeutic application and contributing to the global fight against drug-resistant infections.

Menaquinone-7 (MK-7) is a valuable vitamin K2 produced by Bacillus subtilis. Although many strategies have been adopted to increase the yield of MK-7 in B. subtilis, the effectiveness of these common approaches is not high because long metabolic synthesis pathways and numerous bypass pathways competing for precursors with MK-7 synthesis. Regarding the modification of bypass pathways, studies of common static metabolic engineering method such as knocking out genes involved in side pathway have been reported previously. Since byproductsphenylalanine(Phe), tyrosine (Tyr), tryptophan (Trp), folic acid, dihydroxybenzoate, hydroxybutanone in the MK-7 synthesis pathway are indispensable for cell growth, the complete knockout of the bypass pathway restricts cell growth, resulting in limited increase in MK-7 synthesis. Dynamic regulation via quorum sensing (QS) provides a cost-effective strategy to harmonize cell growth and product synthesis, eliminating the need for pricey inducers. SinR, a transcriptional repressor, is crucial in suppressing biofilm formation, a process closely intertwined with MK-7 biosynthesis. Given this link, we targeted SinR to construct a dynamic regulatory system, aiming to modulate MK-7 production by leveraging SinR's regulatory influence.

A modular PhrC-RapC-SinR QS system is developed to dynamic regulate side pathway of MK-7. In this study, first, we analyzed the SinR-based gene expression regulation system in B. subtilis 168 (BS168). We constructed a promoter library of different abilities, selected suitable promoters from the library, and performed mutation screening on the selected promoters. Furthermore, we constructed a PhrC-RapC-SinR QS system to dynamically control the synthesis of Phe, Tyr, Trp, folic acid, dihydroxybenzoate, hydroxybutanone in MK-7 synthesis in BS168. Cell growth and efficient synthesis of the MK-7 production can be dynamically balanced by this QS system. Using this system to balance cell growth and product fermentation, the MK-7 yield was ultimately increased by 6.27-fold, from 13.95 mg/L to 87.52 mg/L.

In summary, the PhrC-RapC-SinR QS system has been successfully integrated with biocatalytic functions to achieve dynamic metabolic pathway control in BS168, which has potential applicability to a large number of microorganisms to fine-tune gene expression and enhance the production of metabolites.

Cell-free systems are emerging as powerful platforms for synthetic biology with widespread applications in both fundamental research, such as artificial cell construction, and practical uses like recombinant protein production. Among these, cell-free protein synthesis (CFPS) plays a crucial role in gene expression for various downstream applications. However, the development of CFPS systems based on certain chassis, such as Bacillus subtilis, still remains limited due to their low in vitro productivity. Here, we report the development of a highly productive CFPS system derived from an engineered B. subtilis 164T7P strain, which contains a genomic integration of the T7 RNA polymerase gene. This modification allows the preparation of cell extracts that inherently contain T7 RNA polymerase, enabling T7 promoter-based transcription without the supplementation of purified T7 RNA polymerase in CFPS reactions. Through systematic optimization of cell extract preparation and key reaction parameters, we achieved the synthesis of 286 ± 16.7 μg/mL of sfGFP in batch reactions, with yields increasing to over 1100 μg/mL in a semicontinuous format that can replenish substrates and remove inhibitory byproducts. We further demonstrated the system's versatility by using it for two synthetic biology applications: prototyping ribosome binding site (RBS) elements and synthesizing pulcherriminic acid─a bioactive cyclodipeptide. The system successfully characterized RBS performance, with in vitro and in vivo rankings correlating with predicted strengths, and expressed two active biosynthetic enzymes (cyclodipeptide synthase─YvmC and cytochrome P450 enzyme─CypX), leading to the production of pulcherriminic acid. Overall, our B. subtilis-based CFPS system offers a robust platform for high-yield protein synthesis, in vitro prototyping of gene regulatory elements, and natural product biosynthesis, highlighting its broad potential for synthetic biology and biotechnology applications.

To evaluate the effects of Bacillus subtilis ZY1 supplementation, production performance, egg quality, serum parameters, and intestinal health in laying hens were investigated. A total of 240 healthy 59-week-old Hy-Line Brown laying hens were randomly assigned to four groups: a control group (Con) fed a basal diet and three treatment groups supplemented with Bacillus subtilis ZY1 at levels of 0.05 % (LG), 0.1 % (MG), and 0.2 % (HG). The duration of trial lasted eleven weeks, including a one-week pre-feeding phase. The results indicated that dietary supplementation with ZY1 increased the egg laying rate in the LG and HG groups (P < 0.05) as well as improved the qualified egg rate in the LG and MG groups (P < 0.05). Moreover, the LG group also demonstrated superior egg quality and enhanced antioxidant capacity and immune function by decreasing the level of MDA (42.47 %) and improving the content of T-AOC, GSH-Px, CAT, SOD, IgM and IgG (34.31 %, 23.92 %, 37.68 %, 31.64 %, 14.01 %, and 17.84 %, respectively) in serum samples (P < 0.05). The changes in biochemical parameters such as AST, LDH, TG (12.40 %, 13.79 %, and 32.13 % decreased, respectively) and Ca (41.35 % increased) were particularly pronounced in LG groups (P < 0.05), indicating that ZY1 supplementation enhanced metabolic capacity of laying hens (P < 0.05). Furthermore, laying hens in the treatment groups exhibited significantly increased villus height (VH) and an elevated villus height-to-crypt depth ratio (VH/CD) within their duodenal tissues (P < 0.05). These findings suggest that dietary supplementation with ZY1 effectively improves production performance, egg quality, serum parameters, and intestinal health in laying hens; notably, a dosage of 0.05 % ZY1 was identified as the optimal level for these improvements. This study provides valuable insights into optimizing the application of Bacillus subtilis ZY1 in laying hen management practices.

Transition state regulators from Bacillus can control diverse physiological responses such as growth, metabolism, motility, virulence, and sporulation. The AbrB protein is a transcriptional regulator involved in multiple functions during exponential phase and intricated regulatory pathways that control adaptive states differentially. Despite its importance, the AbrB role has not been well characterized during the growth cycle, and its implication in metabolic functions remains elusive, especially in the Bacillus cereus group. In this work, we characterized the role of AbrB on phenotypes such as spreading motility, growth profiles, sporulation, and on activity of core metabolic pathways of Bacillus thuringiensis. For this, a strain with inducible abrB expression was generated in the wild type Bt HD73 background. In vitro evaluations of phenotypic traits demonstrated differences in sporulation and motility, where induction of abrB presumably affected these functions under nutrient-limited media. In addition, AbrB induction during bioreactor fermentations led to higher biomass production and changes dissolved oxygen (DO) profile, which was also accompanied with a delay in sporulation. Based on these results, metabolic pathways such as glycolysis and the Krebs cycle were explored to address the effect of AbrB overproduction on transcription of genes coding for pyruvate dehydrogenase (pdHA), lactate dehydrogenase (ldH), citrate synthase (citZ) and aconitase (citB). Our findings suggest variations in the carbon-flux in the central carbon metabolism due to abrB overexpression. This work contributes to the elucidation of AbrB involvement in regulatory networks of B. thuringiensis, to develop engineering-based strategies to use these bacteria in other biotechnological applications besides as biological control agent.

The use of microorganisms in environmental biotreatment is gaining attention, particularly Bacillus subtilis (B. subtilis), recognized for its effectiveness in wastewater treatment and soil remediation. Its success stems from its diverse biological activities and adaptability, which improve environmental quality and ecological balance. This paper reviews the remediation capabilities and mechanisms of B. subtilis, focusing on its applications in water purification and soil pollution management. B. subtilis facilitates pollutant degradation and adsorption through enzyme production, organic acids, unique cell wall properties, and interactions with other microorganisms. The article addresses current challenges and future directions, emphasizing the need for enhanced cultivation, screening, and genetic engineering of functional strains. Understanding the interactions of these strains with other microorganisms and studying their ecological and toxicological impacts are essential for optimizing microbial remediation, providing both theoretical and practical foundations for bioremediation efforts.

A new generation of farming inputs is being developed, designed to be more environmentally friendly and have fewer negative impacts on consumer health. Bio-based antimicrobial compounds are one such example. These compounds are used against pathogens and stimulate plant immune systems, reducing disease severity. This study evaluated suspensions and hydro-alcoholic extracts of 55 cyanobacteria from the Nostocales order for their antimicrobial effects and growth-promoting activity on tomato plants. Suspensions and extracts of 0.5 and 1 mg mL-1 of two cyanobacteria, Nostoc sp. G-4D and Calothrix sp. G-403 were selected for their disease control capabilities and growth enhancement effects on plants. The GC-MS technique was used to investigate the chemical compounds in the hydroalcoholic extracts of two cyanobacteria species. The analysis shows that the Nostoc sp. G-4D extract contained 28 compounds, accounting for 96.04% of the total composition, while the Calothrix sp. G-403 extract contained 27 compounds, making up 93.62% of the total composition. These findings highlight the rich chemical diversity in the extracts, which might be responsible for the observed bioactivities. The predominant components of the hydroalcoholic extracts of Nostoc sp G-4D and Calothrix sp. G-403 were Hexadecanoic acid, methyl ester (28.29%) and Octadecanoic acid, methyl ester (15.89%) for the former, and Hexadecanoic acid, methyl ester (27.95%) and phytol (10.82%) for the latter.

Coffee quality can be modified with microorganism addition during post-harvest processing. While most studies focus on yeasts and lactic acid bacteria, other species identified in the digestive tract of palm civets might also contribute to the quality of luwak coffee. Bacteria akin to those identified in palm civets' gastrointestinal tract or feces were evaluated for their potential to modify coffee bean composition. Among those, Bacillus subtilis ATCC 6633, Gluconobacter sp. KKP 3751 and Lactiplantibacillus plantarum ATCC 4080 exhibited strong growth in green coffee extract. The use of these bacteria significantly changed the amounts of basic coffee components (taste and aroma precursors), and slightly altered bioactive compound levels in green and roasted beans. The influence of fermentation duration was evaluated using L. plantarum. A stationary growth phase and positive changes regarding phenolic content were achieved after 24 h of fermentation. Overall, the use of bacteria can influence bean composition, offering the potential to create unique coffee products.

The growing demand for sustainable and eco-friendly alternatives in bioprocessing, healthcare, and manufacturing has stimulated significant interest in Bacillus subtilis surface display technology. This innovative platform, leveraging both spore and vegetative cell forms, provides exceptional versatility for a wide spectrum of applications, spanning from green technologies to advanced biomedical innovations. The robustness of spores and the metabolic activity of vegetative cells enable efficient enzyme immobilization, biocatalysis, and biosensor development, facilitating bioremediation, pollutant degradation, and renewable energy generation. Additionally, B. subtilis surface display systems have demonstrated remarkable potential in vaccine development and drug delivery, offering a cost-effective, scalable, and environmentally sustainable alternative to traditional methods. These systems can effectively present antigens or therapeutic molecules, enabling targeted drug delivery and robust immune responses. This review explores recent advancements, challenges, and opportunities in harnessing B. subtilis surface display technology for sustainable biomanufacturing, green innovations, and transformative biomedical applications, emphasizing its role in addressing pressing global challenges in environmental sustainability and healthcare.

Biotechnological research and application of microbial enzyme production have consistently been focal points for scientific inquiry and industrial advancement. In this study, Bacillus subtilis Y4X3 was isolated from saline-alkaline soil in Xinjiang, China. Extracellular enzyme production analysis revealed that B. subtilis Y4X3 can secrete various enzymes, including cellulase, xylanase, protease, and amylase. Sequencing and assembly of the complete genome of this strain revealed a genome size of 4,215,636 bp with 43.51% C + G content, including 4438 coding genes. Genome annotation was performed with databases to predict gene functions in B. subtilis Y4X3, and a variety of genes related to carbohydrate metabolism were identified. A cellulase-encoding gene was subsequently cloned from the genome and heterologously expressed in Escherichia coli. The optimum pH and temperature for the purified cellulase Cel5A were 5.0 and 60 °C, respectively. Stability analysis revealed that Cel5A remained stable at pH 5.0-9.0 and 20-60 °C; after 1 h at pH 9.0, the relative enzyme activity still exceeded 60%. Additionally, Cel5A was positively affected by various metal ions and exhibited good tolerance to multiple chemical reagents. The results indicate that B. subtilis Y4X3 has the potential to produce a variety of industrial enzymes and could serve as a promising candidate for more efficient and cost-effective industrial applications; the characterized thermostable and alkali-resistant cellulase Cel5A also has potential applications in biotechnology and industry.

Amylase activity is a critical biomarker for assessing the freshness of honey. Historically, bees have been considered the sole source of honey amylase. However, recent studies suggest that Bacillus subtilis may also contribute to amylase production in the honey sac of Apis mellifera.

In this study, amylase levels were measured in samples of nectar, honey sac fluid, and honey. The identification of B. subtilis in nectar, honey sac, and honey was evaluated. An in vitro bacterial culture system and a feeding experiment were developed to simulate honey sac conditions.

Our results showed that B. subtilis was detected in all sample groups, with the highest concentration in honey sac samples. Amylase levels in honey sac and honey samples were significantly higher than those in nectar. In the simulation experiment, amylase activity was only observed in cultures containing both B. subtilis and sucrose/nectar; no activity was detected in cultures containing only H2O or no B. subtilis. In the feeding experiment, bees fed sucrose or nectar showed higher amylase activity in their honey sacs than those fed water.

Our data show that B. subtilis can produce amylase and offer potential for more standardized quality assessment of honey.

The production of industrial enzymes requires an efficient expression system with a suitable host. This study investigated the isolated Bacillus subtilis 007 as a host for expressing three enzymes with potential application in the food industry. Firstly, testing the PaprE and P43 promoters and the corresponding 5' untranslated regions revealed great differences in the production of the recently discovered β-galactosidase from Paenibacillus wnnyii. Expression controlled by the PaprE promoter yielded a significantly higher activity of 2515 µkat/L, compared to 56 µkat/L with the P43 promoter. Modifications on the PaprE core promoter region or the spacer, the sequence between the Shine-Dalgarno sequence and the start codon, did not improve β-galactosidase production. Since the aprE 5' untranslated region contributes to a high mRNA stability, it was incorporated into the P43 construct to determine whether mRNA stability is responsible for the differences observed in β-galactosidase production. Interestingly, mRNA stability was significantly improved and led to a nearly 50-fold higher β-galactosidase production of 2756 µkat/L. This strategy was successfully validated by the expression of two other enzymes: the cellobiose-2-epimerase from Caldicellulosiruptor saccharolyticus and the β-glucosidase from Pyrococcus furiosus. These findings underscored the crucial role of post-transcriptional regulation and emphasized mRNA stability as a key role in recombinant enzyme production in B. subtilis 007.

Direct-fed microbials (DFMs) have shown the potential to improve livestock performance and overall health. Extensive research has been conducted to identify new DFMs and understand their mechanisms of action in the gut. Bacillus species are multifunctional spore-forming bacteria that exhibit resilience to harsh conditions, making them ideal candidates for applications in the feed industry and livestock production. This study investigates the mode of action of B. licheniformis and B. subtilis in the rumen using diverse in vitro techniques. Our results revealed that both strains germinated and grew in sterile rumen and intestinal contents from dairy cows and bulls. Gas composition analysis of in vitro cultures in a medium containing 40% rumen fluid demonstrated that germination of B. licheniformis and B. subtilis strains reduced oxygen levels, promoting an anaerobic environment favorable to rumen microbes. Enzymatic activity assays showed that B. licheniformis released sugars from complex substrates and purified polysaccharides in filtered rumen content. Additionally, the combination of B. licheniformis and B. subtilis survived and grew in the presence of a commercial monensin dose in rumen fluid media. The effects of B. licheniformis and B. subtilis on rumen fermentation activity and microbiota were studied using an in vitro batch fermentation assay. In fermenters that received a combination of B. licheniformis and B. subtilis, less CO2 was produced while dry matter degradation and CH4 production was comparable to the control condition, indicating better efficiency of dry matter utilization by the microbiota. The investigation of microbiota composition between supplemented and control fermenters showed no significant effect on alpha and beta diversity. However, the differential analysis highlighted changes in several taxa between the two conditions. Altogether, our data suggests that the administration of these strains of Bacillus could have a beneficial impact on rumen function, and consequently, on health and performance of ruminants.

In biotechnology, B. subtilis is established for heterologous protein production. In addition, the species provides a variety of bioactive metabolites, including the non-ribosomally produced surfactin lipopeptide. However, to control the formation of the target product-forming enzyme, different expression systems could be introduced, including the principle of genetic code expansion by the incorporation of externally supplied non-canonical amino acids.

Integration of an amber stop codon into the srfA operon and additional chromosomal integration of an aminoacyl-tRNA synthetase/tRNA mutant pair from Methanococcus jannaschii enabled site-directed incorporation of the non-canonical amino acid O-methyl-L-tyrosine (OMeY). In different fed-batch bioreactor approaches, OMeY-associated surfactin production was quantified by high-performance thin-layer chromatography (HPTLC). Physiological adaptations of the B. subtilis production strain were analyzed by mass spectrometric proteomics.

Using a surfactin-forming B. subtilis production strain, which enables high cell density fermentation processes, the principle of genetic code expansion was introduced. Accordingly, the biosynthesis of the surfactin-forming non-ribosomal peptide synthetase (NRPS) was linked to the addition of the non-canonical amino acid OMeY. In OMeY-associated fed-batch bioreactor fermentation processes, a maximum surfactin titre of 10.8 g/L was achieved. In addition, the effect of surfactin induction was investigated by mass spectrometric proteome analyses. Among other things, adaptations in the B. subtilis motility towards a more sessile state and increased abundances of surfactin precursor-producing enzymes were detected.

The principle of genetic code expansion enabled a precise control of the surfactin bioproduction as a representative of bioactive secondary metabolites in B. subtilis. This allowed the establishment of inducer-associated regulation at the post-transcriptional level with simultaneous use of the native promoter system. In this way, inductor-dependent control of the production of the target metabolite-forming enzyme could be achieved.

This study investigates a novel antimicrobial peptide AtR905 derived from the endophytic fungus Aspergillus terreus, which was successfully expressed in Bacillus subtilis, purified, and characterized, and highlighted as a promising potential biocontrol agent against various plant pathogens. The results indicated AtR905 exhibited broad-spectrum antimicrobial activities against key pathogens such as Ralstonia solanacearum and Clavibacter michiganensis with very low minimal inhibitory concentrations (MICs). Stability tests confirmed that AtR905 retains its antimicrobial properties under varying thermal, pH, and UV conditions. The Oxford Cup test indicated that AtR905 showed obvious fungicidal activity against six plant pathogenic fungi, especially Rhizoctonia solani and Botrytis cinerea. Additionally, in vivo experimental demonstrated AtR905 could effectively control the B. cinerea on tobacco leaves and R. solanacearum on tomato plants. Scanning electron microscopy revealed significant membrane disruption in bacterial cells treated with AtR905. These findings suggest that AtR905 is a promising candidate for sustainable plant disease management, potentially reducing the reliance on chemical pesticides and mitigating the issue of antibiotic resistance in agricultural settings. Further research is needed to evaluate the long-term field applicability and ecological impacts of AtR905.

Hydrocarbon fuel biofouling and biocorrosion require expensive cleanup of aviation infrastructures unless appropriate sustainment measures are applied. The identification of novel biological control agents offers promising alternatives to the current chemical biocides used in fuel sustainment. In this study, 496 microbial fuel isolates from our in-house repository were screened to identify new endogenously produced antimicrobial compounds. Using agar plug screening, liquid culture growth testing, and Jet A fuel culture assays, the two fuel-isolate strains Pseudomonas protegens #133, and Bacillus subtilis #232 demonstrated promising biocontrol activity against bacteria, yeast, and filamentous fungi. Liquid chromatography-quadrupole time of flight tandem mass spectrometry (LC-QTOF-MS/MS) of #232 culture filtrate identified several common lipopeptide antimicrobials including gageostatin C, gageopeptin B, and miscellaneous macrolactins. In contrast, LC-QTOF-MS/MS identified the siderophore pyochelin as one of the predominant compounds in #133 culture filtrate with previously demonstrated antimicrobial effect. Jet fuel microbial consortium culture testing of #133 culture filtrate including flow-cytometry live/dead cell mechanism determination demonstrated antimicrobial action against Gram-positive bacteria. The study concludes that antimicrobial compounds secreted by #133 have bactericidal effects against Gordonia sp. and cause cell death through bacterial lysis and membrane damage with potential applications in the biocidal treatment of hydrocarbon-based aviation fuels.

Thermostability is a key factor for the industrial application of enzymes. This review categorizes enzymes by their applications and discusses the importance of engineering thermostability for practical use. It summarizes fundamental theories and recent advancements in enzyme thermostability modification, including directed evolution, semi-rational design, and rational design. Directed evolution uses high-throughput screening to generate random mutations, while semi-rational design combines hotspot identification with screening. Rational design focuses on key residues to enhance stability by improving rigidity, foldability, and reducing aggregation. The review also covers rational strategies like engineering folding energy, surface charge, machine learning methods, and consensus design, along with tools that support these approaches. Practical examples are critically assessed to highlight the benefits and limitations of these strategies. Finally, the challenges and potential contributions of artificial intelligence in enzyme thermostability engineering are discussed.

Ribosome pausing slows down translation and can affect protein synthesis. Improving translation efficiency can therefore be of commercial value. In this study, we investigated whether ribosome pausing occurs during production of the α-amylase AmyM by the industrial production organism Bacillus subtilis under repeated batch fermentation conditions.

We began by assessing our ribosome profiling procedure using the antibiotic mupirocin that blocks translation at isoleucine codons. After achieving single codon resolution for ribosome pausing, we determined the genome wide ribosome pausing sites for B. subtilis at 16 h and 64 h growth under batch fermentation. For the highly expressed α-amylase gene amyM several strong ribosome pausing sites were detected, which remained during the long fermentation despite changes in nutrient availability. These pause sites were neither related to proline or rare codons, nor to secondary protein structures. When surveying the genome, an interesting finding was the presence of strong ribosome pausing sites in several toxins genes. These potential ribosome stall sites may prevent inadvertent activity in the cytosol by means of delayed translation.

Expression of the α-amylase gene amyM in B. subtilis is accompanied by several ribosome pausing events. Since these sites can neither be predicted based on codon specificity nor on secondary protein structures, we speculate that secondary mRNA structures are responsible for these translation pausing sites. The detailed information of ribosome pausing sites in amyM provide novel information that can be used in future codon optimization studies aimed at improving the production of this amylase by B. subtilis.

Mycotoxin detoxification by microorganisms offers a specific, economical, and environmentally sustainable alternative to physical/chemical methods. Three strains of B. subtilis, isolated from poultry farm environments and recognized by EFSA as safe in animal nutrition for all animal species, consumers, and the environment, were screened for their ability to remove mycotoxins. All of them demonstrated mycotoxin-dependent removal efficacy, being very effective against ZEA and its analogues (α- and β-ZOL, α- and β-ZAL, and ZAL) achieving up to 100% removal within 24 h under aerobic, anaerobic, and restrictive growth conditions with toxins as the sole carbon source. ZEA removal remained effective across a wide range of pH values (5-8), temperatures (20-40 °C), and at high toxin concentrations (up to 10 µg/mL). Additionally, up to 87% ZEA removal was achieved after 48 h of incubation (30 °C) of the strains in a contaminated liquid food model containing 1 µg/mL of the toxin. Mechanistic studies suggest that ZEA detoxification involves metabolic processes rather than physical adsorption or entrapment into bacterial cells. Enzymatic activities within the bacterial cells or associated with their cell walls likely play a role in the metabolization of the toxin. Interestingly, it has been observed that growth conditions and culture media can influence the metabolization and/or conjugation of the toxin, which can result in the production of various metabolites. Further investigation is needed to identify these metabolites and assess their safety.

Elevated lactate concentrations are implicated in various acute and chronic diseases, such as sepsis and mitochondrial dysfunction, respectively. Conversely, ineffective lactate clearance is associated with poor clinical prognoses and high mortality in these diseases. While several groups have proposed using small molecule inhibitors and enzyme replacement to reduce circulating lactate, there are few practical and effective ways to manage this condition. Recent evidence suggests that lactate is exchanged between the systemic circulation and the gut, allowing bidirectional modulation between the gut microbiota and peripheral tissues. Inspired by these findings, this work seeks to engineer spore-forming probiotic Bacillus subtilis strains to enable intestinal delivery of lactate oxidase as a therapeutic enzyme. After strain optimization, we showed that oral administration of engineered B. subtilis spores to the gut of mice reduced the level of blood lactate in two different mouse models involving exogenous challenge or pharmacologic perturbation without disrupting gut microbiota composition, liver function, or immune homeostasis. Taken together, through the oral delivery of engineered probiotic spores to the gastrointestinal tract, our proof-of-concept study offers a practical strategy to aid in the management of disease states with elevated blood lactate and provides a new approach to "knocking down" circulating metabolites to help understand their roles in host physiological and pathological processes.

In this study, we sequence, assemble, and analyze the genome of endophyte Bacillus amyloliquefaciens Can02R isolated from the roots of the resurrection plant host, Chenopodium album. The assembly of the strain's genome amounts to 3,965,760 bp and contains 3,989 coding sequences, among which synthetic antibiotic clusters and multidrug resistance transporters can be found.

Horizontal gene transfer (HGT) plays a pivotal role in bacterial evolution, shaping the genetic diversity of bacterial populations. It can occur through mechanisms such as conjugation, transduction, and natural transformation. Bacillus subtilis, a model Gram-positive bacterium, serves not only as a robust system for studying HGT but also as a versatile organism with established industrial applications, such as producing industrial enzymes, antibiotics, and essential metabolites. In this study, we characterize a novel method of plasmid transfer, termed Cell-to-Cell Natural Transformation for Plasmid Transfer (CTCNT-P), which efficiently facilitates plasmid transfer between naturally competent B. subtilis strains. This method involves co-culturing donor and recipient cells under antibiotic stress and achieves significantly higher efficiency compared to traditional methods such as Spizizen medium or electroporation-mediated transformation. Importantly, we demonstrate that CTCNT-P is applicable for plasmid transformation in wild B. subtilis isolates from natural environments and other Bacillus species, including Bacillus amyloliquefaciens, Bacillus licheniformis, and Bacillus thuringiensis. The simplicity and efficiency of CTCNT-P highlight its strong potential for industrial applications, including genetic modification of wild Bacillus strains for synthetic biology and the development of biocontrol agents.

The quality of Sichuan paocai in natural fermentation is often inconsistent due to the complexity of its microbial community and environmental influences. To address this, dominant microbial strains were selectively inoculated to improve the product's quality and safety. However, vacuum freeze-drying, commonly used to prepare direct vat set (DVS) starters, can significantly damage strains due to freezing stress. This study aimed to optimize a freeze-drying protection system for Lactiplantibacillus plantarum and Bacillus subtilis to enhance their survival. Using response surface methodology, combinations of cryoprotectants were evaluated. A formulation comprising skim milk powder, glycerol, sucrose, and L-proline significantly improved strain viability after lyophilization, outperforming single cryoprotectants. Further investigation into storage conditions revealed that low temperatures (-20 °C) provided the best preservation for DVS starters. Furthermore, the optimized DVS starters demonstrated excellent fermentation performance in Sichuan paocai, enhancing its color, flavor, and sensory quality compared to natural fermentation. These findings offer a reliable freeze-drying protection strategy for survival and viability of L. plantarum and B. subtilis.

Thiabendazole (TBZ), a recalcitrant fungicide, is frequently applied in postharvest fruit treatment and generates significant volumes of industrial wastewater (WW) that conventional treatment plants cannot handle. This explores a bioelectrochemical system (BES) for TBZ degradation using Tunisian hypersaline sediments (THSs) as inoculum. Four sets of BES, along with biological controls, were tested using THS subjected to different levels of TBZ biostimulation. Sediments underwent one, two, or three biostimulation phases with increasing TBZ concentrations (0, 10, 100, and 300 mg kg-1). Potentiostatic control was applied to BES, polarized at 0.1 V vs. saturated calomel reference electrode (SCE), with a carbon felt working electrode (72 cm2 L-1) and maintained at 25°C. While current production was very low, sediments biostimulated with 100 mg kg-1 kg TBZ produced the highest current density (3.2 mA m-2), a 5-fold increase over untreated sediments (0.6 mA m-2). GC-FID analysis showed >99% TBZ degradation in all reactors. The TBZ half-elimination time from 27 days with biological treatments to 19 days in BES and further to 6 days following biostimulation. Bacterial analysis revealed a substantial microbial community shift after biostimulation, with a reduction in Bacillota (-64%) and an increase in Proteobacteria (+62%), dominated by Pseudomonas (45%) and Marinobacter (16%). These findings provide insight into the selective potential of biostimulation cycles to enhance microbial community composition and improve BES performance for TBZ wastewater treatment.

Cow's milk represents an important protein source. Here, especially casein proteins are important components, which might be a promising source of alternative protein production by microbial expression systems. Nevertheless, caseins are difficult-to-produce proteins, making heterologous production challenging. However, the potential of genome-reduced Bacillus subtilis was applied for the recombinant production of bovine αS1-casein protein.

A plasmid-based gene expression system was established in B. subtilis allowing the production of his-tagged codon-optimized bovine αS1-casein. Upscaling in a fed-batch bioreactor system for high cell-density fermentation processes allowed for efficient recombinant αS1-casein production. After increasing the molecular abundance of the recombinant αS1-casein protein using immobilized metal affinity chromatography, zeta potential and particle size distribution were determined in comparison to native bovine αS1-casein.

Non-sporulating B. subtilis strain BMV9 and genome-reduced B. subtilis strain IIG-Bs-20-5-1 were applied for recombinant αS1-casein production. Casein was detectable only in the insoluble protein fraction of the genome-reduced B. subtilis strain. Subsequent high cell-density fed-batch bioreactor cultivations using strain IIG-Bs-20-5-1 resulted in a volumetric casein titer of 56.9 mg/L and a yield of 1.6 mgcasein/gCDW after reducing the B. subtilis protein content. Comparative analyses of zeta potential and particle size between pre-cleaned recombinant and native αS1-casein showed pH-mediated differences in aggregation behavior.

The study demonstrates the potential of B. subtilis for the recombinant production of bovine αS1-casein and underlines the potential of genome reduction for the bioproduction of difficult-to-produce proteins.

The interactions between beneficial bacteria and pathogens are understudied. Here we investigate the interactions between the probiotic strain Bacillus subtilis PS-216 and the pathogen Salmonella Typhimurium SL1344. We show here that the sporulation of B. subtilis is impaired when it competes with S. Typhimurium in a nutrient-depleted medium. The sporulation impairment in B. subtilis is mediated by the sigma factor B (SigB)-dependent general stress response, as the ΔsigB mutant remains blind to manipulative cues from S. Typhimurium. Furthermore, we show that decreased sporulation frequency in B. subtilis depends on cell-cell contact between the two species involving the S. Typhimurium Type VI Secretion System, whereas B. subtilis uses the SigB-dependent response to trade spore quantity for higher spore quality.

In bacteria, where gene transcription and translation occur concurrently, post-transcriptional regulation is acknowledged to be effective and precise. The 5' untranslated regions (5' UTRs) typically harbor diverse post-transcriptional regulatory elements, like riboswitches, RNA thermometers, small RNAs, and upstream open reading frames, that serve to modulate transcription termination, translation initiation, and mRNA stability. Consequently, exploring 5' UTR-derived regulatory elements is vital for synthetic biology and metabolic engineering. Over the past few years, the investigation of successive mechanisms has facilitated the development of various genetic tools from bacterial 5' UTRs. This review consolidates current understanding of 5' UTR regulatory functions, presents recent progress in 5' UTR-element design and screening, updates the tools and regulatory strategies developed, and highlights the challenges and necessity of establishing reliable bioinformatic analysis methods and non-model bacterial chassis in the future.

A rise in population and societal changes have increased pressure on resources required to meet the growing demand for food and changing dietary preferences. The increasing demand for animal protein is concerning and raises questions regarding sustainability due to its environmental impact. Subsequently, scientists seek alternative proteins, such as microbial proteins (MPs), as an environmentally friendly choice. The production of MPs promotes benefits, including reducing deforestation and CO2 emissions. Several microorganism types, such as bacteria, yeast, fungi, and algae, use a variety of substrates for MP production, from agricultural residues to lignocellulosic biomass. These complex substrates, including lignocellulosic biomass, are converted to fermentable sugar through either chemical, physical, or biological methods. Indeed, fermentation can occur through submerged cultures or other methods. However, this depends on the substrate and microorganisms being utilized. MPs have properties that make them versatile and useful ingredients in various applications. Using residues and lignocellulosic biomass as raw materials for producing MPs offers sustainability, cost-effectiveness, and waste reduction advantages. These properties are consistent with the principles established by green chemistry, which aims to conserve resources effectively and operate sustainably in all areas. This review highlights the importance of studying manufacturing aspects and the characteristics associated with MPs, which can be implemented to solve problems and encourage novel methods in the global food/feed industry.

Bacillus sp. THPS1 is a novel strain isolated from a high-temperature hot spring in Thailand, exhibiting distinctive genomic features that enable adaptation to an extreme environment. This study aimed to characterize the genomic and functional attributes of Bacillus sp. THPS1 to understand its adaptation strategies and evaluate its potential for biotechnological applications. The draft genome is 5.38 Mbp with a GC content of 35.67%, encoding 5606 genes, including those linked to stress response and sporulation, which are essential for survival in high-temperature conditions. Phylogenetic analysis and average nucleotide identity (ANI) values confirmed its classification as a distinct species within the Bacillus genus. Pangenome analysis involving 19 others closely related thermophilic Bacillus species identified 1888 singleton genes associated with heat resistance, sporulation, and specialized metabolism, suggesting adaptation to nutrient-deficient, high-temperature environments. Genomic analysis revealed 12 biosynthetic gene clusters (BGCs), including those for polyketides and non-ribosomal peptides, highlighting its potential for synthesizing secondary metabolites that may facilitate its adaptation. Additionally, the presence of three Siphoviridae phage regions and 96 mobile genetic elements (MGEs) suggests significant genomic plasticity, whereas the existence of five CRISPR arrays implies an advanced defense mechanism against phage infections, contributing to genomic stability. The distinctive genomic features and functional capacities of Bacillus sp. THPS1 make it a promising candidate for biotechnological applications, particularly in the production of heat-stable enzymes and the development of resilient bioformulations.

Anthracnose caused by Colletotrichum scovillei is a significant disease of pepper, including in postharvest stage. Bacillus species represent a potential microbial resource for controlling postharvest plant diseases. Here, a strain HG-8-2 was obtained and identified as Bacillus velezensis through morphological, biochemical, physiological, and molecular analyses. The culture filtrate showed highly antifungal activity against C. scovillei both in vitro and on pepper fruit. Crude lipopeptide extracts, which had excellent stability, could effectively inhibit mycelial growth of C. scovillei with an EC50 value of 28.48 ± 1.45 μg mL-1 and inhibited conidial germination. Pretreatment with the extracts reduced the incidence and lesion size of postharvest anthracnose on pepper fruit. Analysis using propidium iodide staining, malondialdehyde content detection and scanning electron microscope observation suggested that the crude lipopeptide extracts harbored antifungal activity by damaging cell membranes and mycelial structures. The RNA-seq analysis conducted on C. scovillei samples treated with the extracts, as compared to untreated samples, revealed significant alterations in the expression of multiple genes involved in protein biosynthesis. Overall, these results demonstrated that B. velezensis HG-8-2 and its crude lipopeptide extracts exhibit highly antagonistic ability against C. scovillei, thereby offering an effective biological agent for the control of anthracnose in pepper fruit.

Heme is an iron-containing porphyrin compound widely used in the fields of healthcare, food, and medicine. Compared to animal blood extraction, it is more advantageous to develop a microbial cell factory to produce heme. However, heme biosynthesis in microorganisms is tightly regulated, and its accumulation is highly cytotoxic. The current review describes the biosynthetic pathway of free heme, its fermentation production using different engineered bacteria constructed by metabolic engineering, and strategies for further improving heme synthesis. Heme synthetic pathway in Bacillus subtilis was modified utilizing genome-editing technology, resulting in significantly improved heme synthesis and secretion abilities. This technique avoided the use of multiple antibiotics and enhanced the genetic stability of strain. Hence, engineered B. subtilis could be an attractive cell factory for heme production. Further studies should be performed to enhance the expression of heme synthetic module and optimize the expression of heme exporter and fermentation processes, such as iron supply. KEY POINTS: • Strengthening the heme biosynthetic pathway can significantly increase heme production. • Heme exporter overexpression helps to promote heme secretion, thereby further promoting excessive heme synthesis. • Engineered B. subtilis is an attractive alternative for heme production.