Fusarium crown rot (FCR), predominantly caused by the soil-borne pathogen Fusarium pseudograminearum, poses a severe and escalating threat to global wheat (Triticum aestivum L.) production and food security. This review consolidates recent advances in understanding the pathogen’s biology, infection strategies, host–pathogen interactions, and sustainable management practices. F. pseudograminearum deploys trichothecene mycotoxins, particularly deoxynivalenol (DON), to undermine host immunity, with its virulence governed by transcription factors and functional proteins involved in fungal growth, toxin biosynthesis, and stress adaptation. In response, wheat initiates a multilayered defense mechanism, primarily by upregulating genes related to toxin detoxification and transport, hormonal signaling, and structural reinforcement. Integrated disease management (IDM) practices aimed at mitigating disease severity include agronomic interventions, fungicides, biocontrol agents, and breeding efforts supported by quantitative trait locus (QTL) mapping, genome-wide association (GWAS) studies, and genome editing. To address the dynamic nature of F. pseudograminearum populations under changing environmental conditions, future directions should prioritize climate-resilient strategies, integrated multi-omics approaches, drone-based early detection technologies, and global pathogen surveillance systems. This review provides an updated framework to guide sustainable and effective FCR control.

Serotonin (5-hydroxytryptamine, 5-HT) plays a critical role in maintaining intestinal health, regulating mood and sleep, and enhancing crop stress tolerance. Here, a Bacillus licheniformis cell factory was developed for the sustainable and efficient synthesis of 5-HT. First, a tryptophan-producing chassis strain, TRP18, was obtained by blocking competitive pathways, modifying the tryptophan operon, and rewiring carbon metabolic flux, and the Trp titer was increased by 23.94-fold over the starting strain, TRP0. Subsequently, a heterologous 5-HT biosynthetic pathway was established, and tryptophan hydroxylase Luz15 was optimized by protein engineering. Finally, the 5-HT pathway was optimized by a dual-cassette system, and an optimal strain, HT-12, was attained, producing 1.23 g/L 5-HT in a 5-L bioreactor, representing the highest titer of 5-HT from glucose in Bacillus species to date. This study provides an efficient and sustainable process for 5-HT production in B. licheniformis, laying the foundation for industrial-scale manufacturing of 5-HT and other Trp-derived biochemicals.

Bacillus licheniformis DW2 has emerged as a promising microbial chassis for biosynthesis of industrial enzymes, lipopeptides and biochemicals. However, the lack of dynamic control tools to date has limited its expansion in basic research and industrial applications. Here, a well- performing bifunctional dynamic control system was developed for the dynamic up- and down- regulation of genes, as well as the effective biosynthesis of poly-γ-glutamic acid (γ-PGA). Firstly, a dynamic turn-on module was developed in B. licheniformis DW2, including an artificially designed broad-range regulated xylose-inducible expression system with a regulation range of 1.03 ∼ 352.37-fold, and an artificially designed highly sensitive IPTG-inducible expression system that can respond to 0.001 mM IPTG. Subsequently, a dynamic turn-off module based on the ClpXP protease was developed, which could regulate the degradation efficiency of target protein by selecting different ssrA tags. Then, a bifunctional dynamic control system based on the coupling of LacI and ClpXP was established in B. licheniformis for the first time, through combining the dynamic turn- on and off modules, which realized the dynamic up- and down- regulation of the expression of rfp and gfp genes simultaneously. Finally, it was successfully applied to dynamically optimize the metabolic flux of γ-PGA, increasing the yields of γ-PGA by 33.51 %. This work not only developed a well-performance bifunctional dynamic control tool for gene expression regulation, but also offered a strong support for the efficient production of diversified products by B. licheniformis.

Alkaline protease has been commercially used in the areas of detergents, food and agriculture, and improving the performance and production of alkaline protease serves as an important role in promoting its market expansion. Here, an alkaline protease AE0 from Alkalihalobacillus clausii FYX was firstly characterized in Bacillus licheniformis DW2△aprE, the optimal temperature and pH of AE0 were 60 °C and 10.5, the Km and Kcat values for casein were 17.25 mg/mL and 60.51 s-1, respectively, as well as the specific activity was 21,365.93 U/mg. Subsequently, six mutants (G113I, H118D, T141Y, S151A, N167S and Q185S) were obtained through semi-rational design, and G113I exhibited the most optimal performance with a specific activity of 28,150.64 U/mg. Furthermore, the double mutant AE0DM1 and triple mutant AE0MM2 were attained, and their specific activities reached 31,026.32 U/mg and 31,868.56 U/mg, respectively. Concurrently, through the evaluation of thermal stability and measurement of reaction kinetic parameters, G113I was advantageous for enhancing the thermal stability of AE0, while H118D and N167S were more beneficial for enhancing the catalytic efficiency. In addition, the enzyme activity of AE0MM2 produced by strain DW2△aprE/RC0-AE0MM2 was increased by 178.5 % through promoter engineering, reached 36,685.33 U/mL, which also showed the wonderful performance on enzymatic hydrolysis of soybean meal to enhance its utilization rate. Taken together, this work provided an alkaline protease with improved thermal stability and catalytic efficiency, as well as an efficient expression system of alkaline protease for industrial application.

Ectoine is a high-value protective agent with extensive applications in the fields of fine chemicals and biopharmaceuticals, and it is naturally synthesized by Halomonas in extreme environment, however, the current production level cannot meet the growing market demand. In this study, we aimed to develop an efficient and environmentally friendly ectoine production process using Bacillus licheniformis as the host organism. Through introducing ectoine synthetase gene cluster ectABC from Halomonas elongate, as well as optimizing ectABCHs expression by promoter and 5’-UTR optimization, ectoine titer was increased to 0.55 g/L. Furthermore, subsequent introduction of exogenous phosphoenolpyruvate carboxylase PPCEC and down-regulated expression of phosphoenolpyruvate carboxykinase PCK optimized the carbon flux through C4 anaplerotic pathway, and further benefited ectoine synthesis. Furthermore, the carbon flux towards aspartic acid accumulation was increased through optimization of glyoxylate and TCA cycles, accompanied with introducing lysCT311ICg and asdCg, and blocking by-products pathways, ectoine titer produced by B. licheniformis ECT12 was 2.00 g/L. Moreover, NADPH supply was enhanced by overexpression of exogenous NADH kinase Pos5Sc, and ectoine transportation was improved by introducing compatible solute transporter ProP from Escherichia coli, and the resulting B. licheniformis ECT14 was able to produce 2.60 g/L ectoine. Last but not the least, the ectoine yield of 3.29 g/L was attained in a 5-L fermenter. Taken together, this study not only established B. licheniformis as a framework for sustainable production of ectoine, but also paved the way for achieving the industrial production of ectoine and aspartic acid derivatives in the future.

Protocatechuic acid (PCA) is a natural phenolic acid with various biological activities, which is widely used in the fields of pharmaceuticals, functional foods and biobased plastics. Microbial fermentation provides a green and sustainable process for PCA production. However, the current yield of PCA by microorganisms remain limited. Here, a Bacillus licheniformis cell factory was developed for green and sustainable efficient synthesis of PCA. Firstly, a heterologous PCA biosynthetic pathway was established in B. licheniformis DW2 through the screening and expression optimization of 3-dehydroshikimate (3-DHS) dehydratase, and obtained a starting strain PCA6 with a PCA titer of 0.58 g/L. Subsequently, the conversion of intracellular PCA into catechol was unexpectedly discovered and blocked, correspondingly identified an endogenous PCA decarboxylase complex BsdBCD, and excavated a novel transcription factor BsdA that responds to PCA activation. The biosynthesis level of PCA was significantly enhanced by engineering B. licheniformis, such as weakening competitive pathway, enhancing precursor supply, strengthening 3-DHS synthesis flux, identifying PCA transporter MDR and promoting PCA efflux, and obtained an optimal strain PCA22 with a PCA titer of 17.83 g/L. Finally, PCA22 produced 21.68 g/L PCA, and a yield of 0.54 g/g from glucose via optimizing fermentation process, which represented the highest yield of PCA from glucose to date. This study provided a green and sustainable biotechnology for the efficient synthesis of PCA, and promoting the further development of green chemical industry.

River-lake confluence zones, characterized by unique hydrodynamic conditions, are critical areas for pollutant transformation. Nevertheless, degradation of microplastics (MP) mediated by multi-trophic microbial communities remains poorly understood under such complex hydrodynamic disturbances. This study investigated the characteristics of multi-trophic microbiota of the microplastome and explored their roles in MP degradation across four distinct hydrodynamic zones, i.e., maximum velocity zone (Z1), flow buffer zone (Z2), flow deflection zone (Z3), and flow reestablishment zone (Z4), in a river-lake confluence. A pronounced spatial heterogeneity in MP abundance and available nutrients was revealed among the four flow zones, with Z3 exhibiting the most intense MP degradation. Additional microcosm experiments demonstrated that microbial MP degradation was primarily driven by the enriched degrading bacteria and fungi, facilitated by multi-trophic microbial interactions. Furthermore, in situ analysis revealed that both bottom-up and top-down effects occurred across all flow zones, with their intensity being positively correlated with the degree of MP degradation. Thereby, nutrient availability driven by hydrodynamics stimulated the growth of MP degrading bacteria and fungi through a bottom-up effect. The increase in the relative abundance of MP degrading bacteria, concurrent with enhanced protozoan predation on bacteria, suggested that this top-down control operated through the selective predation of protozoa on non-MP degrading bacteria. Across the entire river-lake confluence zone, directional flow fluctuations were identified as the paramount environmental factor through the causal effect model, explaining >50% of the variance in bottom-up and top-down effects. Our study demonstrates how hydrodynamics governs MP degradation via multi-trophic microbial interactions, advancing our fundamental understanding of MP fate in aquatic ecosystems.

Bacterial infections are a leading cause of global health loss. The prerequisites for bacteria to colonize and establish systemic infection in the host are environmental adaptability and the expression of virulence factors. Phosphorus constitutes the fifth most important element in terms of its cellular content, which is pivotal for DNA replication, metabolism, signal transmission, and microbial cell composition. The phosphate (Pho) regulon is a well-established unique mechanism in response to inorganic phosphate (Pi) starvation. The Pho regulon is strongly related to bacterial pathogenicity, except for the simple regulatory system for Pho balance. The PhoBR two-component regulatory system of the Pho regulon has documented the effects of affecting virulence in microbes. This review emphasizes the impact of the absence of PhoB and virulence-related gene regulation by PhoB on pathogenicity in common pathogens of Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica serovar Typhimurium, Vibrio cholerae, etc. Collectively, Pho regulon is a regulatory network connecting Pho homeostasis with bacterial virulence. This study may offer valuable information for understanding the regulation of bacterial virulence by the Pho regulon, providing novel insights for the development of antimicrobial strategies.

Lactic acid, an important organic acid, commonly exists in a variety of foods. During food processing, lactic acid may undergo dehydration and condensation with proteins. This study investigated the effect of lactylation on the sensitization of bovine beta-lactoglobulin during food processing. First, we screened 19 lactylation sites on beta-lactoglobulin through mass spectrometry. Comparing the specific IgE/IgG epitopes of beta-lactoglobulin, we found that lactylation masks it. At the same time, the structure of beta-lactoglobulin is destroyed after binding to lactic acid. Animal experiment results show that the levels of antibodies (IgE and IgG1) and Th2-type cytokines (IL-4 and IL-13) in vivo induced by lactated beta-lactoglobulin are significantly reduced. All results indicate that the allergenicity of beta-lactoglobulin is reduced after lactylation. In conclusion, this study provides valuable insights into the molecular mechanisms underlying the reduction of beta-lactoglobulin allergenicity by lactylation and lays a solid foundation for the application of lactylation in hypoallergenic foods.

In view of the extensive potential applications of chitinase (ChiA) in various fields such as agriculture, environmental protection, medicine, and biotechnology, the development of a high-yielding strain capable of producing chitinase with enhanced activity holds significant importance. The objective of this study was to utilize the extracellular chitinase from Bacillus thuringiensis as the target, and Bacillus licheniformis as the expression host to achieve heterologous expression of ChiA with enhanced activity. Initially, through structural analysis and molecular dynamics simulation, we identified key amino acids to improve the enzymatic performance of chitinase, and the specific activity of chitinase mutant D116N/E118N was 48

Introduction This study aimed to investigate the microbial characteristics of yak uteri collected using intrauterine cotton swabs (CS) during different reproductive stages and the correlation of these microbial characteristics with reproductive status. Methods We used a macrogenomic approach to analyze the functional aspects of different microorganisms in samples collected during the pre-estrus, estrus, late estrus, and diestrus stages. Results The results revealed the presence of 1293 microbial genera and 3401 microbial species in the uteri of yaks at different reproductive stages. The dominant bacterial species varied across the different periods, with Micrococcus and Proteus being dominant during pre-estrus; Pseudomonas , Clostridium , Flavobacterium , Bacillus , and Staphylococcus during estrus; Acinetobacter , Bacillus and Proteus during late estrus; and Pseudomonas , Escherichia coli , and Proteus during diestrus. Discussion The primary functions of these bacteria are enriched in various metabolic pathways, including carbohydrate and amino acid metabolism, intracellular transport and secretion, post-translational protein modification, and drug resistance. These findings suggest that the microbial diversity in the uterus of yaks plays a crucial role in reproductive regulation and can help prevent reproductive tract-related diseases.

Establishment of a green and sustainable process for production of the antioxidant hydroxytyrosol using metabolically engineered Bacillus licheniformis .

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

Disulfide bonds in proteins have strongly influence on the folding efficiency by constraining the conformational space. The inefficient disulfide bond formation of proteins is the main limiting factor of enzyme activity and stability. This study aimed to increase the activity of disulfide-bond-containing proteins via promoting disulfide bonds formation in Bacillus licheniformis. Initially, the glutamate decarboxylase GAD from Escherichia coli was selected as the model protein and introduced into the B. licheniformis. Then, the disulfide isomerase and oxidoreductase from different sources were excavated and overexpressed successively to improve the catalytic efficiency of GAD. The final engineered B. licheniformis showed significantly improved GAD specific activity (from 10.4 U/mg to 80.0 U/mg), which also presented perfect adaptability for other disulfide-bond-containing proteins, for instance, UDP-glucosyltransferase from Arabidopsis thaliana. Taken together, our work demon-strated that the activity of GAD in B. licheniformis was regulated by the disulfide bonds formation status and provided a promising platform for the expression of disulfide-bond-containing proteins.

During the production of nattokinase (NK) by Bacillus species, certain by-products such as poly-γ-glutamic acid (γ-PGA) are simultaneously synthesized. The impact of γ-PGA synthesis on NK production remains unclear. In this study, we knocked out the pgsC gene, a component of the γ-PGA synthetase cluster (pgsBCA), and constructed a novel recombinant strain, Bacillus licheniformis BL11. Next, we compared the fed-batch fermentation profiles of BL11 and its parental strain BL10, conducted transcriptional analysis, and measured intracellular ATP content. We also optimized glucose-feeding strategies under varying oxygen supply conditions. Our results indicated that the utilization rates of glucose and soybean meal were both improved in the pgsC-deficient strain BL11, and NK activity was enhanced. Furthermore, the transcriptional levels of genes involved in glycolysis and the TCA cycle were relatively upregulated in BL11. The maximal NK activity reached 2522.2 FU/mL at 54 h of fermentation using a constant glucose-feeding rate of 5.0 g/(L·h) under high oxygen supply conditions. The newly developed recombinant strain B. licheniformis BL11, along with the optimized feeding strategy, shows promise for large-scale NK production.

Owing to the feature of strong & alpha;-glucosidase inhibitory activity, 1-deoxynojirimycin (1-DNJ) has broad appli-cation prospects in areas of functional food, biomedicine, etc., and this research wants to construct an efficient strain for 1-DNJ production, basing on Bacillus amyloliquefaciens HZ-12. Firstly, using the temperature-sensitive shuttle plasmid T2 (2)-Ori, gene ptsG in phosphotransferase system (PTS) was weakened by homologous recombination, and non-PTS pathway was strengthened by deleting its repressor gene iolR, and 1-DNJ yield of resultant strain HZ-S2 was increased by 4.27-fold, reached 110.72 mg/L. Then, to increase precursor fructose-6 -phosphate (F-6-P) supply, phosphofructokinase was weaken, fructose phosphatase GlpX and 6-phosphate glucose isomerase Pgi were strengthened by promoter replacement, moreover, regulator gene nanR was deleted, 1-DNJ yield was further increased to 267.37 mg/L by 2.41-fold. Subsequently, promoter of 1-DNJ synthetase cluster was optimized, as well as 5 & PRIME;-UTRs of downstream genes in synthetase cluster, and 1-DNJ produced by the final strain reached 478.62 mg/L. Last but not the least, 1-DNJ yield of 1632.50 mg/L was attained in 3 L fermenter, which was the highest yield of 1-DNJ reported to date. Taken together, our results demonstrated that metabolic engineering was an effective strategy for 1-DNJ synthesis, this research laid a foundation for industrialization of functional food and drugs based on 1-DNJ.

Poly-γ-glutamic acid (γ-PGA) is a natural polymer with various applications, and its high-viscosity hinders oxygen transmission and improvement of synthesis level. Vitreoscilla hemoglobin (VHB) has been introduced into various hosts as oxygen carrier, however, its expression strength and contact efficiency with oxygen hindered efficient oxygen transfer and metabolite synthesis. Here, we want to optimize the expression cassette of VHB for γ-PGA production. Firstly, our results implied that γ-PGA yields were enhanced when introducing twin-arginine translocation (Tat) signal peptides (SPYwbN, SPPhoD and SPTorA) into VHB expression cassette, and the best performance was attained by SPYwbN from Bacillus subtilis, the γ-PGA yield of which was 18.53% higher than that of control strain, and intracellular ATP content and oxygen transfer coefficient (KLa) were increased by 29.71% and 73.12%, respectively, indicating that VHB mediated by SPYwbN benefited oxygen transfer and ATP generation for γ-PGA synthesis. Furthermore, four promoters were screened, and P vgb was proven as the more suitable promoter for VHB expression and γ-PGA synthesis, and γ-PGA yield of attaining strain WX/pPvgb-YwbN-Vgb was further increased to 40.59 g/L by 10.18%. Finally, WX/pPvgb-YwbN-Vgb was cultivated in 3 L fermentor for fed-batch fermentation, and 46.39 g/L γ-PGA was attained by glucose feeding, increased by 49.26% compared with the initial yield (31.01 g/L). Taken together, this study has attained an efficient VHB expression cassette for oxygen transfer and γ-PGA synthesis, which could also be applied in the production of other metabolites.

Terminators serve as the regulatory role in gene transcription termination; however, few researches about terminator optimization have been conducted, which leads to the lack of available and universal terminator for gene expression regulation in Bacillus. To solve this problem and expand synthetic biology toolbox of Bacillus licheniformis, the terminator T1 of endogenous α-amylase gene (amyL) was characterized in this research, with a termination efficiency of 87.81%. Then, we explored and optimized the termination strength of terminator T1 from four aspects: the distance between stop codon and terminator, GC content at the bottom of stem structure, loop size, and U-tract length, and the best terminator T24 was attained by combination optimization strategy, which termination efficiency was increased to 97.97%, better than the commonly used terminator T7 (T7P) from Escherichia coli. Finally, terminator T24 was applied to protein expression, which, respectively, led to 33.00%, 25.93%, and 11.78% increases of green fluorescence intensity, red fluorescence intensity, and keratinase activity, indicating its universality in protein expression. Taken together, this research not only expands a plug-and-play synthetic biology toolbox in B. licheniformis but also provides a reference for the artificial design of versatile intrinsic terminator.

Poly-γ-glutamic acid (γ-PGA) is a multifunctional biopolymer mainly produced by Bacillus. The cofactor specificity of enzymes plays a critical role in regulating metabolic process and metabolite production. Here, we present a novel approach for switching cofactor specificity of glutamate dehydrogenase RocG from nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide (NADH) to improve γ-PGA production. Firstly, 3D structural modeling and molecular docking were performed to predict the binding modes of NADH and NADPH. Several site-specific mutants based on the conventional and Random Accelerated Molecular Dynamics simulations were obtained to alter cofactor specificity. Then, the effects of RocG variants overexpressions on γ-PGA production were evaluated. Compared to the wild-type, the mutant RocGD276E showed highest increase in γ-PGA yield, increased by 40.50%. Meanwhile, yields of main by-products acetoin and 2,3-butandieol were decreased by 21.70% and 16.53%, respectively. Finally, the results of enzymatic properties confirmed that glutamate dehydrogenase mutant RocGD276E exhibited the higher affinity for NADH, caused a shift in coenzyme preference from NADPH to NADH, with a catalytic efficiency comparable with NADPH-dependent RocG. Taken together, this research demonstrated that switching the cofactor preference of glutamate dehydrogenase via rational design was an effective strategy for high-level production of γ-PGA in Bacillus licheniformis.