ACS Synthetic Biology
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2026
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IF 4.5
Yunlong Wu,Yuhao Sun,Huan Miao,Xiaole Xia,Ling Gao
The biosynthesis of flavin adenine dinucleotide (FAD), an essential redox coenzyme, is limited by inefficient substrate utilization and low catalytic activity. To address this, an “Uptake-Catalysis” system was constructed in Bacillus subtilis for FAD synthesis from riboflavin (RF). First, the Streptomyces davawensis transporter (RibM) was coexpressed with native FAD synthase (BsFADs) at a 2:1 ratio, which increased RF consumption to 51%. Subsequently, the isothermal compressibility perturbation engineering (ICPE) strategy was adopted, generating the optimum mutant BsFADsP61W+R202Y, which increased catalytic efficiency (kcat/Km) toward RF and FMN by 83% and 73%, respectively. Mechanistic analysis revealed that this improvement was due to a 39% shortening of RF substrate channel. Finally, FAD titer peaked at 563 mg/L with a conversion rate (cr-FAD) of 92% in shaking-flask fermentation optimization. This work achieved a 225-fold increase in FAD production, validating the effectiveness of the uptake-catalysis synergistic strategy, thereby establishing an efficient and scalable synthesis paradigm.
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JOURNAL OF HAZARDOUS MATERIALS
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2026
·
IF 10.6
Baiyun Jin,Fan Ye,Wei zhou,Guoyu Xiang,Zhoucheng Li,Yu Ding,Wentao Ma,Yuanzhu Mi
Non-covalent interactions predominated by hydrogen bonding are of vital importance to the demulsification of oil-water emulsions. This study reports a Bacillus subtilis -inspired ionic liquid demulsifier (MEB-12) with multiple hydrogen-bonding sites for the treatment of oil-in-water (O/W) emulsions. The two hydrophobic “tails” of this demulsifier facilitate rapid migration within the emulsion and enable precise localization to the oil droplets, while the four symmetric hydrophilic head groups form a multivalent hydrophilic domain. This domain outcompetes interfacial asphaltenes by forming stronger multidentate hydrogen bonds with water molecules, mediated by its abundant ether, ester, and bromide hydrogen-bonding sites, thereby destabilizing the emulsion film. When treating O/W emulsions with an oil content of 0.2% at ambient temperature, MEB-12 at a concentration of 10 mg/L achieved a light transmittance (T %) of 98.4% and an oil removal efficiency (ORE) of 99.95%, demonstrating excellent demulsification performance at low dosage. Meanwhile, MEB-12 exhibited remarkable tolerance toward high salinity. To investigate the competitive adsorption mechanism between demulsifier and asphaltenes, zeta potential, dynamic interfacial tension, droplet coalescence time, and three-phase contact angle were employed as characterization techniques. Additionally, density functional theory was utilized to analyze the electrostatic potential on the molecular surface. Meanwhile, the independent gradient model based on Hirshfeld partition analysis and bond critical point were adopted to explore hydrogen bonding interactions. These characterization and computational simulation results demonstrated that MEB-12 exhibited excellent interfacial activity. Specifically, MEB-12 triggered the reconfiguration of the oil-water interfacial film via non-covalent interactions, thereby disintegrating the rigid film, promoting the coalescence of emulsified droplets, and achieving efficient demulsification.
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Journal of Agriculture and Food Research
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2026
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IF 7.2
Zhuoyu Li,Yuchen Li,Nengwu Wang,Guanhong Wu,Xinying Li,Jinqin Deng,Xin Wang,Yan Zhang,Zhaoming Dong
The silkworm ( Bombyx mori ), a well-established model organism in the order Lepidoptera, is widely used to investigate insect immunity. Currently, our understanding of the innate immunity of silkworm is primarily based on genomic and transcriptomic studies. To explore potential antimicrobial proteins/peptides (AMPs), this study infected silkworm pupae with the probiotic Bacillus subtilis and collected hemolymph for proteomic analysis. A total of 669 proteins were identified, of which 214 were significantly changed after infection. GO and KEGG analyses revealed several immune- and metabolism-related processes and pathways, while protein interaction analysis identified clusters associated with immune-related functions as well as clusters associated with protein synthesis. Finally, we mapped the immune pathways of silkworm pupae in response to bacterial infection and found that immune recognition proteins were primarily upregulated in the early stage of infection, whereas immune effector proteins were upregulated in the late stages. This study provides a comprehensive proteomic profile of immune-related proteins in pupal hemolymph and valuable insights for the development and application of AMPs in the silkworm.
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iScience
·
2021
·
IF 5.458
Manlu Zhu,Haoyan Mu,Fei Han,Qian Wang,Xiongfeng Dai
Summary Tight coordination between transcription and translation has long been recognized as the hallmark of gene expression in bacteria. In Escherichia coli cells, disruption of the transcription-translation coordination leads to the loss of transcription processivity via triggering Rho-mediated premature transcription termination. Here we quantitatively characterize the transcription and translation kinetics in Gram-positive model bacterium Bacillus subtilis . We found that the speed of transcription elongation is much faster than that of translation elongation in B. subtilis under various growth conditions. Moreover, a Rho-independent loss of transcription processivity occurs constitutively in several genes/operons but is not subject to translational control. When the transcription elongation is decelerated under poor nutrients, low temperature, or nucleotide depletion, the loss of transcription processivity is strongly enhanced, suggesting that its degree is modulated by the speed of transcription elongation. Our study reveals distinct design principles of gene expression in E. coli and B. subtilis .
研究领域:
微生物学
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分子生物学
·
基因表达
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JOURNAL OF HAZARDOUS MATERIALS
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2026
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IF 10.6
Yijun Mo,Jialin Huang,Zihan Xiao,Miaoyue Zhang,Bo Wu,Xiaofan Yang,Meng Zhang,Jean Louis Morel,Stéphanie Ouvrard,Chao Jin,Rongliang Qiu
Accurately predicting microbial transport under environmental stress in subsurface and aquatic environments is essential for assessing microbial risks, from modeling pathogen spread to optimizing in-situ bioremediation. While the biological foundations of microbial heavy metal tolerance are established, the effects of toxic heavy metals like cadmium (Cd) on microbial-mineral interactions and subsequent transport mechanisms remain poorly understood. To address these challenges, we explored how Cd stress reconfigures the deposition behavior of Bacillus subtilis in porous media. We report, for the first time, that Cd pre-exposure induces a “stress memory” that attenuates the acute escape response from toxic surfaces, thereby enhancing deposition. We employed a multi-scale approach, integrating column experiments, quartz crystal microbalance with dissipation (QCM-D), whole-genome resequencing, and proteomics. This integrated analysis determined that the history-dependent shift from active avoidance to tolerant colonization may be driven by enhanced EPS secretion (Increased by 100.0–139.0%) and a reprogrammed signal transduction pathway (increased from 0 to 5). Consequently, B. subtilis subjected to higher prior Cd stress exhibited more attenuated migration responses, resulting in greater deposition and colonization while encountering Cd on the contact surface, with maximum deposited mass being 49.1–91.2% higher than that of the unstressed cells. These findings highlight the necessity of incorporating microbial active behavior and stress adaptation strategies into existing transport theory and provide insights for the prevention and control of pathogenic bacteria in polluted environments.
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Bioresources and Bioprocessing
·
2025
·Vol. 12
·期 1
·1-18
·
IF 5.1
Zhang Rui,Wang Han,Wang Li,Zheng Zhiming
Menaquinone-7, a form of vitamin K 2 , plays a critical role in the treatment of hemorrhagic diseases caused by vitamin K deficiency and in the prevention of bone fractures. Microbial fermentation has emerged as a promising method for MK-7 production due to its high product optical purity and significant physiological activity. However, the current production efficiency of MK-7 remains insufficient to meet industrial demands. In this study, we employed a combination of single-factor experiments, Plackett-Burman design, steepest ascent experiments, and Box-Behnken design to optimize the fermentation medium for MK-7 production by Bacillus subtilis BS - Δ ackA . Through regression analysis and consideration of practical production constraints, the optimal fermentation medium parameters were determined as follows: 20 g/L sucrose, 20.7 g/L glycerol, 47.3 g/L soy peptone, 4 g/L yeast extract, and 1.9 g/L KH 2 PO 4 , 0.1 g/L MgSO 4 ·7 H 2 O. Under these optimized conditions, the MK-7 yield reached 154.6 ± 1.32 mg/L. The experimental results demonstrated excellent stability and reliability, aligning closely with the model predictions. This study significantly enhances MK-7 production at the shake-flask fermentation level, providing valuable insights for large-scale industrial applications.
研究领域:
微生物发酵
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生物技术
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生物工程
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应用微生物学
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营养科学
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Heliyon
·
2024
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IF 3.4
Jingya Zhang,Yifan Wu,Wei Li,Honglin Xie,Jingyan Li,Yongqiang Miao,Zengqi Yang,Yefei Zhou,Xinglong Wang
The increased rate of antibiotic resistance strongly limits the resolution of Salmonella enterica serovar Typhimurium (S. Typhimurium) infection. Therefore, new strategies to control bacterial infections are urgently needed. Bacillus subtilis (B. subtilis) and its metabolites are desirable antibacterial agents. Here, we aimed to evaluate the antibacterial activity of the novel B. subtilis strain GXYX (No: PRJNA940956) crude lipopeptide against S . Typhimurium. In vitro, GXYX crude lipopeptides affected S . Typhimurium biofilm formation and swimming and attenuated the adhesion and invasion abilities of S . Typhimurium toward BHK-21 cells; in addition, it inhibited the mRNA expression of the filA , filC , csgA , and csgB genes, which are related to the adhesion and invasion ability of S . Typhimurium. In vivo, pretreatment with GXYX crude lipopeptide via intragastric administration improved the survival rate by 30%, which was related to reductions in organ bacterial loads and clinical signs in mice. Intragastric administration of GXYX crude lipopeptide significantly downregulated the mRNA levels of TNF-α, IL-1β, IL-12 and IL-6 in response to S . Typhimurium-induced inflammation compared with intraperitoneal injection. Moreover, it significantly improved the intestinal barrier-related gene (ZO-1, claudin-1, occludin-1) mRNA levels in intestinal tissue damaged by S. Typhimurium infection. In conclusion, GXYX crude lipopeptides were effective at reducing S . Typhimurium colonization, laying a foundation for the further development of novel antibacterial agents.
研究领域:
微生物学
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免疫学
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分子生物学
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Fermentation-Basel
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2025
·Vol. 11
·期 3
·119
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IF 3.3
Liang Wu,Yong Xu
Bacillus subtilis(B. subtilis) has booming application prospects in the feed industry as a value-added probiotic. Considering the intermediate and end saccharides of glucan and xylan digestion during lignocellulose biorefinery processing, the various digested saccharides were detected to disclose the bio-uptake and fermenting characteristics by solo or co-fermentation, especially with respect to five monosaccharides, cellobiose, and functional food additive—xylo-oligosaccharides (XOS).B. subtiliscan directly ferment XOS without xylose, achieving a cell yield of 0.71 g/g, while it hardly utilizes cellobiose except glucose co-fermentation. With a 5 g/L glucose addition and co-fermentation, bacterial cell yield was improved to 0.70 g/g. In total,B. subtilishad a 0.56–0.90 g/g cell yield from corncob-digested saccharides and the highest saccharide-uptaking rate of 98%. This study provides critical insights and experimental data to advance lignocellulosic biorefinery technologies, particularly for probiotics production and simplification of enzymatic hydrolysis, as well as the potential application in high value-added XOS biopurification.
研究领域:
生物工程
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微生物学
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BIOTECHNOLOGY AND BIOENGINEERING
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2020
·Vol. 117
·期 10
·2957-2968
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IF 4.002
Shan Liu,Juan Wang,Zhiguang Zhu,Ting Shi,Yi-Heng P. Job Zhang
Secretory production of recombinant proteins provides a simple approach to the production and purification of target proteins in the enzyme industry. We developed a combined strategy for the secretory production of three large-size heterologous enzymes with a special focus on 83-kDa isoamylase (IA) from an archaeon Sulfolobus tokodaii in a bacterium Bacillus subtilis . First, a secretory protein of the B. subtilis family 5 glycoside hydrolase endoglucanase (Cel5) was used as a fusion partner, along with the NprB signal peptide, to facilitate secretory production of IA. This secretory partner strategy was effective for the secretion of two other large enzymes: family 9 glycoside hydrolase from Clostridium phytofermentas and cellodextrin phosphorylase from Clostridium thermocellum . Second, the secretion of Cel5-IA was improved by directed evolution with two novel double-layer Petri-dish-based high-throughput screening (HTS) methods. The high-sensitivity HTS relied on the detection of high-activity Cel5 on the carboxymethylcellulose/Congo-red assay. The second modest-sensitivity HTS focused on the detection of low-activity IA on the amylodextrin-I 2 assay. After six rounds of HTS, a secretory Cel5-IA level was increased to 234 mg/L, 155 times the wild-type IA with the NprB signal peptide only. This combinatory strategy could be useful to enhance the secretory production of large-size heterologous proteins in B. subtilis .
研究领域:
微生物学
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生物技术
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酶工程
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分子生物学
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Microorganisms
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2025
·Vol. 13
·期 3
·536
·
IF 4.2
Lina Yang,Can Tang,Yan Cui,Jianhua Zhang
Menaquinone-7 (MK-7) is recognized for its important biological activity, andBacillus subtilisis the preferred strain for its fermentative production. However, the limited phenotypic diversity among high-yielding strains complicates the development of rapid screening methods. To address this, we utilized the effect of MK-7 on transmembrane potential to develop a high-throughput screening (HTS) strategy for efficiently identifying strains with improved MK-7 production. Among various membrane potential fluorescent dyes tested, Rhodamine 123 was selected for quantifying intracellular MK-7 levels due to its effective staining and minimal impact on cell growth. By optimizing pretreatment protocols and staining conditions, we established an HTS protocol that combines fluorescence-activated cell sorting with HPLC to identify strains with increased MK-7 production. A linear correlation was observed between mean MK-7 content and average fluorescence intensity (R2= 0.9646). This approach was applied to mutant libraries generated through atmospheric room temperature plasma mutagenesis. After three cycles of mutagenesis and screening, the mutant AR03-27 was identified, showing an 85.65% increase in MK-7 yield compared to the original SJTU2 strain. Resequencing analysis revealed that the top three mutants contained mutations in genes related to membrane transport, suggesting their potential role in enhancing MK-7 yield.
研究领域:
微生物学
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生物化学
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合成生物学
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BIOTECHNOLOGY LETTERS
·
2017
·Vol. 39
·期 8
·1229-1235
·
IF 1.73
Wang Qianqian,Xu Yixiang,Xu Jiaqi,Wang Xudong,Shen Chen,Zhang Yan,Liu Xiufeng,Yu Boyang,Zhang Jian
Objectives To characterize glycosyltransferases from Bacillus subtilis ATCC 6633 and investigate their substrate specificity towards plant polyphenols. Results Among the cloned and expressed six UDP-glycosyltransferases (BsGT1-6), BsGT-1 showed activity with a wide range of polyphenols: morin, quercetin, alizarin, rehin, curcumin and aloe emodin. The gene of BsGT-1 has an ORF of 1206 bp encoding 402 amino acids. The recombinant enzyme was purified to homogeneity by Ni–NTA affinity chromatograph, and its biochemical characteristics were identified by HPLC–UV/MS, 1 H-NMR and 13 C-NMR. BsGT-1 has an MW of approx. 46 kDa as indicated by SDS-PAGE; its activity was optimal at 40 °C and pH 8.5. The Km value of BsGT-1 towards morin was 110 μM. Conclusions BsGT-1 from B. subtilis was cloned. It had high catalytic capabilities towards polyphenols which would make it feasible for the structural modification of polyphenols.
研究领域:
生物化学
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分子生物学
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酶学
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MICROCHIMICA ACTA
·
2025
·Vol. 192
·期 11
·1-13
·
IF 5.3
Wu Chaoqian,Wang Yanlin,Li Nana,Zhang Haoqing,Ge Tida,Yu Shaoning
Bacillus subtilis (BS) effectively suppresses soilborne pathogens and enhances crop resistance through various mechanisms. However, its efficacy is significantly influenced by effective inter-root colonization rates, among other factors, necessitating precise quantitative monitoring for optimal application. Conventional detection methods are often time-consuming and technically demanding, limiting their application in the field. To address this challenge, we developed a dual-recognition biosensing platform. Specifically, tridentate-functionalized aptamer was utilized for rapid BS enrichment and secondary recognition achieved using fragment crystallizable mannose-binding lectin (FcMBL)-modified nanozyme (MOF-818). The rigid scaffold of MOF-818 ensured superior catechol oxidase activity and environmental stability, facilitating sensitive and robust online quantification of BS in complex soil matrices. The linear detection range of the platform was 10 2 –10 7 CFU/mL, and the detection limit was 12 CFU/mL. This work provides a reliable tool for real-time monitoring of BS in agricultural systems, facilitating its efficient use in biocontrol and soil health management. Graphical
A dual-recognition specific colorimetric sensing platform for detecting BS in soil
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