Bioresources and Bioprocessing
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2022
·Vol. 9
·期 1
·1-12
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IF 4.983
Ye Jing,Li Yunjie,Bai Yuqing,Zhang Ting,Jiang Wei,Shi Ting,Wu Zijian,Zhang Yi-Heng P. Job
To mimic the Escherichia coli T7 protein expression system, we developed a facile T7 promoter-based protein expression system in an industrial microorganism Bacillus subtilis. This system has two parts: a new B. subtilis strain SCK22 and a plasmid pHT7. To construct strain SCK22, the T7 RNA polymerase gene was inserted into the chromosome, and several genes, such as two major protease genes, a spore generation-related gene, and a fermentation foam generation-related gene, were knocked out to facilitate good expression in high-density cell fermentation. The gene of a target protein can be subcloned into plasmid pHT7, where the gene of the target protein was under tight control of the T7 promoter with a ribosome binding site (RBS) sequence of B. subtilis (i.e., AAGGAGG). A few recombinant proteins (i.e., green fluorescent protein, α-glucan phosphorylase, inositol monophosphatase, phosphoglucomutase, and 4-α-glucanotransferase) were expressed with approximately 25–40% expression levels relative to the cellular total proteins estimated by SDS-PAGE by using B. subtilis SCK22/pHT7-derived plasmid. A fed-batch high-cell density fermentation was conducted in a 5-L fermenter, producing up to 4.78 g/L inositol monophosphatase. This expression system has a few advantageous features, such as, wide applicability for recombinant proteins, high protein expression level, easy genetic operation, high transformation efficiency, good genetic stability, and suitability for high-cell density fermentation.
研究领域:
微生物学
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生物技术
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分子生物学
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基因工程
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高影响因子
Journal of Fungi
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2026
·Vol. 12
·期 3
·
IF 4.3
Yihua Liao,Jiayi Shen,Tian Yang,Huijuan Peng,Tingwei Qi,Yan Li,Chengcheng Li
Fusariumhead blight (FHB), caused byFusarium graminearum, is a fungal disease that severely affects wheat. The mycotoxins it produces, such as deoxynivalenol (DON), pose serious risks to human and animal health. In this study, a biocontrol strain, LYH8, was isolated from local sources in Jingzhou, Hubei Province. Plate confrontation assays demonstrated that LYH8 effectively inhibited the mycelial growth ofF. graminearum, with an inhibition rate of 43%, and induced morphological abnormalities such as hyphal swelling and shrinkage. Based on16S rRNAandgyrBgene sequencing and phylogenetic analysis, LYH8 was identified asBacillus velezensis. In vivo experiments showed that disease severity in wheat coleoptiles and spikes was significantly reduced by treatment with LYH8 by 75–85%, and the accumulation of DON and its deoxynivalenol-3-glucoside (D3G) in grains was decreased by 20–22%. Further transcriptome analysis revealed that it affects pathogen growth by regulating amino acid biosynthesis, ribosomal biosynthesis, carbon metabolism pathways, and the catalytic activities of related genes. In summary, LYH8 significantly controlled FHB through multiple mechanisms, including inhibiting mycelial growth, reducing infection, and blocking toxin synthesis, demonstrating strong biocontrol potential.
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INDUSTRIAL CROPS AND PRODUCTS
·
2026
·
IF 6.4
Qiangbo Zhao,Ruhan Xia,Pengxiang Han,Jiangyuan Han,Weihua Zhao,Xiangyan Zhou,Fuqiang Xu,Wenzhi Yao,Jianhe Wei,Miaoyin Dong,Mengfei Li
Astragalus mongholicus is a well-known traditional Chinese herbal medicine with a long history of medicinal use, while root rot caused by Fusarium oxysporum is a highly destructive soil-borne diseases that leads to yield and quality reduction. Although previous studies have demonstrated that the biocontrol strategy based on microbial agents could effectively inhibit root rot in crops, the biocontrol efficiency and potential mechanism in A. mongholicus have been rarely investigated. In this study, a novel strain GNRP-01A was isolated from the rhizosphere of wild A. mongolicus and exhibited significant biocontrol efficacy (77.8 %) against A. mongolicus root rot, which was identified as Bacillus subtilis based on morphology, 16S rRNA, and whole-genome sequence analyses. The B. subtilis GNRP-01A genome contained multiple biosynthetic gene clusters encoding secondary metabolites ( e.g., surfactin, subtilosin A, bacillibactin) and the genes that promote plant growth ( e.g., amylase, protease, cellulase). Additionally, GC-MS analysis identified 13 volatile organic compounds (VOCs), among which 2-dodecanone, 2-tridecanone, and 6-methyl-2-heptanone showed strong antifungal activities with inhibition rates of 81.53 %, 89.17 %, and 91.86 %, respectively. Moreover, the expression levels of genes associated with plant induced systemic resistance (ISR , e.g. , AmCHI2 , AmPAD4 , AmPDF2.2 and AmPR-10 ) were significantly up-regulated in F. oxysporum + GNRP-01A treated plants, indicating the activation of ISR of A. mongolicus . Collectively, B. subtilis GNRP-01 A controls A. mongholicus root rot through synergistic strategy involving the production of antifungal metabolites and VOCs, activation of plant ISR. These results provide new insights into the biocontrol mechanism of B. subtilis against A. mongholicus root rot, as well as support the potential applications of B. subtilis GNRP-01A as an effective biocontrol agent for root rot prevention and management in medicinal plants.
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Journal of Proteomics
·
2023
·
IF 3.3
Bianxia Li,Zhuocheng Jin,Fan Yang,Huanan Li,Jiashu Liu,Zhengbing Jiang
The wide distribution of laccases in nature makes them involved in different biological processes . However, little information is known about how laccase participates in the defense machinery of bacteria against oxidative stress . The present study aimed to elucidate the oxidative stress response mechanism of Bacillus pumilus ZB1 and the functional role of bacterial laccase in stress defense. The oxidative stress caused by methyl methanesulfonate (MMS) significantly induced laccase activity and its transcript level. The morphological analysis revealed that the defense of B. pumilus ZB1 against oxidative stress was activated. Based on the proteomic study, 114 differentially expressed proteins (DEPs) were up-regulated and 79 DEPs were down-regulated. In COG analysis , 66.40% DEPs were classified into the category “Metabolism”. We confirmed that laccase was up-regulated in response to MMS stress and its functional annotation was related to “Secondary metabolites biosynthesis , transport and catabolism”. Based on protein-protein interaction prediction, two up-regulated DEPs (YcnJ and GabP) showed interaction with laccase and contributed to the formation of laccase stability and adaptability. The overexpressed laccase might improve the antioxidative property of B. pumilus ZB1. These findings provide an insight and the guidelines for better exploitation of bioremediation using bacterial laccase. Significance Bacillus pumilus is a gram-positive bacterium that has the potential for many applications, such as bioremediation. The expression of bacterial laccase is significantly influenced by oxidative stress, while the underlying mechanism of laccase overexpression in bacteria has not been fully studied. Elucidation of the biological process may benefit the bioremediation using bacteria in the future. In this study, the differentially expressed proteins were analyzed using a TMT-labeling proteomic approach when B. pumilus was treated with methyl methanesulfonate (MMS). Reactive oxygen species induced by MMS activated the secondary metabolites biosynthesis, transport, and catabolism in B. pumilus , including laccase overexpression. Moreover, the simultaneously up-regulated YcnJ and GabP may benefit the synthesis and the stability of laccase, then improve the antioxidative property of B. pumilus against environmental stress. Our findings advance the understanding of the adaptive mechanism of B. pumilus to environmental conditions.
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高影响因子
Foods
·
2023
·Vol. 12
·期 18
·3350
·
IF 5.2
Yuhan Wang,Hongjuan Mo,Zhihong Hu,Bingjie Liu,Zhiqian Zhang,Yaowei Fang,Xiaoyue Hou,Shu Liu,Guang Yang
Chitooligosaccharides (COS), a high-value chitosan derivative, have many applications in food, pharmaceuticals, cosmetics and agriculture owing to their unique biological activities. Chitosanase, which catalyzes the hydrolysis of chitosan, can cleave β-1,4 linkages to produce COS. In this study, a chitosanase-producingBacillus paramycoidesBP-N07 was isolated from marine mud samples. The chitosanase enzyme (BpCSN) activity was 2648.66 ± 20.45 U/mL at 52 h and was able to effectively degrade chitosan. The molecular weight of purifiedBpCSN was approximately 37 kDa. The yield and enzyme activity ofBpCSN were 0.41 mg/mL and 8133.17 ± 47.83 U/mg, respectively. The optimum temperature and pH ofBpCSN were 50 °C and 6.0, respectively. The results of the high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC) of chitosan treated withBpCSN for 3 h showed that it is an endo-chitosanase, and the main degradation products were chitobiose, chitotriose and chitotetraose.BpCSN was used for the preparation of oligosaccharides: 1.0 mg enzyme converted 10.0 g chitosan with 2% acetic acid into oligosaccharides in 3 h at 50 °C. In summary, this paper reports thatBpCSN has wide adaptability to temperature and pH and high activity for hydrolyzing chitosan substrates. Thus,BpCSN is a chitosan decomposer that can be used for producing chitooligosaccharides industrially.Keywords:Bacillus paramycoides;chitosanase;chitosan;chitosan oligosaccharides;enzymatic properties
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Toxins
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2023
·Vol. 15
·期 8
·488
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IF 4.2
Xue Liu,Na Wu,Mingyu Zhang,Feng Xue,Qing Xu
Zearalenone (ZEN) is a widespread mycotoxin found in grain and feed, presenting a serious threat to animal and human health. This study investigated the ability of the novel strain B73, isolated from petroleum-contaminated soil, to detoxify ZEN. B73 was identified asBacillus spizizeniithrough physiological and biochemical tests, and further confirmed based on the 16S rRNA gene sequence and the complete genome sequence.B. spizizeniiB73 was capable of degrading up to 99.3% of ZEN at a concentration of 10 μg/mL in a minimal medium (pH = 7.0) within 8 h at 37 °C via HPLC-UV. In addition,B. spizizeniiB73 was used to treat ZEN-contaminated wheat bran, dried distillers grains (DDGS), and corn meal, whereby the respective degradation rates reached 96.32%, 98.73%, and 80.31% after 36 h of treatment. HPLC-Q-Exactive-MS/MS analysis revealed one of the degradation products to have the formula C17H24O4.B. spizizeniiB73 is a novel strain isolated from petroleum-contaminated soil, and the extracellular enzymes secreted by this strain show a remarkable ability to degrade ZEN.Keywords:zearalenone;Bacillus spizizenii;detoxification;cell extract;enzymeKey Contribution:Our study demonstrates thatBacillus spizizeniiB73 is capable of optimally degrading ZEN at 37 °C, pH 7.0, and with a 4% inoculation amount. After 36 h of degradation, ZEN degradation rates in wheat bran, corn meal, and DDGS were found to be 96.32%, 98.73%, and 80.31%, respectively, when the medium contained 50% grain content.
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高影响因子
BIOLOGICAL CONTROL
·
2023
·
IF 4.2
Chengxian Wang,Xingling Zhao,Kai Wu,Chengyue Liang,Jing Liu,Hong Yang,Changmei Wang,Bin Yang,Fang Yin,Wudi Zhang
Medicine plants such as Panax notoginseng (Sanqi) frequently experience replant failure owing to the accumulation of soilborne pathogens and autotoxins in continuous cropping soil. Bacillus sp. is a commonly used biocontrol agent to combat soilborne pathogens, but further research is needed to understand its impact on controlling Sanqi root rot and the underlying mechanisms. Here, we isolated and screened a Bacillus strain, B19, from Sanqi rhizosphere soil that exhibited potential for inhibiting Sanqi root rot pathogens. We then tested the strain's effect on the incidence of Sanqi root rot in continuous cropping soil, along with its motility/chemotaxis and biofilm formation ability (i.e., potential for root colonization). We also analyzed the strain's genomic characteristics, including potential secondary metabolite synthesis gene clusters. Our results showed that the strain B19 exhibited high inhibitory activity against fungal pathogens ( F. solani and F. oxysporum ) and could metabolize saponins as a sole carbon source. The application of B19 to continuous cropping soil significantly reduced the incidence of root rot in Sanqi seedlings. B19 was identified as B. velezensis through morphological characteristics, 16S rRNA and gyrA gene sequence analysis, and genome-level annotation. Notably, B19′s genome encodes many genes associated with functions such as motility/chemotaxis and biofilm formation, as well as 13 biosynthetic gene clusters that could encode secondary metabolites, including surfactin, fengycin, and siderophore. Additionally, B19 demonstrated the capability to produce volatile organic compounds that inhibit pathogenic fungi. The motility/chemotaxis and biofilm-forming ability of B19 were enhanced by saponins. Collectively, these properties could enhance B19′s biocontrol ability, thereby reducing the incidence of root rot.
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JOURNAL OF APPLIED MICROBIOLOGY
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2023
·
IF 4
Tang Xi,Cai Yi-Fan,Yu Xiao-Mei,Zhou Wen-Wen
AimsThis study aimed to screen a bacterial strain with high detoxifying capability for aflatoxin B1 (AFB1), verify its biotransformation efficiency, and detoxification process.Methods and resultsThree hundred and fifty samples collected from different environmental niche were screened using coumarin as the sole carbon source. HPLC was used to detect residues of AFB1, and 16S rRNA sequencing was performed on the isolated strain with highest AFB1 removal ratio for identification. The detoxified products of this strain were tested for toxicity in E. coli as well as LO2, Caco-2 and HaCaT human cell lines. HPLC-MS was applied to further confirm AFB1 removal and detoxification process.ConclusionsWe identified a strain from plant leaf designated as DT with high AFB1-detoxifying ability that is highly homologous to Bacillus aryabhattai. The optimum detoxification conditions of this strain were 37°C and pH 8.0, resulting in 82.92% removal ratio of 2 μg mL−1 AFB1 in 72 h. The detoxified products were nontoxic for E. coli and significantly less toxic for the LO2, Caco-2 and HaCaT human cell lines. HPLC-MS analysis also confirmed the significant drop of the AFB1 characteristic peak. Two possible metabolic products, C19H15O8 (m/z 371) and C19H19O8 (m/z 375), were observed by mass spectrometry. Potential biotransformation pathway was based on the cleavage of double bond in the terminal furan of AFB1. These generated components had different chemical structures with AFB1, manifesting that the attenuation of AFB1 toxicity would be attributed to the destruction of lactone structure of AFB1 during conversion process.
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ACS Sustainable Chemistry & Engineering
·
2023
·
IF 8.4
Guangzheng Jiang,Wenhui Xu,Xiaoxin Huang,Zhenqing Dai,Chunxia Zhou,Pengzhi Hong,Chengyong Li
The surface-enhanced Raman scattering (SERS) technique has demonstrated great advantages, such as high sensitivity and minimal equipment requirement. However, its application is largely hindered by high-expense SERS substrates. Here, we develop a low-cost and easily accessible SERS platform based on natural cuttlebone-derived organic matrix (CDOM) for rapid and portable detection of the Bacillus cereus spore biomarker, 2,6-pyridinedicarboxylic acid (DPA). The natural porous structure of CDOM helps achieve a significant load of Ag nanoparticles (AgNPs). By adjusting the amount of NaOH in Tollens’ reagent, more uniform AgNPs can be obtained on the surface of CDOM. The pyridine N atom or carboxylic acid O atom of DPA acts on the AgNP surface to produce the characteristic peak of SERS. Meanwhile, internal standard materials are added to resist the instability caused by natural materials. The SERS substrate realized the quantitative detection of DPA within 30 min. The linear range is from 40 to 1000 nM, and the detection limit is 8.62 nM. The sensor can detect bacterial spores in foods, such as milk. This method can be used as a reliable idea and an example for the practical application of biologically derived materials.
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FOOD CHEMISTRY
·
2019
·
IF 5.399
Jiaxi Chu,Haizhen Zhao,Zhaoxin Lu,Fengxia Lu,Xiaomei Bie,Chong Zhang
Millet bran was fermented with Bacillus natto and the changes of structural, physicochemical and functional properties of its dietary fiber were investigated. Results showed that B. natto fermentation enhanced soluble DF content from 2.3% to 13.2%, and soluble DF/insoluble DF ratio from 3.1% to 19.9%. SEM and FTIR assay indicated that fermentation led to the degradation of cellulose and hemicellulose, thereby forming more porous and loose structure and polysaccharides. The binding capacities such as water and oil holding capacity, swelling capacity as well as cholesterol, bile salts, nitrite ion and glucose adsorption capacity were improved, while cation exchange capacity was not significantly changed. The total phenolic content and DPPH free radical scavenging capacity increased significantly. Overall, fermentation of millet bran by B. natto improved the structural and functional properties of its DF, which could be applied as a functional ingredient in food products.
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Catalysts
·
2019
·Vol. 9
·期 9
·734
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IF 3.444
Jian Zhang,Yan Shao,Weiwei Wang
Curcumin is a naturally occurring polyphenolic compound that is commonly used in both medicine and food additives, but its low aqueous solubility and poor bioavailability hinder further clinical applications. For assessing the effect of the glycosylation of curcumin on its aqueous solubility, two glycosyltransferase genes (BsGT1andBsGT2) were cloned from the genome of the strainBacillus subtilisATCC 6633 and over-expressed inEscherichia coli.Then, the two glycosyltransferases were purified, and their glycosylation capacity toward curcumin and its two analogues was verified. The results showed that bothBsGT1andBsGT2could convert curcumin and its two analogues into their glucosidic derivatives. Then, the structures of the derivatives were characterized as curcumin 4′-O-β-D-glucoside and two new curcumin analogue monoglucosides namely, curcumoid-O-α-D-glucoside (2a) and 3-pentadienone-O-α-D-glucoside (3a) by nuclear magnetic resonance (NMR) spectroscopy. Subsequently, the dissolvability of curcumin 4′-O-β-D-glucoside was measured to be 18.78 mg/L, while its aglycone could not be determined. Furthermore, the optimal catalyzing conditions and kinetic parameters ofBsGT1andBsGT2toward curcumin were determined, which showed that the Kcat value ofBsGT1was about 2.6-fold higher than that ofBsGT2, indicating that curcumin is more favored forBsGT2. Our findings effectively apply the enzymatic approach to obtain glucoside derivatives with enhanced solubility.Keywords:curcumin 4′-O-β-D-glucoside;glycosyltransferases;Bacillus subtilisATCC 6633
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LWT-FOOD SCIENCE AND TECHNOLOGY
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2018
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IF 3.129
Zhen Qin,Sa Luo,Yun Li,Qiming Chen,Yongjun Qiu,Liming Zhao,Lihua Jiang,Jiachun Zhou
Chitooligosaccharides are widely used in functional food, medicaments and agriculture. Controllable conversion of chitosan to chitooligosaccharides with desired degree of polymerization (DP) is of great value. Herein, a novel glycoside hydrolase family 46 chitosanase (BaCsn46A) was cloned and expressed from Bacillus amyloliquefaciens. BaCsn46A exhibited good enzymatic properties with high specific activity (1031.2 U/mg) under optimal hydrolysis conditions of pH 6.0 and 50 °C. BaCsn46A is an endo-type chitosanase, which could be utilized to yield abundant chitooligosaccharides. Furthermore, the preparation of high degree of polymerization chitooligosaccharides (DP 5–10) was investigated by BaCsn46A in an enzymatic reactor with a subsequent series of membranes. The maximum concentration of total chitooligosaccharides (DP 2–10) was 28.11 g/L (93.70% yield). The proportion of high degree of polymerization chitooligosaccharides (DP 5–10) occupied 56.17% (w/w) of total chitooligosaccharides. This study provided a cleaner production process for the controllable preparation of chitooligosaccharides with specific degree of polymerization.
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危险品化学品经营许可证（带存储）