INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2018
·
IF 3.909
Shu-Ping Zou,Xiu-Ling Xuan,Zhi-Jian Wang,Yu-Guo Zheng
Epoxide hydrolase-mediated biocatalysis has a great prospective in the biosynthesis of optically pure epoxides. The present work targets toward the thermo-stabilization of epoxide hydrolase by covalent conjugation with polysaccharide. An epoxide hydrolase from Agrobacterium radiobacter (ArEH) was modified by covalent coupling to seven oxidized polysaccharides with different chemical structures and characteristics. Among all conjugates, ArEH with ficoll exhibited both the highest specific activity (494 U/mg) and half-life at 60 °C ( t 1/2 = 183 min). The conjugated enzyme also displayed wider optimum pH and temperature ranges, higher catalytic number ( k cat ), and catalytic efficiency ( k cat / K m ) as compared to its native counterpart. The conjugation significantly decreased the enthalpy of activation (Δ H *), free energy of transition state binding (Δ G * E − T ), and free energy of activation (Δ G *) for epichlorohydrin hydrolysis. Moreover, the conjugated ArEH showed enhanced thermal stability as evidenced by its longer half-life ( t 1/2 ), lower thermal deactivation constant ( k d ), and higher D values at 50–70 °C. Furthermore, the enzymatic activity of conjugated ArEH elevated significantly by Ca 2+ and it showed increased tolerance against Co 2+ , Fe 3+ and EDTA inhibitors. Finally, fluorescence and circular dichroism spectroscopic analysis were performed to confirm the noticeable conformational changes in ArEH structure after conjugation with ficoll, which could be responsible for the observed catalytic and stability enhancement.
研究领域:
生物化学
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酶工程
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DOI
高影响因子
AMB Express
·
2019
·Vol. 9
·期 1
·1-9
·
IF 2.226
Zhao Shuxue,Hu Chunhui,Guo Lizhong,Li Kuiran,Yu Hao
A 3-hydroxypyridine degrading bacterium, designated strain DW-1, was isolated from petroleum contaminated soil in Liao River China. 16S rRNA-based phylogenetic analysis indicates that strain DW-1 belongs to genus Agrobacterium. The optimal cultivation temperature and pH for strain DW-1 with 3-hydroxypyridine were 30 °C and 8.0, respectively. Under optimal conditions, strain DW-1 could completely degrade up to 1500 mg/L of 3-hydroxypyridine in 66 h. The 3-hydroxypyridine degradation pathway of strain DW-1 was suggested by HPLC and LC–MS analysis. The first reaction of 3-hydroxypyridine degradation in strain DW-1 was α-hydroxylation so that the major metabolite 2,5-dihydroxypyridine was produced, and then 2,5-dihydroxypyridine was transformed by a Fe2+-dependent dioxygenase to form N-formylmaleamic acid. N-Formylmaleamic acid will be transformed to maleic acid and fumaric acid through maleamic acid. This is the first report of the 3-hydroxypyridine degradation pathway and the utilization of 3-hydroxypyridine by a Agrobacterium sp. It may be potentially used for the bioremediation of environments polluted with 3-hydroxypyridine.
研究领域:
微生物学
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环境科学
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PubMed
高影响因子
TRANSGENIC RESEARCH
·
2025
·Vol. 34
·期 1
·1-17
·
IF 2
Yang Yanhui,Lu Jun,Xue Tianrui,Cao Zhenli,Li Mingjie,Zhang Zhongyi,Wang Rong,Wu Tongyu
The Agrobacterium tumefaciens mediated transformation is the prevailing methodology for plant genetic manipulation; however, A. tumefaciens overgrowth is a common constraint in the process. Exploring auxotrophic A. tumefaciens could reduce overgrowth and enhance plant transformation efficiency. The ILVC gene, which encodes the ketol-acid isomeroreductase, is critical for Valine (Val) and isoleucine (Ile) biosynthesis in some microorganisms. However, its function in A. tumefaciens is unclear. To ascertain the function of ILVC and generate an auxotrophic A. tumefaciens , this study employed an allelic exchange to disrupt the ILVC in A. tumefaciens strain GV3101. This resulted in the loss of ketol-acid isomeroreductase activity and the prevention of Val and Ile biosynthesis, creating a dual-auxotrophic GV3101 ∆ILVC . Transient expression assays in Nicotiana benthamiana transformation demonstrated that the GV3101 ∆ILVC was capable of T-DNA transfer. Moreover, stable genetic transformation analysis in N. benthamiana indicated that the introduction of GV3101 ∆ILVC led to a reduction in overgrowth within infected plant tissues. Additionally, an enhancement in transformation efficiency was observed with the prolongation of the co-cultivation time of the explant-infected strain. This study revealed the function of ILVC and explored a dual-auxotrophic A. tumefaciens for Val and Ile, potentially broadening the utilization of auxotrophic strains in plant genetic transformation.
研究领域:
植物学
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微生物学
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遗传学
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DOI
PubMed
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
·
2017
·
IF 3.807
Wenjun Yu,Rongshui Wang,Haiyan Huang,Huijun Xie,Shuning Wang
Agrobacterium tumefaciens S33 can grow with nicotine as the sole source of carbon, nitrogen, and energy via a novel hybrid of the pyridine pathway and the pyrrolidine pathway. Characterization of the enzymes involved in the hybrid pathway is important for understanding its biochemical mechanism. Here, we report that the molybdenum-containing nicotine dehydrogenase (NdhAB), which catalyzes the initial step of nicotine degradation, is located in the periplasm of strain S33, while the 6-hydroxynicotine oxidase and 6-hydroxypseudooxynicoine oxidase are in the cytoplasm. This is consistent with the fact that NdhA has a Tat signal peptide. Interestingly, an open reading frame (ORF) adjacent to the ndhAB gene was verified to encode a copper-containing electron carrier, pseudoazurin (Paz), which has a signal peptide typical of bacterial Paz proteins. Both were transported into the periplasm after being produced in the cytoplasm. We purified NdhAB from the periplasmic fraction of strain S33 and found that with Paz as the physiological electron acceptor, NdhAB catalyzed the hydroxylation of nicotine at a specific rate of 110.52 ± 8.09 μmol · min−1 · mg of protein−1, where the oxygen atom in the hydroxyl group of the product 6-hydroxynicotine was derived from H2O. The apparent Km values for nicotine and Paz were 1.64 ± 0.07 μM and 3.61 ± 0.23 μM, respectively. NAD(P)+, O2, and ferredoxin could not serve as electron acceptors. Disruption of the paz gene disabled the strain for nicotine degradation, indicating that Paz is required for nicotine catabolism in the strain. These findings help our understanding of electron transfer during nicotine degradation in bacteria.IMPORTANCE Nicotine is a toxic and addictive N-heterocyclic aromatic alkaloid produced in tobacco. Its catabolism in organisms and degradation in tobacco wastes have become major concerns for human health and the environment. Bacteria usually decompose nicotine using the classical strategy of hydroxylating the pyridine ring with the help of activated oxygen by nicotine dehydrogenase, which binds one molybdopterin, two [2Fe2S] clusters, and usually one flavin adenine dinucleotide (FAD) as well. However, the physiological electron acceptor for the reaction is still unknown. In this study, we found that the two-component nicotine dehydrogenase from Agrobacterium tumefaciens S33, naturally lacking an FAD-binding domain, is located in the periplasmic space and uses a copper-containing electron carrier, pseudoazurin, as its physiological electron acceptor. We report here the role of pseudoazurin in a reaction catalyzed by a molybdopterin-containing hydroxylase occurring in the periplasmic space. These results provide new biochemical knowledge on microbial degradation of N-heterocyclic aromatic compounds.
研究领域:
微生物学
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生物化学
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环境科学
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分子生物学
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PubMed
高影响因子
BIOTECHNOLOGY AND APPLIED BIOCHEMISTRY
·
2020
·Vol. 68
·期 1
·71-81
·
IF 1.638
Zeping Sun,Jing Lv,Chaofan Ji,Huipeng Liang,Shengjie Li,Zhaoxia Yang,Wenhuan Xu,Sufang Zhang,Xinping Lin
Rhodosporidium toruloides has been reported as a potential biotechnological microorganism to produce carotenoids. The most commonly used molecular and genetic manipulation methods based on Agrobacterium -mediated transformation (ATMT). However, this method was of relatively lower transformation efficiency. In this study, we optimized the ATMT method for R. toruloides on account of the promoter on T-DNA, the ratio of A. tumefaciens to R. toruloides NP11, acetosyringone concentration, cocultivation temperature and time, and a transformation efficiency of 2,369 cells per 10 5 recipient cells was obtained and was 24 times as that of the previous report. With this optimized method, four redder mutants and four yellower mutants were selected out with torularhodin and β-carotene production preference, respectively. The highest torularhodin production was 1,638.15 µg/g dry cell weight in A1-13. The yellower mutants were found to divert the metabolic flux from torularhodin and torulene to γ-carotene and β-carotene, and the proportion of γ-carotene and β-carotene were all over 92%. TAIL-PCR was carried out to found T-DNA insertion in these mutants, and insertion hotspot was found. RT-qPCR results showed that CTA 1 genes in these mutants were closely related to the synthesis of total carotenoids, especially torularhodin, and was a potenial metabolic engineering site in the future.
研究领域:
微生物学
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分子生物学
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生物技术
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PubMed
高影响因子
JOURNAL OF APPLIED MICROBIOLOGY
·
2019
·Vol. 127
·期 6
·1716-1726
·
IF 2.683
Cheng R.,Wang L.,Li J.,Fu R.,Wang S.,Zhang J.
AimsTo purify and characterize an exopolysaccharide (EPS) from an Agrobacterium strain ZCC3656 with high EPS‐secreting performance and investigate its anti‐inflammatory activity using lipopolysaccharide (LPS)‐induced macrophage cells in an acute liver injury mouse model.Methods and ResultsTwelve rhizobial strains were compared for EPS fermentation production in modified M9 salts supplemented with mannitol or sucrose as the sole carbon source. Agrobacterium sp. ZCC3656 exhibited the highest EPS yield (21·1 g l−1) and was characterized for EPS production by carbon source utilization, time course fermentation and serial subcultivation assays. The EPS, designated Riclin, was purified by deproteinization using the Sevag method. The combined results of gel permeation chromatography, monosaccharide composition, methylation analysis and nuclear magnetic resonance analyses indicated that Riclin is a succinoglycan‐like polysaccharide comprised of glucose, galactose, succinate and pyruvate at a ratio of 7·8. : 1·0 : 0·9 : 1·1 and has an molecular weight of approximately 2·5 × 106 Da. Riclin inhibited TNF‐α, IL‐1β and IL‐6 expression in LPS‐stimulated RAW 264.7 macrophage cells in a dose‐dependent manner. In addition, Riclin pretreatment increased the survival rate of D‐Gal/LPS treated mice, inhibited serum ALT and AST activities and reduced the production of the inflammatory mediators TNF‐α, IL‐1β and IL‐6.ConclusionsAgrobacterium sp. ZCC3656 is a highly stable EPS‐producing strain. The EPS Riclin from ZCC3656 is a succinoglycan‐type polysaccharide that is noncytotoxic and exhibits remarkable anti‐inflammatory effects in vivo and in vitro.Significance and Impact of the StudySuccinoglycans are well known for good rheological properties and their physiological interactions with plants. However, their potential activity towards mammals has received little attention. Our study revealed that the succinoglycan Riclin exhibited excellent anti‐inflammatory activities and could be considered as a promising reagent in anti‐inflammatory treatment.
研究领域:
微生物学
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免疫学
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药理学
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PubMed
高影响因子
Foods
·
2024
·Vol. 13
·期 17
·2729
·
IF 4.7
Xiaoqing Xu,Lingling Du,Meng Wang,Ran Zhang,Junjie Shan,Yu Qiao,Qing Peng,Bo Shi
Agrobacteriumsp. are notable for their ability to produce substantial amounts of exopolysaccharides. Our study identified an exopolysaccharide (Galacan, 4982.327 kDa) fromAgrobacteriumsp. FN01. Galacan is a heteropolysaccharide primarily composed of glucose and galactose at a molar ratio of 25:1. The FT-IR results suggested that Galacan had typical absorption peaks of polysaccharide. The results of periodate oxidation, Smith degradation, and NMR confirmed the presence of structural units, such as β-D-Galp(→, →3)β-D-Galp(1→, →2,3)β-D-Glcp(1→, β-D-Glcp(1→, and →2)β-D-Glcp(1→. Galacan demonstrated significant biological activities. In experiments conducted with zebrafish, it facilitated the proliferation ofLactobacillus brevisin the intestinal tract, suggesting potential prebiotic properties. Moreover, in vivo studies revealed its antihyperglycemic effects, as evidenced by significant reductions in blood glucose levels and enhanced fluorescence intensity of pancreatic β cells in a streptozotocin (STZ)-induced hyperglycemic zebrafish model. Additionally, antiaging assays demonstrated Galacan’s ability to inhibit β-galactosidase activity and enhance telomerase activity in a hydrogen peroxide (HP)-induced aging zebrafish model. These findings emphasized the potential of Galacan as a natural prebiotic with promising applications in diabetes prevention and antiaging interventions.
研究领域:
微生物学
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生物化学
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药理学
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分子生物学
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水生生物学
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DOI
PubMed
高影响因子
JOURNAL OF APPLIED MICROBIOLOGY
·
2022
·Vol. 133
·期 5
·3161-3175
·
IF 4.059
Pan Deng,Wang Xing‐Yun,Zhou Jin‐Wei,Yang Liang,Khan Abbas,Wei Dong‐Qing,Li Jun‐Jian,Jia Ai‐Qun
AimsIn the current study the anti‐virulence and anti‐biofilm activities of the cinnamic acid derivative, 3‐methoxycinnamic acid, was investigated against Agrobacterium tumefaciens.Methods and ResultsBased on the disc diffusion test and β‐galactosidase activity assay, 3‐methoxycinnamic acid was shown to interfere with the quorum sensing (QS) system of A. tumefaciens. Crystal violet staining assay, phenol‐sulfuric acid method, Bradford protein assay and confocal laser scanning microscopy (CLSM) revealed that the biofilm formation of A. tumefaciens was inhibited after the treatment of 3‐methoxycinnamic acid. Employing high‐performance liquid chromatography (HPLC) analysis of culture supernatant revealed that the production of 3‐oxo‐octanoylhomoserine lactone (3‐oxo‐C8‐HSL) decreased concentration‐dependently after treatment with 3‐methoxycinnamic acid. Swimming and chemotaxis assays also indicated that 3‐methoxycinnamic acid had a good effect on reducing the motility and chemotaxis of A. tumefaciens. In addition, the RT‐qPCR, molecular docking and simulations further demonstrated that 3‐methoxycinnamic acid could competitively inhibit the binding of 3‐oxo‐C8‐HSL to TraR and down‐regulate virulence‐related genes.Conclusions3‐Methoxycinnamic acid is proved to have good anti‐virulence and anti‐biofilm activities against A. tumefaciens.Significance and Impact of the StudyThis is the first study that investigates the anti‐virulence and anti‐biofilm activities of 3‐methoxycinnamic acid against A. tumefaciens. With its potential QS‐related virulence and biofilm inhibitory activities, 3‐methoxycinnamic acid is expected to be developed as a potent pesticide or adjuvant for the prevention and treatment of crown gall caused by A. tumefaciens.
研究领域:
微生物学
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植物病理学
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药理学
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PubMed
高影响因子
MOLECULAR BREEDING
·
2026
·Vol. 46
·期 3
·20
·
IF 3.6
Lin Fan,Ma Hongping,Chen Jianhui,Liang Shuang,Wu Zhijun,Fan Haili,Chen Shenglong,Kong Xiangqi,Zhang Tong,Hu Hongyan,Wang Hong
Genetic transformation has been a crucial technology for plant molecular biology research and molecular design breeding, particularly in modern agriculture, and currently gene function validation is mostly conducted on model plants, which severely limits the development of non-model plants such as medicinal plants. Polygonum cuspidatum Sieb. et Zucc. plays a significant role in the treatment of diabetes, cancer, respiratory and cardiovascular diseases, however, its optimal regeneration and gene transformation system is still not available. Here, we at first establish an efficient regeneration system for P. cuspidatum as: stems are the most suitable explants for callus induction and regeneration of buds, and N1 medium (MS medium with 1.0 mg/L 1-naphthylacetic acid (NAA), 0.1 mg/L kinetin (KT)) is the most suitable regeneration medium. Then based on this regeneration system, an Agrobacterium tumefaciens -mediated genetic transformation system via callus regeneration (CR) and organ regeneration (OR) is established and optimized as: the stems are immersed in the A. tumefaciens resuspension medium (1/4 MS medium added with 0.02% Silwet L-77) and vacuum infiltrated for 10 min, the optimal co-culture time is 5 days, and the most suitable screening antibiotic is 100 mg/L hygromycin (Hyg). In addition, the practicality of our introduced transformation system is confirmed through the experimental verification of the reporter gene DsRED and two candidate genes: stilbene synthase ( PcSTS) and MYB transcription factor ( PcMYB62) . In summary, our established system can not only efficiently regenerate and genetically transform P. cuspidatum , a typical non-model but highly valuable plant, but it could also serve as a reference for the construction of other non-model plant transformation systems.
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高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
·
2019
·
IF 3.571
Ruiqi Bao,Ning Gao,Jing Lv,Chaofan Ji,Huipeng Liang,Shengjie Li,Chenxu Yu,Zhenyu Wang,Xinping Lin
Nine transformants of Rhodosporidium toruloides with significant changes in the carotenoid profile were obtained by Agrobacterium tumefaciens-mediated transformation, including a white, three red, and four yellow mutants. A red mutant A1-15-BRQ that showed a high torularhodin production was selected for culture condition optimization. Results indicated that the torularhodin yield was boosted with glucose as the carbon source, at a carbon/nitrogen ratio of 22, a loading volume of 75 mL, and 28 °C. The torularhodin yield of 21.3 mg/L consisting of 94.4% total carotenoids was obtained by Box–Behnken design experiments. The torularhodin yield was 17.0-fold higher than that of the wild type, with time shortened from 9 to 3 days. This study reports an effective strategy for improving torularhodin production and provides a candidate R. toruloides strain for highly selective production of torularhodin.
研究领域:
微生物学
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生物技术
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代谢工程
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DOI
PubMed
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
·
2019
·
IF 4.077
Rongshui Wang,Jihong Yi,Jinmeng Shang,Wenjun Yu,Zhifeng Li,Haiyan Huang,Huijun Xie,Shuning Wang
Agrobacterium tumefaciens S33 degrades nicotine via a novel hybrid of the pyridine and the pyrrolidine pathways. The hybrid pathway consists of at least six steps involved in oxidoreductive reactions before the N-heterocycle can be broken down. Collectively, the six steps allow electron transfer from nicotine and its intermediates to the final acceptor O2 via the electron transport chain (ETC). 6-Hydroxypseudooxynicotine oxidase, renamed 6-hydroxypseudooxynicotine dehydrogenase in this study, has been characterized as catalyzing the fourth step using the artificial electron acceptor 2,6-dichlorophenolindophenol. Here, we used biochemical, genetic, and liquid chromatography-mass spectrometry (LC-MS) analyses to determine that 6-hydroxypseudooxynicotine dehydrogenase utilizes the electron transfer flavoprotein (EtfAB) as the physiological electron acceptor to catalyze the dehydrogenation of pseudooxynicotine, an analogue of the true substrate 6-hydroxypseudooxynicotine, in vivo, into 3-succinoyl-semialdehyde-pyridine. NAD(P)+, O2, and ferredoxin could not function as electron acceptors. The oxygen atom in the aldehyde group of the product 3-succinoyl-semialdehyde-pyridine was verified to be derived from H2O. Disruption of the etfAB genes in the nicotine-degrading gene cluster decreased the growth rate of A. tumefaciens S33 on nicotine but not on 6-hydroxy-3-succinoylpyridine, an intermediate downstream of the hybrid pathway, indicating the requirement of EtfAB for efficient nicotine degradation. The electrons were found to be further transferred from the reduced EtfAB to coenzyme Q by the catalysis of electron transfer flavoprotein:ubiquinone oxidoreductase. These results aid in an in-depth understanding of the electron transfer process and energy metabolism involved in the nicotine oxidation and provide novel insights into nicotine catabolism in bacteria.IMPORTANCE Nicotine has been studied as a model for toxic N-heterocyclic aromatic compounds. Microorganisms can catabolize nicotine via various pathways and conserve energy from its oxidation. Although several oxidoreductases have been characterized to participate in nicotine degradation, the electron transfer involved in these processes is poorly understood. In this study, we found that 6-hydroxypseudooxynicotine dehydrogenase, a key enzyme in the hybrid pyridine and pyrrolidine pathway for nicotine degradation in Agrobacterium tumefaciens S33, utilizes EtfAB as a physiological electron acceptor. Catalyzed by the membrane-associated electron transfer flavoprotein:ubiquinone oxidoreductase, the electrons are transferred from the reduced EtfAB to coenzyme Q, which then could enter into the classic ETC. Thus, the route for electron transport from the substrate to O2 could be constructed, by which ATP can be further sythesized via chemiosmosis to support the baterial growth. These findings provide new knowledge regarding the catabolism of N-heterocyclic aromatic compounds in microorganisms.
研究领域:
微生物学
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生物化学
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分子生物学
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环境科学
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DOI
PubMed
高影响因子
Plant Methods
·
2018
·Vol. 14
·期 1
·1-8
·
IF 4.269
Alagarsamy Karthikeyan,Shamala Lubobi Ferdinand,Wei Shu
Background Camellia sinensis var. sinensis is widely grown for tea beverages that possess significant health promoting effects. Studies on tea plant genetics and breeding are hindered due to its recalcitrance to Agrobacterium -mediated genetic transformation. Among the possible reasons, oxidation of phenolics released from explant tissues and bactericidal effects of tea polyphenols during the process of transformation play a role in the plant recalcitrance. The aim of the present study was to alleviate the harmful effects of phenolic compounds using in - planta transformation. Results Two-month old seedlings of tea cultivar “Nong Kangzao” were infected at the hypocotyl with wild type Agrobacterium rhizogenes and maintained in an environment of high humidity. 88.3% of infected plants developed hairy roots at the wounded site after 2 months of infection. Our data indicated that transgenic hairy root induction of tea can be achieved using A. rhizogenes following the optimized protocol. Conclusion With this method, composite tea plants containing wild-type shoots with transgenic roots can be generated for “in root” gene functional characterization and root-shoot interaction studies. Moreover, this method can be applied to improve the root system of composite tea plants for a better resistance to abiotic and biotic stresses.
研究领域:
植物生物技术
·
分子遗传学
·
农学
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DOI
PubMed
高影响因子
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