APPLIED AND ENVIRONMENTAL MICROBIOLOGY
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2018
·Vol. 84
·期 14
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IF 3.633
Yan Zhang,Feng Liu,Na Xu,Yin-Qi Wu,Yu-Cong Zheng,Qian Zhao,Guoqiang Lin,Hui-Lei Yu,Jian-He Xu
Two Baeyer-Villiger monooxygenases (BVMOs), designated BoBVMO and AmBVMO, were discovered from Bradyrhizobium oligotrophicum and Aeromicrobium marinum, respectively. Both monooxygenases displayed novel features for catalyzing the asymmetric sulfoxidation of bulky and pharmaceutically relevant thioethers. Evolutionary relationship and sequence analysis revealed that the two BVMOs belong to the family of typical type I BVMOs and the subtype ethionamide monooxygenase. Both BVMOs are active toward medium- and long-chain aliphatic ketones as well as various thioether substrates but are ineffective toward cyclohexanone, aromatic ketones, and other typical BVMO substrates. BoBVMO and AmBVMO showed the highest activities (0.117 and 0.025 U/mg protein, respectively) toward thioanisole among the tested substrates. Furthermore, these BVMOs exhibited distinct activity and excellent stereoselectivity toward bulky and prochiral prazole thioethers, which is a unique feature of this family of BVMOs. No native enzyme has been reported for the asymmetric sulfoxidation of bulky prazole thioethers into chiral sulfoxides. The identification of BoBVMO and AmBVMO provides an important scaffold for discovering enzymes capable of asymmetrically oxidizing bulky thioether substrates by genome mining.IMPORTANCE Baeyer-Villiger monooxygenases (BVMOs) are valuable enzyme catalysts that are an alternative to the chemical Baeyer-Villiger oxidation reaction. Although BVMOs display broad substrate ranges, no native enzymes were reported to have activity toward the asymmetric oxidation of bulky prazole-like thioether substrates. Herein, we report the discovery of two type I BVMOs from Bradyrhizobium oligotrophicum (BoBVMO) and Aeromicrobium marinum (AmBVMO) which are able to catalyze the asymmetric sulfoxidation of bulky prazole thioethers (proton pump inhibitors [PPIs], a group of drugs whose main action is a pronounced and long-lasting reduction of gastric acid production). Efficient catalysis of omeprazole oxidation by BoBVMO was developed, indicating that this enzyme is a promising biocatalyst for the synthesis of bulky and pharmaceutically relevant chiral sulfoxide drugs. These results demonstrate that the newly identified enzymes are suitable templates for the discovery of more and better thioether-converting BVMOs.
研究领域:
酶学
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生物催化
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药物化学
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微生物学
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DOI
PubMed
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
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2024
·Vol. 90
·期 8
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IF 3.9
Yating Li,Tong Xu,Yanqin Tu,Tong Li,Yifeng Wei,Yan Zhou
Phloroglucinol (1,3,5-trihydroxybenzene) is a key intermediate in the degradation of polyphenols such as flavonoids and hydrolysable tannins and can be used by certain bacteria as a carbon and energy source for growth. The identification of enzymes that participate in the fermentation of phloroglucinol to acetate and butyrate in Clostridia was recently reported. In this study, we present the discovery and characterization of a novel metabolic pathway for phloroglucinol degradation in the bacterium Collinsella sp. zg1085, from marmot respiratory tract. In both the Clostridial and Collinsella pathways, phloroglucinol is first reduced to dihydrophoroglucinol by the NADPH-dependent phloroglucinol reductase (PGR), followed by ring opening to form (S)-3-hydroxy-5-oxohexanoate by a Mn2+-dependent dihydrophloroglucinol cyclohydrolase (DPGC). In the Collinsella pathway, (S)-3-hydroxy-5-oxohexanoate is then cleaved to form malonate semialdehyde and acetone by a newly identified aldolase (HOHA). Finally, a NADP+-dependent malonate-semialdehyde dehydrogenase converts malonate semialdehyde to CO2 and acetyl-CoA, an intermediate in carbon and energy metabolism. Recombinant expression of the Collinsella PGR, DPGC, and HOHA in E. coli enabled the conversion of phloroglucinol into acetone, providing support for the proposed pathway. Experiments with Olsenella profusa, another bacterium containing the gene cluster of interest, show that the PGR, DPGC, HOHA, and MSDH are induced by phloroglucinol. Our findings add to the variety of metabolic pathways for the degradation of phloroglucinol, a widely distributed phenolic compound, in the anaerobic microbiome.
研究领域:
微生物学
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生物化学
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代谢途径
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酶学
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产品关联
DOI
PubMed
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
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2024
·Vol. 90
·期 9
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IF 3.9
Yuan Li,Shu-Fang Li,Lin Zhang,Ya-Ping Xue,Yu-Guo Zheng
Hydroxysteroid dehydrogenases (HSDHs) catalyze the oxidation/reduction of hydroxyl/keto groups of steroids with high regio- or stereoselectivity, playing an essential role in producing optically pure chemicals. In this work, a novel approach was developed to simultaneously improve the stability and activity of 7β-hydroxysteroid dehydrogenase (7β-HSDH) by combining B-factor analysis and computer-aided prediction. Several advantageous mutants were identified, and the most promising variant, S51Y/P202Y, exhibited 2.3-fold improvements in catalytic activity, 3.3-fold in half-life at 40°C, and 4.7-fold in catalytic efficiency (kcat/Km), respectively. Structural modeling analysis showed that the shortened reversible oxidation reaction catalytic distance and the strengthened residue interactions compared to the wild type were attributed to the improved stability and activity of the obtained mutants. To synthesize ursodeoxycholic acid cost-effectively by mutant S51Y/P202Y, a NAD-kinase was employed to facilitate the substitution of nicotinamide adenine dinucleotide phosphate (NADP+) with nicotinamide adenine dinucleotide (NAD+) in the whole-cell catalysis system. The substrate 7-ketolithocholic acid (100 mM) was converted completely in 0.5 h, achieving a space-time yield of 1,887.3 g L−1 d−1. This work provided a general target-oriented strategy for obtaining stable and highly active dehydrogenase for efficient biosynthesis.
研究领域:
生物技术
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酶工程
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生物化学
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化学生物学
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产品关联
DOI
PubMed
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
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2024
·Vol. 90
·期 9
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IF 3.9
Wenmin Chai,Xianbing Mao,Chunfeng Li,Liancai Zhu,Zongyi He,Bochu Wang
Parasites can manipulate host behavior to facilitate parasite transmission. One such host–pathogen interaction occurs between the fungus Ophiocordyceps sinensis and the ghost moth Thitarodes xiaojinensis. O. sinensis is involved in the mummification process of infected host larvae. However, the underlying molecular and chemical mechanism for this phenomenon is unknown. We characterized the small molecules regulating host behaviors and the altered metabolites in infected and mummified host larvae. Lipid-related metabolites, such as phosphatidylcholine, were identified in infected and mummified larvae. Decreased levels of the neurotransmitter acetylcholine (ACh) and elevated choline levels were observed in the brains of both the infected and mummified larvae. The aberrant activity of acetylcholinesterase (AChE) and relative mRNA expression of ACE2 (acetylcholinesterase) may mediate the altered transformation between ACh and choline, leading to the brain dysfunction of mummified larvae. Caspofungin treatment inhibited the mummification of infected larvae and the activity of AChE. These findings indicate the importance of ACh in the mummification of host larvae after O. sinensis infection.
研究领域:
真菌学
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神经生物学
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代谢组学
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DOI
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
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2024
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IF 3.9
Xuemeng Ji,Dancai Fan,Jin Wang,Bowei Zhang,Yaozhong Hu,Huan Lv,Jing Wu,Yi Sun,Jingmin Liu,Yan Zhang,Shuo Wang
Cronobacter sakazakii poses a significant threat, particularly to neonates and infants. Despite its strong pathogenicity, understanding of C. sakazakii biofilms and their role in infections remains limited. This study investigates the roles of HmsP and c-di-GMP in biofilm formation and identifies key genetic and proteomic elements involved. Gene knockout experiments reveal that HmsP and c-di-GMP are linked to biofilm formation in C. sakazakii. Comparative proteomic profiling identifies the lysozyme inhibitor protein LprI, which is downregulated in hmsP knockouts and upregulated in c-di-GMP knockouts, as a potential biofilm formation factor. Further investigation of the lprI knockout strain shows significantly reduced biofilm formation and decreased virulence in a rat infection model. Additionally, LprI is demonstrated to bind extracellular DNA, suggesting a role in anchoring C. sakazakii within the biofilm matrix. These findings enhance our understanding of the molecular mechanisms underlying biofilm formation and virulence in C. sakazakii, offering potential targets for therapeutic intervention and food production settings.
研究领域:
微生物学
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分子生物学
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感染病学
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DOI
PubMed
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
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2023
·Vol. 89
·期 4
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IF 4.4
Xuling Jiang,Wanqing Wei,Yaozhong Cui,Wei Song,Yingying Li,Xiulai Chen,Cong Gao,Jia Liu,Liang Guo,Liming Liu,Jing Wu
Pyrrolidone is a high value-added monomer and an important active drug intermediate. However, the efficient enzymatic synthesis of pyrrolidone remains a challenge. Here, we developed and reconstructed a three-enzyme cascade pathway using Escherichia coli BL21(DE3) for the production of pyrrolidone from l-glutamate (l-Glu). The carnitine-CoA ligase from Escherichia coli (EcCaiC) at a low expression level and with a low activity is regarded as the rate-limiting enzyme. Here, we obtained the best EcCaiCF380M/N430D double mutant with a kcat/Km value 1.5 times higher than that of the wild type via mechanism-based protein engineering. For this, we (i) eliminated the steric hindrance of the loop ring to improve the precatalytic conformation of the adenylation intermediate and (ii) fixed the hinge region to stabilize the closed conformation of the enzyme. Furthermore, ribosome-binding site (RBS) optimization led to an increase in the expression level of EcCaiCF380M/N430D, which was then cloned into the plasmid pET-EcCaiCF380M/N430D-DegoPPK2. Finally, under optimal induction and transformation conditions, 16.62 g/L of pyrrolidone was generated from 30 g/L l-Glu (batch feeding) within 24 h with a molar conversion rate of 95.2% and the highest productivity ever obtained, to our knowledge (0.69 g/L/h). Our findings demonstrate a strategy that is potentially attractive for the industrial production of pyrrolidone.IMPORTANCE This study developed a three-enzyme cascade pathway for the production of pyrrolidone from l-Glu. The catalytic efficiency of carnitine CoA ligase from Escherichia coli (EcCaiC) was improved by mechanism-based protein engineering, and the titer of pyrrolidone was further increased by ribosome-binding site (RBS), induction conditions, and conversion conditions optimization. Finally, we efficiently produced pyrrolidone by one pot in vivo with 95.2% conversion and 0.69 g/L/h productivity. Our study provides a new possibility for the industrial production of enzymatic synthesis of pyrrolidone.
研究领域:
生物化学
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合成生物学
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酶工程
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DOI
PubMed
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
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2017
·Vol. 83
·期 10
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IF 3.807
Yaojie Gao,Gudan Xu,Pan Wu,Jin Liu,You-sheng Cai,Zixin Deng,Wenqing Chen
2′-Chloropentostatin (2′-Cl PTN, 2′-chloro-2′-deoxycoformycin) and 2′-amino-2′-deoxyadenosine (2′-amino dA) are two adenosine-derived nucleoside antibiotics coproduced by Actinomadura sp. strain ATCC 39365. 2′-Cl PTN is a potent adenosine deaminase (ADA) inhibitor featuring an intriguing 1,3-diazepine ring, as well as a chlorination at C-2′ of ribose, and 2′-amino dA is an adenosine analog showing bioactivity against RNA-type virus infection. However, the biosynthetic logic of them has remained poorly understood. Here, we report the identification of a single gene cluster (ada) essential for the biosynthesis of 2′-Cl PTN and 2′-amino dA. Further systematic genetic investigations suggest that 2′-Cl PTN and 2′-amino dA are biosynthesized by independent pathways. Moreover, we provide evidence that a predicted cation/H+ antiporter, AdaE, is involved in the chlorination step during 2′-Cl PTN biosynthesis. Notably, we demonstrate that 2′-amino dA biosynthesis is initiated by a Nudix hydrolase, AdaJ, catalyzing the hydrolysis of ATP. Finally, we reveal that the host ADA (designated ADA1), capable of converting adenosine/2′-amino dA to inosine/2′-amino dI, is not very sensitive to the powerful ADA inhibitor pentostatin. These findings provide a basis for the further rational pathway engineering of 2′-Cl PTN and 2′-amino dA production.IMPORTANCE 2′-Cl PTN/PTN and 2′-amino dA have captivated the great interests of scientists, owing to their unusual chemical structures and remarkable bioactivities. However, the precise logic for their biosynthesis has been elusive for decades. Actually, the identification and elucidation of their biosynthetic pathways not only enrich the biochemical repertoire of novel enzymatic reactions but may also lay solid foundations for the pathway engineering and combinatorial biosynthesis of this family of purine nucleoside antibiotics to generate novel hybrid analogs with improved features.
研究领域:
微生物学
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分子生物学
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药物化学
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DOI
PubMed
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
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2019
·Vol. 85
·期 12
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IF 4.077
Xinyu Lu,Weiwei Wang,Lige Zhang,Haiyang Hu,Ping Xu,Tao Wei,Hongzhi Tang
N,N-Dimethylformamide (DMF) is one of the most common xenobiotic chemicals, and it can be easily emitted into the environment, where it causes harm to human beings. Herein, an efficient DMF-degrading strain, DM1, was isolated and identified as Methylobacterium sp. This strain can use DMF as the sole source of carbon and nitrogen. Whole-genome sequencing of strain DM1 revealed that it has a 5.66-Mbp chromosome and a 200-kbp megaplasmid. The plasmid pLVM1 specifically harbors the genes essential for the initial steps of DMF degradation, and the chromosome carries the genes facilitating subsequent methylotrophic metabolism. Through analysis of the transcriptome sequencing data, the complete mineralization pathway and redundant gene clusters of DMF degradation were elucidated. The dimethylformamidase (DMFase) gene was heterologously expressed, and DMFase was purified and characterized. Plasmid pLVM1 is catabolically crucial for DMF utilization, as evidenced by the phenotype identification of the plasmid-free strain. This study systematically elucidates the molecular mechanisms of DMF degradation by Methylobacterium.IMPORTANCE DMF is a hazardous pollutant that has been used in the chemical industry, pharmaceutical manufacturing, and agriculture. Biodegradation as a method for removing DMF has received increasing attention. Here, we identified an efficient DMF degrader, Methylobacterium sp. strain DM1, and characterized the complete DMF mineralization pathway and enzymatic properties of DMFase in this strain. This study provides insights into the molecular mechanisms and evolutionary advantage of DMF degradation facilitated by plasmid pLVM1 and redundant genes in strain DM1, suggesting the emergence of new ecotypes of Methylobacterium.
研究领域:
环境微生物学
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生物化学
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DOI
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
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2022
·Vol. 88
·期 6
·
IF 5.005
Ke Huang,Wei Liu,Yuanhe Li,Sha Zeng,Fang-Jie Zhao
Methylarsenate (MAs(V)) is a product of microbial arsenic (As) biomethylation and has also been widely used as an herbicide. Some microbes are able to reduce nontoxic MAs(V) to highly toxic methylarsenite (MAs(III)) possibly as an antibiotic. The mechanism of MAs(V) reduction in microbes has not been elucidated. Here, we found that the bacterium Enterobacter sp. CZ-1 isolated from an As-contaminated paddy soil has a strong ability to reduce MAs(V) to MAs(III). Using a MAs(III)-responsive biosensor to detect MAs(V) reduction in E. coli Trans5α transformants of a genomic library of Enterobacter sp. CZ-1, we identified gshA, encoding a glutamate-cysteine ligase, as a key gene involved in MAs(V) reduction. Heterologous expression of gshA increased the biosynthesis of glutathione (GSH) and MAs(V) reduction in E. coli Trans5α. Deletion of gshA in Enterobacter sp. CZ-1 abolished its ability to synthesize GSH and decreased its MAs(V) reduction ability markedly, which could be restored by supplementation of exogenous GSH. In the presence of MAs(V), Enterobacter sp. CZ-1 was able to inhibit the growth of Bacillus subtilis 168; this ability was lost in the gshA-deleted mutant. In addition, deletion of gshA greatly decreased the reduction of arsenate to arsenite. These results indicate that GSH plays an important role in MAs(V) reduction to generate MAs(III) as an antibiotic.IMPORTANCE Arsenic is a ubiquitous environmental toxin. Some microbes detoxify inorganic arsenic through biomethylation, generating relatively nontoxic pentavalent methylated arsenicals, such as methylarsenate. Methylarsenate has also been widely used as an herbicide. Surprisingly, some microbes reduce methylarsenate to highly toxic methylarsenite possibly to use the latter as an antibiotic. How microbes reduce methylarsenate to methylarsenite is unknown. Here, we show that gshA encoding a glutamate-cysteine ligase in the glutathione biosynthesis pathway is involved in methylarsenate reduction in Enterobacter sp. CZ-1. Our study provides new insights into the crucial role of glutathione in the transformation of a common arsenic compound to a natural antibiotic.
研究领域:
微生物学
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环境科学
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毒理学
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DOI
PubMed
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
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2019
·Vol. 85
·期 23
·
IF 4.077
Bingbing Ma,Qianwen Wang,Haruo Ikeda,Chunfang Zhang,Lian-Hua Xu
Our previous study showed that CYP105D7, a substrate-promiscuous P450, catalyzes the hydroxylation of 1-deoxypentalenic acid, diclofenac, naringenin, and compactin. In this study, 14 steroid compounds were screened using recombinant Escherichia coli cells harboring genes encoding CYP105D7 and redox partners (Pdx/Pdr, RhFRED, and FdxH/FprD), and the screening identified steroid A-ring 2β- and D-ring 16β-hydroxylation activity. Wild-type CYP105D7 was able to catalyze the hydroxylation of five steroids (testosterone, progesterone, 4-androstene-3,17-dione, adrenosterone, and cortisone) with low (<10%) conversion rates. Structure-guided site-directed mutagenesis of arginine residues around the substrate entrance and active site showed that the R70A and R190A single mutants and an R70A/R190A double mutant exhibited greatly enhanced conversion rates for steroid hydroxylation. For the conversion of testosterone in particular, the R70A/R190A mutant's kcat/Km values increased 1.35-fold and the in vivo conversion rates increased significantly by almost 9-fold with high regio- and stereoselectivity. Molecular docking analysis revealed that when Arg70 and Arg190 were replaced with alanine, the volume of the substrate access and binding pocket increased 1.08-fold, which might facilitate improvement of the hydroxylation efficiency of steroids.IMPORTANCE Cytochrome P450 monooxygenases (P450s) are able to introduce oxygen atoms into nonreactive hydrocarbon compounds under mild conditions, thereby offering significant advantages compared to chemical catalysts. Promiscuous P450s with broad substrate specificity and reaction diversity have significant potential for applications in various fields, including synthetic biology. The study of the function, molecular mechanisms, and rational engineering of substrate-promiscuous P450s from microbial sources is important to fulfill this potential. Here, we present a microbial substrate-promiscuous P450, CYP105D7, which can catalyze hydroxylation of steroids. The loss of the bulky side chains of Arg70 and Arg190 in the active site and substrate entrance resulted in an up to 9-fold increase in the substrate conversion rate. These findings will support future rational and semirational engineering of P450s for applications as biocatalysts.
研究领域:
生物化学
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合成生物学
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酶工程
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DOI
PubMed
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
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2016
·Vol. 82
·期 2
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IF 3.823
Hong-Jun Chao,Yan-Fei Chen,Ti Fang,Ying Xu,Wei E. Huang,Ning-Yi Zhou
In addition to growing on p-cresol, Pseudomonas putida NCIMB 9866 is the only reported strain capable of aerobically growing on 2,4-xylenol, which is listed as a priority pollutant by the U.S. Environmental Protection Agency. Several enzymes involved in the oxidation of the para-methyl group, as well as the corresponding genes, have previously been reported. The enzyme catalyzing oxidation of the catabolic intermediate 4-hydroxyisophthalate to the ring cleavage substrate protocatechuate was also purified from strain NCIMB 9866, but its genetic determinant is still unavailable. In this study, the gene hipH, encoding 4-hydroxyisophthalate hydroxylase, from strain NCIMB 9866 was cloned by transposon mutagenesis. Purified recombinant HipH-His6 was found to be a dimer protein with a molecular mass of approximately 110 kDa. HipH-His6 catalyzed the hydroxylation of 4-hydroxyisophthalate to protocatechuate with a specific activity of 1.54 U mg−1 and showed apparent Km values of 11.40 ± 3.05 μM for 4-hydroxyisophthalate with NADPH and 11.23 ± 2.43 μM with NADH and similar Km values for NADPH and NADH (64.31 ± 13.16 and 72.76 ± 12.06 μM, respectively). The identity of protocatechuate generated from 4-hydroxyisophthalate hydroxylation by HipH-His6 has also been confirmed by high-performance liquid chromatography and mass spectrometry. Gene transcriptional analysis, gene knockout, and complementation indicated that hipH is essential for 2,4-xylenol catabolism but not for p-cresol catabolism in this strain. This fills a gap in our understanding of the gene that encodes a critical step in 2,4-xylenol catabolism and also provides another example of biochemical and genetic diversity of microbial catabolism of structurally similar compounds.
研究领域:
微生物学
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环境科学
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生物化学
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遗传学
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DOI
PubMed
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
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2023
·Vol. 89
·期 7
·
IF 4.4
Lu Zhang,Xiaoqing Xu,Yunfei Sun,Yuan Huang,Zhou Yang
Heavy metal pollution in waters causes serious stress to aquatic ecosystems. Several autotrophs with strong tolerance are extensively used to adsorb heavy metals, but their use may be limited by the specific conditions of polluted waters due to their single nutrition mode. By contrast, mixotrophs possess strong environmental adaptability due to their plastic metabolic modes. However, studies focusing on mixotroph’s resistance and its underlying mechanism in response to heavy metals and their bioremediation potentials are currently lacking. In this study, we investigated the population, phytophysiological, and transcriptomic (RNA-Seq) responses of a typical and common mixotrophic organism, Ochromonas, to cadmium exposure and then evaluated their capacity to remove cadmium under mixotrophic condition. Compared with autotrophy, mixotrophic Ochromonas enhanced photosynthetic performances under short-time cadmium exposure and subsequently shifted to stronger resistance with increasing exposure time. Transcriptomic analyses suggested that the genes related to photosynthesis, ATP production, ECM components, and scavenging of reactive oxygen species and damaged organelles were upregulated to boost mixotrophic Ochromonas resistance to cadmium. Consequently, the harm of metal exposure was eventually reduced and cellular stability was maintained. Approximately, 70% of cadmium at 2.4 mg L−1 cadmium was removed by mixotrophic Ochromonas in the end, benefiting from upregulated genes associated with the transport of metal ions. Hence, mixotrophic Ochromonas tolerance to cadmium can be attributed to multiple pathways of energy metabolism and effective transport of metal ions. Collectively, this study advanced a better understanding of the unique mechanism of heavy metal resistance in mixotrophs and their potential use in recovering cadmium-contaminated aquatic ecosystems.IMPORTANCE Mixotrophs widely living in aquatic ecosystems possess unique ecological roles and strong environmental adaptability due to their plastic metabolic modes; however, little is known about their underlying resistance mechanism and bioremediation potential in response to environmental stresses. For the first time, this work investigated how mixotrophs respond to metal pollutants through physiological, population dynamics, and transcriptional regulation, and highlighted the unique underlying mechanism of mixotrophs to resist and remove heavy metal, thereby advancing our understanding of the potentials of mixotrophs in recovering metal-contaminated aquatic environments. These unique properties in mixotrophs are essential for the long-term functional stability of aquatic ecosystems.
研究领域:
环境科学
·
分子生物学
✓ 已验证
DOI
高影响因子