INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
·
2016
·Vol. 17
·期 4
·493
·
IF 3.257
Yongqiang Li,Jianwei Liu,Mei Lu,Zhiqing Ma,Chongling Cai,Yonghong Wang,Xing Zhang
Carboxylesterasesare an important class of detoxification enzymes involved in insecticide resistance in insects. A subgroup ofHelicoverpa armigeraesterases, known as Clade 001, was implicated in organophosphate and pyrethroid insecticide resistance due to their overabundance in resistant strains. In this work, a novel carboxylesterasegene001DofH. armigerafrom China was cloned, which has an open reading frame of 1665 nucleotides encoding 554 amino acid residues. We used a series of fusion proteins to successfully express carboxylesterase 001D inEscherichia coli. Three different fusion proteins were generated and tested. The enzyme kinetic assay towards 1-naphthyl acetate showed all three purified fusion proteins are active with aKcat between 0.35 and 2.29 s−1, and aKm between 7.61 and 19.72 μM. The HPLC assay showed all three purified fusion proteins had low but measurable hydrolase activity towardsβ-cypermethrin and fenvalerate insecticides (specific activities ranging from 0.13 to 0.67 μM·min−1·(μM−1·protein)). The enzyme was stable up to 40 °C and at pH 6.0–11.0. The results imply that carboxylesterase 001D is involved in detoxification, and this moderate insecticide hydrolysis may suggest that overexpression of the gene to enhance insecticide sequestration is necessary to allow carboxylesterases to confer resistance to these insecticides inH. armigera.Keywords:Helicoverpaarmigera;carboxylesterase;pyrethroid;heterologous expression
研究领域:
昆虫学
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分子生物学
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DOI
PubMed
高影响因子
PeerJ
·
2025
·
IF 2.4
Shuping Zhang,Zhenhua Liu,Jia Li,Qian Liu,Yongqing Zhang,Gaobin Pu
Background Geraniol 10-hydroxylase (G10H) is a cytochrome P450 monooxygenase involved in regulation, which is involved in the biosynthesis of monoterpene. However, G10H is not characterized at the enzymatic mechanism and regulatory function in Lonicera japonica . Methods and Results A gene related to the biosynthesis of monoterpenoid, geraniol 10-hydroxylase, has been cloned from the medicinal plant Lonicera japonica . The gene, LjG10H , encodes a peptide of 498 amino acids with a predicted molecular weight of 54.45 kDa. LjG10H shares a homology of 72.93–83.90% with G10H from other plants. Phylogenetic analysis suggests that the protein encoded by this gene belongs to the cytochrome P450 monooxygenase family. Tissue-specific expression analysis revealed that LjG10H is most highly expressed in flowers. Through heterologous expression in E. coli , the LjG10H protein was purified and its catalytic activity was studied. The results show that the enzyme can catalyze the hydroxylation of geraniol to 10-hydroxygeraniol. Additionally, analysis of Lonicera japonica seedlings with silenced LjG10H revealed a reduction in monoterpenoid content. Conclusions This study demonstrates that LjG10H plays an important role in the biosynthetic pathway of iridoids. This is the first article that ascribes G10H to be associated with the biosynthetic pathway of iridoid. This study provides a theoretical basis for the functional mechanism of LjG10H in regulating iridoid synthesis and provides a valuable resource for molecular breeding studies.
研究领域:
植物学
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分子生物学
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药用植物研究
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DOI
PubMed
高影响因子
Metabolites
·
2025
·Vol. 15
·期 1
·45
·
IF 3.7
Zhe Liu,Fengxu Xiao,Yupeng Zhang,Jiawei Lu,Youran Li,Guiyang Shi
Purpose: This study aimed to utilize genetically engineeredBacillus licheniformisfor the production of ergothioneine (EGT). Given the value of EGT and the application ofBacillus licheniformisin enzyme preparation production, we cloned the key enzymes (EanA and EanB) fromChlorbium limicola. Through gene alignment, new ergothioneine synthase genes (EanAN and EanBN) were identified and then expressed inBacillus licheniformisto construct strains. Additionally, we investigated the factors influencing the yield of EGT and made a comparison withEscherichia coli. Methods: The relevant genes were cloned and transferred intoBacillus licheniformis. Fermentation experiments were conducted under different conditions for yield analysis, and the stability of this bacterium was also evaluated simultaneously. Results: The constructed strains were capable of producing EGT. Specifically, the yield of the EanANBN strain reached (643.8 ± 135) mg/L, and its stability was suitable for continuous production. Conclusions: Genetically engineeredBacillus licheniformisdemonstrates potential in the industrial-scale production of EGT. Compared withEscherichia coli, it has advantages, thus opening up new possibilities for the application and market supply of EGT.
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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DOI
PubMed
高影响因子
PEST MANAGEMENT SCIENCE
·
2022
·Vol. 78
·期 5
·2052-2064
·
IF 4.462
Hao Guo,Xin-Lin Gong,Guo-Cheng Li,Bao-Tong Mo,Nan-Ji Jiang,Ling-Qiao Huang,Chen-Zhu Wang
BACKGROUND The fall armyworm, Spodoptera frugiperda (J. E. Smith), is a polyphagous moth species that is spreading all around the globe. It uses ( Z )-9-tetradecenyl acetate (Z9-14:Ac) and ( Z )-7-dodecenyl acetate ( Z 7-12:Ac) (100:3.9) as essential sex pheromone components. However, our understanding of the molecular basis of pheromone detection of S. frugiperda is still incomplete. RESULTS Herein, we identified six PRs , i.e. SfruOR6 , 11 , 13 , 16 , 56 , and 62 , by transcriptome sequencing. Subsequently, we heterologously expressed them in Drosophila OR67d neurons and determined their response spectra with a large panel of sex pheromones and analogs. Among them, SfruOR13-expressing neurons strongly respond to the major sex pheromone component Z9-14:Ac, but also comparably to ( Z , E )-9,12-tetradecadienyl acetate (Z9,E12-14:Ac) and weakly to ( Z )-9-dodecenyl acetate (Z9-12:Ac). Both SfruOR56 and SfruOR62 are specifically tuned to the minor sex pheromone component Z7-12:Ac with varying intensities and sensitivities. In addition, SfruOR6 is activated only by Z9,E12-14:Ac, and SfruOR16 by both ( Z )-9-tetradecenol (Z9-14:OH) and ( Z )-9-tetradecenal (Z9-14:Ald). However, the OR67d neurons expressing SfruOR11 remain silent to all compounds tested, a phenomenon commonly found in the OR11 clade of Noctuidae species. Next, using single sensillum recording, we characterized four sensilla types on the antennae of males, namely A, B, C and D types that are tuned to the ligands of PRs, thereby confirming that S. frugiperda uses both SfruOR56 and SfruOR62 to detect Z7-12:Ac. Finally, using wind tunnel assay, we demonstrate that both Z9,E12-14:Ac and Z9-14:OH act as antagonists to the sex pheromone. CONCLUSION We have deorphanized five PRs and characterized four types of sensilla responsible for the detection of pheromone compounds, providing insights into the peripheral encoding of sex pheromones in S. frugiperda .
研究领域:
昆虫学
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分子生物学
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DOI
PubMed
高影响因子
Bioresources and Bioprocessing
·
2024
·Vol. 11
·期 1
·1-12
·
IF 4.3
Fu Xin,Lin Kexin,Zhang Xiaodong,Guo Zhiyong,Kang Lixin,Li Aitao
Unspecific peroxygenases (UPOs) are glycosylated enzymes that provide an efficient method for oxyfunctionalizing a variety of substrates using only hydrogen peroxide (H2O2) as the oxygen donor. However, their poor heterologous expression has hindered their practical application. Here, a novel UPO from Marasmius fiardii PR910 (MfiUPO) was identified and heterologously expressed in Pichia pastoris. By employing a two-copy expression cassette, the protein titer reached 1.18 g L−1 in a 5 L bioreactor, marking the highest record. The glycoprotein rMfiUPO exhibited a smeared band in the 40 to 55 kDa range and demonstrated hydroxylation, epoxidation and alcohol oxidation. Moreover, the peroxidative activity was enhanced by 150% after exposure to 50% (v/v) acetone for 40 h. A semi-preparative production of 4-OH-β-ionone on a 100 mL scale resulted in a 54.2% isolated yield with 95% purity. With its high expression level, rMfiUPO is a promising candidate as an excellent parental template for enhancing desirable traits such as increased stability and selectivity through directed evolution, thereby meeting the necessary criteria for practical application.
研究领域:
生物化学
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酶工程
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合成生物学
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DOI
PubMed
高影响因子
MOLECULES
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2022
·Vol. 27
·期 15
·4720
·
IF 4.927
Xinjun Yu,Zengchao Jiang,Xiaodan Xu,Changyi Huang,Zheyi Yao,Xiao Yang,Yinjun Zhang,Dongsheng Wang,Chun Wei,Xuwei Zhuang
Chitin, the second richest polymer in nature, is composed of the monomer N-acetylglucosamine (GlcNAc), which has numerous functions and is widely applied in the medical, food, and chemical industries. However, due to the highly crystalline configuration and low accessibility in water of the chitin resources, such as shrimp and crab shells, the chitin is difficult utilize, and the traditional chemical method causes serious environment pollution and a waste of resources. In the present study, three genes encoding chitinolytic enzymes, including the N-acetylglucosaminidase fromOstrinia furnacalis(OfHex1), endo-chitinase fromTrichoderma viride(TvChi1), and multifunctional chitinase fromChitinolyticbacter meiyuanensis(CmChi1), were expressed in thePichia pastorissystem, and the positive transformants with multiple copies were isolated by the PTVA (post-transformational vector amplification) method, respectively. The three recombinants OfHex1, TvChi1, and CmChi1 were induced by methanol and purified by the chitin affinity adsorption method. The purified recombinants OfHex1 and TvChi1 were characterized, and they were further used together for degrading chitin from shrimp and crab shells to produce GlcNAc through liquid-assisted grinding (LAG) under a water-less condition. The substrate chitin concentration reached up to 300 g/L, and the highest yield of the product GlcNAc reached up to 61.3 g/L using the mechano-enzymatic method. A yield rate of up to 102.2 g GlcNAc per 1 g enzyme was obtained.Keywords:chitinase;shrimp and crab shell;N-acetylglucosamine;mechanoenzymology;heterologous expression
研究领域:
生物化学
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生物技术
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环境科学
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DOI
PubMed
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2024
·
IF 5.7
Chen-Lu Xu,Chen-Yuan Zhu,Yang-Nan Li,Jian Gao,Ye-Wang Zhang
A novel heparinase III from Pedobacter schmidteae (PsHep-III) with high activity and good stability was successfully cloned, expressed, and characterized. PsHep-III displayed the highest specific activity ever reported of 192.8 U mg–1 using heparin as the substrate. It was stable at 25 °C with a half-life of 323 h in an aqueous solution. PsHep-III was employed for the depolymerization of heparin, and the enzymatic hydrolyzed products were analyzed with gel permeation chromatography and high-performance liquid chromatography. PsHep-III can break glycosidic bonds in heparin like →4]GlcNAc/GlcNAc6S/GlcNS/GlcNS6S/GlcN/GlcN6S(1 → 4)ΔUA/ΔUA2S[1 → and efficiently digest heparin into seven disaccharides including N-acetylated, N-sulfated, and N-unsubstituted modification, with molecular masses of 503, 605, 563, 563, 665, 360, and 563 Da, respectively. These results indicated that PsHep-III with broad substrate specificity could be combined with heparinase I to overcome the low selectivity at the N-acetylated modification binding sites of heparinase I. This work will contribute to the application of PsHep-III for characterizing heparin and producing low-molecular-weight heparin effectively.
研究领域:
生物化学
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分子生物学
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制药工程
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DOI
PubMed
高影响因子
Frontiers in Microbiology
·
2018
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IF 4.019
Peng Qinying,Gao Guixi,Lü Jin,Long Qingshan,Chen Xuefei,Zhang Fei,Xu Min,Liu Kai,Wang Yemin,Deng Zixin,Li Zhiyong,Tao Meifeng
Streptomyces lividans is a suitable host for the heterologous expression of biosynthetic gene clusters (BGCs) from actinomycetes to discover “cryptic” secondary metabolites. To improve the heterologous expression of BGCs, herein we optimized S. lividans strain SBT5 via the stepwise integration of three global regulatory genes and two codon-optimized multi-drug efflux pump genes and deletion of a negative regulatory gene, yielding four engineered strains. All optimization steps were observed to promote the heterologous production of polyketides, non-ribosomal peptides, and hybrid antibiotics. The production increments of these optimization steps were additional, so that the antibiotic yields were several times or even dozens of times higher than the parent strain SBT5 when the final optimized strain, S. lividans LJ1018, was used as the heterologous expression host. The heterologous production of these antibiotics in S. lividans LJ1018 and GX28 was also much higher than in the strains from which the BGCs were isolated. S. lividans LJ1018 and GX28 markedly promoted the heterologous production of secondary metabolites, without requiring manipulation of gene expression components such as promoters on individual gene clusters. Therefore, these strains are well-suited as heterologous expression hosts for secondary metabolic BGCs. In addition, we successfully conducted high-throughput library expression and functional screening (LEXAS) of one bacterial artificial chromosome library and two cosmid libraries of three Streptomyces genomes using S. lividans GX28 as the library-expression host. The LEXAS experiments identified clones carrying intact BGCs sufficient for the heterologous production of piericidin A1, murayaquinone, actinomycin D, and dehydrorabelomycin. Notably, due to lower antibiotic production, the piericidin A1 BGC had been overlooked in a previous LEXAS screening using S. lividans SBT5 as the expression host. These results demonstrate the feasibility and superiority of S. lividans GX28 as a host for high-throughput screening of genomic libraries to mine cryptic BGCs and bioactive compounds.
研究领域:
微生物学
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合成生物学
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药物开发
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DOI
PubMed
高影响因子
LWT-FOOD SCIENCE AND TECHNOLOGY
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2022
·
IF 6.056
Shuangping Liu,Hongli Yao,Mengfei Sun,Zhilei Zhou,Jian Mao
High content of biogenic amines in fermented food has adverse effects on the human body. Previous studies showed that biogenic amines could be reduced in some fermented foods using enzymes. However, this process was barely explored in alcoholic beverages. In this study, potential biogenic amine degarding genes were cloned from Saccharopolyspora hirsuta F1902 and Saccharopolyspora hordei F2002, and expressed in E. coli to study the enzymatic activities of copper-containing amine oxidases named as CaoA Shi and CaoA Sho in huangjiu . The degradation ratio of total biogenic amines by CaoA Shi (31.72%) was higher than that of CaoA Sho (24.37%). CaoA Shi and CaoA Sho had the highest degradation ratios of histamine, phenylethylamine, followed by tyramine, tryptamine or putrescine, and the smallest was cadaverine. The results indicated that CaoA Shi had better biodegradability of biogenic amines than CaoA Sho in huangjiu (P < 0.05), and the two enzymes had the same amine substrate characteristics. These results provide new insights for the reduction of biogenic amines using amine oxidases, which is useful not only for huangjiu brewing industry, but also for other fermented food industries.
研究领域:
食品科学
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微生物学
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酶学
·
饮料技术
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DOI
高影响因子
ACS Biomaterials Science & Engineering
·
2023
·
IF 5.8
Chang Liu,Qing Yang,Hong Lin,Limin Cao,Kaiqiang Wang,Jianxin Sui
Nowadays, obesity severely impacts human health and is the fifth leading risk factor that leads to death globally. Pancreatic lipase (PL) inhibitors have attracted extensive attention owing to their role in effective prevention and treatment of obesity. Here, a shark-derived single-domain antibody fusion protein was used to inhibit PL for the first time. After biopanning, the heterologous expression system pET28a-SUMO-D2 was constructed using the method of double restriction enzyme digestion and T4 ligase to achieve the soluble expression of the PL-specific antibody gene D2. According to the calculation of protein concentration, the final expression of fusion protein PL-D2S was 1.183 mg per liter of Luria Bertani broth. The binding ability of the soluble fusion protein PL-D2S to PL was identified. Enzyme-linked immunosorbent assay results showed that the fusion protein PL-D2S exhibited a strong binding affinity to PL. The experimental results of PL inhibition of PL-D2S in vitro showed that the fusion protein could significantly inhibit the activity of PL, with an IC50 of 404 μg/mL. Our study shows that the fusion protein PL-D2S is a potential PL inhibitor to prevent and treat obesity.
研究领域:
分子生物学
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生物化学
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药学
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DOI
PubMed
高影响因子
STARCH-STARKE
·
2025
·
IF 2.7
Zixuan Lin,Tingting Hu,Lin Lin,Tao Li,Yan Yan,Wei Wei,Dongzhi Wei
Although α-amylases are critical biocatalysts in starch processing, fungal α-amylase diversity remains largely untapped. This study presents the first systematic characterization of an α-amylase from Penicillium brevicompactum NRRL864. Maximum α-amylase activity of 195 U/mL was obtained at pH 6.0 and 55°C under optimized fermentation conditions. These conditions consisted of 0.75% starch, 3% peptone, 0.5% NaCl, incubation at 20°C for 7 days. A novel α-amylase gene (DF) was identified via phylogenetic analysis and heterologously expressed in Escherichia coli . The gene encodes a 551-amino-acid α-amylase with an open reading frame of 1656 bp, showing 68.36% sequence identity to the α-amylase from Penicillium expansum . The DF enzyme displayed a specific activity of 489.4 U/mg under optimal conditions of 50°C and pH 6.0, and a 124.2% activity enhancement with 1 mM Ca 2 ⁺, indicating Ca 2 ⁺-dependence. The activity of DF was inhibited by a range of chemical reagents, including EDTA and urea. Substrate specificity assays confirmed that the DF enzyme exhibited optimal activity in hydrolyzing soluble starch. DF-mediated saccharification of corn starch yielded a reducing sugar concentration of 317.9 µg/mL within 70 min, confirmed by thin-layer chromatography. Beyond identifying P. brevicompactum as a novel α-amylase source, this work demonstrates DF as a Ca 2 ⁺-responsive GH13 enzyme with significant potential for industrial applications in biofuel and sweetener production.
研究领域:
生物技术
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酶学
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微生物学
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工业生物催化
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分子生物学
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产品关联
DOI
高影响因子
TETRAHEDRON
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2022
·
IF 2.388
Li Rao,Hu-Cheng Shi,Yi Zou
A new fungal nonribosomal peptide-polyketide hybrid synthase , namely CogA, was discovered by genome mining from fungus Cordyceps gunnii . Its ability to synthesize 4-hydroxyl 2-pyrrolidinone alkaloid ( 1 ) via nonenzyme-catalyzed N-1-C-2 closure was confirmed by heterologous expression of it in Aspergillus nidulans . Further engineered biosynthesis of the fluoro-substituted 2-pyrrolidinone alkaloid analogues ( 4 and 5 ) has also been achieved in vivo .
研究领域:
真菌学
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合成生物学
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生物化学
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DOI
高影响因子
危险品化学品经营许可证（带存储）