Biotechnology Journal
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2023
·Vol. 18
·期 9
·2300027
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IF 4.7
Jian-Miao Xu,Zhou-Sheng Wu,Ke-Ji Zhao,Zhi-Jie Xi,Liu-Yu Wang,Feng Cheng,Ya-Ping Xue,Yu-Guo Zheng
Background Biocatalytic production of L-phosphinothricin (L-PPT) is currently the most promising method. In this work, we use an Escherichia coli strain coexpressing of D-amino acid oxidase and catalase ( E. coli DAAO-CAT) to oxidation biocatalytic D-PPT to PPO, then use the second E. coli strain coexpressing glutamate dehydrogenase and formate dehydrogenase ( E. coli GluDH-FDH) to reduce biocatalytic PPO to L-PPT. Main Methods and Major Results We compared the effects of different concentrations of IPTG or lactose on protein expression and enzyme activity in 5 L fermenter. The best induction conditions for E. coli DAAO-CAT were 0.05 mM IPTG, induction for 18 h at 28°C. The specific enzyme activities of DAAO and CAT were 153.20 U g −1 and 896.23 U g −1 , respectively. The optimal induction conditions for E. coli GluDH-FDH were 0.2 mM IPTG, induction for 19 h at 28°C. The specific enzyme activities of GluDH and FDH were 41.72 U g −1 and 109.70 U g −1 , respectively. The 200 mM D-PPT was biocatalyzed by E. coli DAAO-CAT for 4 h with space-time yield of 9.0 g·L −1 ·h −1 and conversion rate of over 99.0%. Then 220 mM PPO was converted to L-PPT by E. coli GluDH-FDH for 3 h with space-time yield of 14.5 g·L −1 ·h −1 and conversion rate of over 99.0%. To our knowledge, this is the most efficient biocatalytic reaction for L-PPT production. Conclusions and Implications We found that IPTG has advantages compared with lactose in the enzyme activity and biomass of E. coli DAAO-CAT and E. coli GluDH-FDH, and IPTG is more environmentally friendly. Our data implicated that IPTG can replace lactose in terms of economic feasibility and effectiveness for scaled-up industrial fermentations. Graphical and Lay Summary A two-step enzymatic method was developed for the biological preparation of L-PPT from D-PPT. In the first step, we constructed a recombinant E. coli BL21(DE3)/pCDFDuet-DAAO-CAT that utilized D-amino acid oxidase (DAAO) to catalyze the conversion of D-PPT to PPO, and catalase to degrade the hydrogen peroxide (H 2 O 2 ) produced during the reaction. Subsequently, in the second step, we constructed another recombinant E. coli BL21(DE3)/pETDuet-LcGluDH-PseFDH that employed glutamate dehydrogenase (GluDH) for the reductive amination of PPO to L-PPT, with NADH supplied by formate dehydrogenase (FDH).
研究领域:
生物化学
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微生物学
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PubMed
高影响因子
APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
·
2011
·Vol. 165
·期 2
·666-675
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IF 1.879
Chen Sheng,Liu Zhiguo,Chen Jian,Wu Jing
Escherichia coli is one of the most commonly used host strains for recombinant protein production. More and more research works on the production of recombinant protein indicate that extracellular production throughout a culture medium is more convenient and attractive compared to intracellular production. In present work, inducing temperature and isopropyl β- d -1-thiogalactopyranoside (IPTG) concentration were investigated to decrease the formation of inclusion body and increase the amount of soluble recombinant cutinase initially. Enzyme activity in the culture medium reached to 118.9 U/ml at 64 h of culture, and no inclusion body was detected in cytoplasm under the inducement condition of 0.2 mM IPTG and 30°C. In addition, it was found that a large amount of cutinase had been accumulated in periplasm since 16-h cultivation under the same inducement condition. Therefore, glycine and surfactant sodium taurodeoxycholate (TDOC) were further used to promote the leakage of recombinant cutinase from periplasm. Supplied with 100 mM glycine and 1 mM TDOC, the amount of cutinase in periplasm decreased remarkably, and the activity in the culture medium reached to 146.2 and 149.2 U/ml after 54 h of culturing, respectively.
研究领域:
生物工程
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分子生物学
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BIOTECHNOLOGY & BIOTECHNOLOGICAL EQUIPMENT
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2020
·
IF 1.186
Shan Yuan,Rubing Zhang,Yujin Cao,Jing Guo,Mo Xian,Wei Liu
Phloroglucinol (PG) is an important chemical that has been produced in engineered E. coli. Low yield and high cost are the bottlenecks that limit the industrialisation and popularisation of PG biosynthesis. In this study, a new expression system (pNEW vector) was developed for PG biosynthesis by using inexpensive p-isopropylbenzoate (cumate) as the inducer, and the copy number control element (rop) was added to optimise the original pNEW vector, which not only promoted the PG yield, but also reduced the leakage expression. Compared to traditional IPTG inducible expression system, 2-fold enhancement of PG yield was obtained with optimised expression system and the cost of inducer was reduced to 1/9. These results provide technical support for the biosynthesis of PG and reference for the selection of induced expression systems for other chemical biosynthesis.
研究领域:
生物技术
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生物化工
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代谢工程
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高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
·
2023
·
IF 6.1
Guili Zhao,Wenjuan Zhang,Xiaodong Fu,Xin Xie,Song Bai,Xiangyang Li
The viral protein genome-linked protein (VPg) of telosma mosaic virus (TeMV) plays an important role in viral reproduction. In this study, the expression conditions of TeMV VPg were explored. A series of novel benzenesulfonamide derivatives were synthesized. The binding sites of the target compounds and TeMV VPg were studied by molecular docking, and the interaction was verified by microscale thermophoresis. The study revealed that the optimal expression conditions for TeMV VPg were in Escherichia coli Rosetta with IPTG concentration of 0.8 mM and induction temperature of 25 °C. Compounds A4, A6, A9, A16, and A17 exhibited excellent binding affinity to TeMV VPg, with Kd values of 0.23, 0.034, 0.19, 0.086, and 0.22 μM, respectively. LYS 121 is the key amino acid site. Compounds A9 inhibited the expression of TeMV VPg in Nicotiana benthamiana. The results suggested that TeMV VPg is a potential antiviral target to screen anti-TeMV compounds.
研究领域:
病毒学
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药物化学
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分子生物学
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植物病理学
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高影响因子
Current Protocols
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2025
·Vol. 5
·期 10
·e70241
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IF 2.2
Yiming Zhao,Jingjun Hong
Chromatin remodeling is a process of regulating gene expression levels by altering chromatin structure, facilitated by chromatin remodeling factors. ISWI (Imitation SWItch), a type of chromatin remodeling factor, plays a crucial role in epigenetic regulatory processes, such as cell differentiation and development. It primarily achieves remodeling effects by promoting the direct sliding of nucleosomes and their relocation on DNA. To study the function and structural characteristics of ISWI, this basic protocol describes a simple and rapid procedure for the large-scale expression and purification of soluble Ct ISWI 77-1038 and Ct ATPase 77-722 proteins in Escherichia coli ( E. coli ). The procedure provides high yields (∼20 mg) and is followed by in vitro measurement of ATP hydrolysis activity. © 2025 Wiley Periodicals LLC. Basic Protocol : Expression, purification, and ATP hydrolysis activity measurement of Ct ISWI and Ct ATPase
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INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2025
·
IF 8.5
Xianglong Li,Haoxian Huang,Yachuan Yuan,Mingdong Wang,Xuanyu Cao,Yanbin Feng,Song Xue
Excessive CO 2 emissions have led to increasingly severe climate and environmental challenges. Within the framework of carbon neutrality, biological carbon fixation is a promising approach to improve the ecological environment and optimize energy systems. Salicylic acid decarboxylase (SAD) catalyzes the conversion of CO 2 and phenolic compounds into salicylic acid derivatives. However, the efficiency of CO 2 fixation in this process is low and is limited by the difficulty of product release during carboxylation. In this study, the engineered SAD, with the tunnel microenvironment remodeled to increase the turnover number ( k cat ), exhibited heightened CO 2 fixation efficiency. The mutant K23A/Y64T introduces a new tunnel architecture that indirectly enlarges the bottleneck radius of the native tunnel. These changes in the tunnel microenvironment facilitated product release, which was supported by evidence from both the catalytic activity and random acceleration molecular dynamics (RAMD). Kinetic analysis revealed that K23A/Y64T exhibited a 26-fold increase in catalytic efficiency ( k cat / K m ) with a K m consistent with that of the wild type, and the CO 2 conversion efficiency increased 14-fold. Dynamic cross-correlation matrices (DCCM) illustrated that α1 with K23A and loop5 with Y64T performed anticorrelated movements, enabling a change in the tunnel microenvironment between the two regions. Unlike conventional approaches targeting substrate–enzyme interactions, this study demonstrates that protein tunnel remodeling can intrinsically enhance carboxylation activity and holds potential for broad substrate applicability.
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TOXICON
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2025
·
IF 2.4
Ranran Ma,Fred Mwabulili,Weiwei Peng,Weibin Ma,Yuhui Yang,Yanli Xie
Fungal mycotoxins pose a serious threat to human and animal health, especially due to their co-occurrence in food and feed, causing synergistic toxicity. Current enzymatic detoxification strategies often depend on redox mediators, limiting enzyme diversity and restricting practical application. In this study, we characterized PsCopA, a novel multicopper oxidase from Pseudomonas sp., heterologously expressed in Escherichia coli , which efficiently degraded aflatoxin B 1 and zearalenone simultaneously without mediators. PsCopA showed optimal activity at 50°C (pH 7.0) for AFB 1 and 60°C (pH 8.0) for ZEN and retained over 80% activity after 2 h incubation across 20-80°C. UPLC-MS/MS analysis identified AFQ 1 and β-zearalanol as degradation products, both exhibiting significantly reduced hepatotoxicity and oxidative stress in zebrafish, even under co-exposure, suggesting effective mitigation of synergistic toxicity. Notably, PsCopA removed over 90% of toxins from contaminated cornmeal and peanut meal. These findings demonstrate PsCopA has a promising prospect for industrial application in food and feed detoxification.
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BIOCHEMICAL ENGINEERING JOURNAL
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2025
·
IF 3.8
Xue-Chun Li,Hong-Jie Xie,Zheng-Xiong Zhou,Bin Sun,Jun-Jie Lin,Xiao-Dan Li,Ren Wang
1-aminocyclopropane-1-carboxylic acid (ACC), a crucial intermediate in the ethylene biosynthetic pathway, plays a decisive role in regulating plant developmental processes and stress responses. In heterologous biosynthesis systems, ACC biosynthesis is inhibited due to its antibacterial activity. Enhancing ACC export confers advantages to ACC-producing biotechnological platforms and microbial cell factories. While bacterial transporters for canonical amino acids in Corynebacterium glutamicum and Escherichia coli have been extensively studied, their roles in ACC transport remain unexplored. To identify the exporters mediating ACC transport in E. coli , relevant experiments were performed in this study. The results showed that the Leucine-exporter LeuE acts as a high-efficiency ACC exporter in E. coli . Subsequently, structural modeling coupled with site-directed mutagenesis revealed that L160 is a critical residue in determining the ACC transport specificity of LeuE. The L160Y mutation enhanced E. coli tolerance to ACC, elevating the threshold from 50 mM to 100 mM. Structural modeling predicted that the L160Y mutation-induced compaction reduced the transporter channel diameter by 28% (from 9.551 Å to 6.876 Å), a change that may enhance the binding affinity of ACC. Notably, a significant reduction in intracellular ACC levels was observed in both the LeuE-overexpressing and LeuE (L160Y)-overexpressing strains. These findings enable large-scale biosynthesis of ACC and its derivatives via engineered microbial systems, thereby facilitating cost-effective development of ACC-based pharmaceuticals and agrochemicals.
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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代谢工程
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转运生理学
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ACS Catalysis
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2025
·
IF 13.1
Ruyue Dong,Jiaying Wang,Jian Tian,Guoshun Xu,Ziqi Liang,Xing Qin,Xiaolu Wang,Xiaoqing Liu,Huiying Luo,Bin Yao,Yaru Wang,Tao Tu
Elucidating enzyme behavior and engineering highly efficient biocatalysts remain fundamental challenges in biocatalysis. Despite advances in enzyme engineering, significant optimization opportunities persist. Herein, we introduce the Evolutionary-Structural Tuning of Enzyme Efficiency Modules (ESTEEM) strategy, which integrates evolutionary principles with structural scaffold analysis to accelerate enzyme design. Using cytochrome P450 monooxygenase (P450s)─enzymes with deeply buried active sites and intricate substrate tunnels─as models, we identified key nonconserved residues (I67 and Q83) within the substrate tunnel. These distal positions critically influence tunnel architecture, and their mutation dramatically enhanced catalytic efficiency. The optimal double-variant exhibited a 106-fold increase in total turnover number without compromising stability while simultaneously broadening the substrate scope. Through adaptive steered molecular dynamics and hydrogen–deuterium exchange mass spectrometry, we uncovered a tunnel-mediated tuning mechanism: activity differences between variants and wild type were governed by a rigidity-flexibility balance in the scaffold, which modulates catalytic efficiency by stabilizing tunnel geometry. Experimental validation and retrospective analysis confirmed the robustness of the ESTEEM strategy. This study establishes how tunnel mutations stabilize substrate transport pathways to enhance catalysis, providing a framework for engineering enzymes specialized activity toward substrates requiring deeply recessed active sites─advancing applications in biocatalysis and synthetic biology.
研究领域:
生物催化
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酶工程
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结构生物学
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合成生物学
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计算生物学
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高影响因子
BIOTECHNOLOGY AND BIOENGINEERING
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2025
·
IF 3.6
Man Zhao,Mengying Yu,Huiru Yuan,Yiting Shen,Zhiqiang Liu,Yuguo Zheng
Carcinine, a valuable imidazole dipeptide with antioxidant and therapeutic properties, faces biosynthesis challenges due to enzyme aggregation and substrate inhibition. In this study, an integrated strategy combining linker peptide engineering and immobilization was applied to address these challenges and enhance carcinine production. Rational design of linker peptides (D 5 , L 2 , L 3 ) in the sfp-Ebony fusion protein enabled its highest soluble expression in WSL 2 E strain, achieving 93.1% conversion efficiency—3.5-fold higher catalytic efficiency than WSGE strain. Response surface methodology optimized sodium alginate-polyvinyl alcohol (SA-PVA) immobilization parameters (5% PVA, 3% SA, 2.3% CaCl₂), yielding excellent immobilized WSL 2 E@SA-PVA cells with 95.93% activity recovery. Structural characterization by scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FT-IR), and X-ray diffraction (XRD) confirmed the formation of a porous SA-PVA matrix that protected cells from harsh conditions. The immobilized biocatalyst exhibited superior operational stability (retaining > 80% activity after 7 cycles) and storage stability (maintaining 44.89% activity after 14 days at 4°C). Fed-batch scale-up (50 mL) achieved a record carcinine titer of 71.13 mM, mitigating the inhibitory effect of high substrate concentrations through phased substrate feeding. This study provides a scalable biocatalytic platform for industrial carcinine production, effectively addressing key bottlenecks in biocatalyst stability and substrate tolerance.
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MICROCHEMICAL JOURNAL
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2025
·
IF 5.1
Mengjie Lin,Wenxin Chen,Cunshan Zhang,Jiewei He,Lina Wu,Ailin Liu,Xinhua Lin,Liqing Lin
Specific-Species enzyme detection serves as a crucial method in foodborne pathogen surveillance. Particularly, endogenous β-galactosidase (β-Gal) enzyme has been widely applied to determine the concentration of Escherichia coli ( E. coli ). The main challenges include reducing analysis time and improving detection sensitivity. A novel strategy for biosensing β-Gal activity and E. coli are developed based on BSA‑manganese dioxide nanosheets (BSA-MnO 2 NS) with the oxidase-mimicking activity to regulate the fluorescence of thiamine. BSA-MnO 2 NS can catalyze the oxidation of non-fluorescent thiamine to generate strong fluorescent dehydrothiamine. When β-Gal enzymes is introduced, p -aminophenyl-β-D-galactopyranoside (PAPG) could be hydrolyzed to form para-aminophenol (PAG), which efficiently reduced BSA-MnO 2 NS into Mn 2+ , resulting in the loss of their oxidase-like activity and the decrease of dehydrothiamine fluorescence. Since endogenous β-Gal in E. coli cells can directly hydrolyze PAPG without destroying bacteria, which greatly reduced the analysis time. The reduction product PAG leads to MnO 2 NS decomposition and thus reduced dehydrothiamine fluorescence. According to this mechanism, fluorescence biosensor for β-Gal activity and E. coli are constructed utilizing thiamine luminescence modulated by BSA-MnO 2 NS, without requiring an additional oxidizer. β-Gal and E. coli showed good linearity in the range of 0.01–2.0 μg/mL and 1.0 × 10 2 –1.0 × 10 4 CFU/mL, and the detection limits reached 8.55 ng/mL and 89.45 CFU/mL, respectively. The proposed enzyme-nanozyme cascade reaction significantly improved the sensitivity. It is highly specific and can be used to distinguish E. coli from other pathogenic bacteria. The strategy has also been successfully applied to the detection of E. coli in real samples.
研究领域:
生物传感
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微生物学
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纳米生物学
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分子诊断
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食品安全检测
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酶学
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高影响因子
Science Immunology
·
2025
·Vol. 10
·期 114
·
IF 16.3
Zhi Li,Jing Wang,Juan Du,Jun Li,Yanan Song,Junji Zhu,Ziyi Li,Wei Zhou,Shiyun Chen,Lijie Yang,Maohui Feng,Xiaolian Cai,Katryn J. Stacey,Wuhan Xiao
Processes that control tissue inflammation are essential to restore homeostasis after infection. The transcription factor NF-κB plays a central role in coordinating inflammation, but the mechanisms that regulate NF-κB signaling are not fully understood. Here, we identify OCEL1 ( occludin/ELL domain containing 1 ) as a negative regulator of NF-κB signaling. In the absence of infection, human OCEL1 bound to the LZ domain of NEMO (NF-κB essential modulator) and inhibited TRAF6-mediated K63-linked polyubiquitination, suppressing NF-κB signaling. During infection, OCEL1-mediated negative regulation of NF-κB signaling was impaired by FK506-binding protein bacterial peptidyl-prolyl cis/trans isomerases, which bound to OCEL1 in an amino-terminal palindromic proline–rich element (PPE) and promoted its degradation. Mice expressing a mutant version of the human OCEL1 PPE had dampened inflammation and increased susceptibility to Pseudomonas aeruginosa infection. Thus, the PPE of human OCEL1 senses bacterial infection, and its degradation releases the suppression of NF-κB signaling and promotes inflammation.
研究领域:
免疫学
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分子生物学
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传染病学
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信号转导
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高影响因子
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