Catalysts
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2020
·Vol. 10
·期 9
·947
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IF 3.52
Hongjie Hui,Yajun Bai,Tai-Ping Fan,Xiaohui Zheng,Yujie Cai
Putrescine, a biogenic amine, is a highly valued compound in medicine, industry, and agriculture. In this study, we report a whole-cell biocatalytic method inEscherichia colifor the production of putrescine, using L-arginine as the substrate. L-arginine decarboxylase and agmatine ureohydrolase were co-expressed to produce putrescine from L-arginine. Ten plasmids with different copy numbers and ordering of genes were constructed to balance the expression of the two enzymes, and the best strain was pACYCDuet-speB-speA. The optimal concentration of L-arginine was determined to be 20 mM for this strain. The optimum pH of the biotransformation was 9.5, and the optimum temperature was 45 °C; under these conditions, the yield of putrescine was 98%. This whole-cell biocatalytic method appeared to have great potential for the production of putrescine.Keywords:L-arginine;putrescine;Escherichia coli;whole-cell catalysis;co-expression
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INDUSTRIAL CROPS AND PRODUCTS
·
2020
·
IF 4.244
Yin Zhou,Qifeng Yao,Tong Zhang,Xiaoqiang Chen,Zhengqi Wu,Ni Zhang,Yundong Shao,Yong Cheng
The effect of green tea polysaccharide conjugate (gTPC) on the growth of E. coli was evaluated. gTPC has optimal antibacterial concentrations for E. coli DH5α (4.00 mg/mL) and 25,922 (1.00 mg/mL), with the antibacterial rates of 55.75 % and 50.65 % respectively. The results of scanning electron microscopy (SEM) and transmission electron microscope (TEM) indicated that gTPC-treated E. coli cells showed obvious morphological changes, part of the cell wall became blurred, and the leakage of cytoplasmic content were observed. The proportion of the propidium iodide (PI) permeable E. coli cells treated using 0.50–2.00 mg/mL gTPC was between 8.79 %–9.49 % as well as 14.10 % (4.00 mg/mL gTPC) and 15.10 % (6.00 mg/mL gTPC). Reactive oxygen species (ROS) production of 1.00–4.00 mg/mL gTPC treated E. coli increased by 26.57 %–38.19 % with a significant increase level ( p < 0.01). The outer membrane disruption played a key role in the antibacterial activity of gTPC against E. coli . This research result will open a new application field of tea polysaccharide conjugates.
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BIOELECTROCHEMISTRY
·
2020
·
IF 4.722
Yafei Chen,Yajie Yang,Yu Wang,Ye Peng,Jinmei Nie,Guanyue Gao,Jinfang Zhi
Mycotoxin contamination in food and feed is a global concern because mycotoxin contamination can cause both acute and chronic health effects in humans and animals. In the present work, an Escherichia coli -based biosensor is described for the toxicity assessment of aflatoxin B 1 (AFB 1 ) and zearalenone (ZEN). In this electrochemical biosensor, E. coli is used as the signal recognition element, p -benzoquinone is used as the mediator, and a two-step reaction procedure has been developed to separate the mediator from the mycotoxins. The current value of the as-prepared microbial biosensor exhibits a linear decrease with concentrations of AFB 1 and ZEN in the range of 0.01–0.3 and 0.05–0.5 μg/mL, with detection limits reaching 1 and 6 ng/mL, respectively. The IC 25 values obtained by the present method are 0.25 and 0.40 μg/mL for AFB 1 and ZEN, which shows that the cytotoxicity of AFB 1 to E. coli is more severe than the cytotoxicity of ZEN to E. coli . The combined toxic effect of these two mycotoxins has also been explored, and synergistic biotoxicity has been observed. Moreover, the biosensor is successfully applied to the toxicity evaluation of mycotoxins in real samples, including peanut and corn oils. This work could provide new insight into mycotoxin and microorganism interactions and could establish a new approach for future mycotoxin detection.
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APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
·
2019
·Vol. 103
·期 9
·3715-3725
·
IF 3.67
Wang Chengcheng,Zheng Pu,Chen Pengcheng
Raspberry ketone is an important ingredient in the flavor and fragrance industries. Due to its low content in fruits and vegetables, the production of natural raspberry ketone using heterologous synthesis in microbial strains is recently attracting increased attention. In this work, a heterologous pathway to produce raspberry ketone from p -coumaric acid, including 4-coumarate: CoA ligase (4CL), benzalacetone synthase (BAS), and raspberry ketone/zingerone synthase (RZS1) from plants, was successfully assembled in Escherichia coli . When the RZS1 gene was introduced into E. coli and co-expressed with two other genes, the intermediate 4-hydroxybenzylidene acetone in the pathway was almost completely transformed into a raspberry ketone. Substituting TB medium for M9 medium increased raspberry ketone titers by 3–4 times. Furthermore, the heterologous pathway was partitioned into two modules; module one produced p -coumaroyl-CoA from p -coumaric acid by 4CL, and module two produced raspberry ketone from coumaroyl-CoA by the action of BAS and RZS1. Optimizing the balanced expression of the two modules, it was shown that moderate expression of module one and high expression of module two was the best combination to enhance raspberry ketone production. The engineered strain CZ-8 reached 90.97 mg/l of raspberry ketone, which was 12 times higher than previously reported. In addition, the preferred approach of the heterologous pathway was related to the heterologous genes from different sources; for example, 4CL from Arabidopsis thaliana seemed to be more suitable for raspberry ketone production than that from Petroselinum crispum . This work paves an alternative way for future economic production of natural raspberry ketone.
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APPLIED AND ENVIRONMENTAL MICROBIOLOGY
·
2018
·Vol. 84
·期 20
·
IF 3.633
Haiquan Yang,Xiao Lu,Jinyuan Hu,Yuan Chen,Wei Shen,Long Liu
Escherichia coli is one of the most widely used host microorganisms for recombinant protein expression and metabolic engineering, but it cannot efficiently secrete recombinant proteins to extracellular space. Here, extracellular protein secretion was enhanced in E. coli by deleting two d,d-carboxypeptidase genes (dacA and dacB, single and double deletions) to perturb the cell wall peptidoglycan network. Deletion of dacA and dacB enhanced the accumulation of intracellular soluble peptidoglycan in E. coli and affected cell morphology, resulting in a more irregular cell shape and the appearance of transparent bulges. Deletion of dacA and dacB appears to disrupt the normal rigid structure, presumably due to perturbation and destruction of the cell wall peptidoglycan network. The extracellular green fluorescent protein (GFP) fluorescence intensity of deletion mutants was increased by >2.0-fold compared with that of control cells, and that of the double deletion mutant was increased by 2.7-fold. Extracellular recombinant fibroblast growth factor receptor 2 (FGFR2) and collagen E4 secretion in deletion mutants was also enhanced compared with that in the control cells. Additionally, the extracellular recombinant amylase activity of single-deletion mutants BL21 ΔdacA pETDuet-amyk and BL21 ΔdacB pETDuet-amyk was increased 2.5- and 3.1-fold, respectively. The extracellular distribution of α-galactosidase by deletion mutants was also increased by >2.0-fold. Deletion of dacA and dacB increased outer membrane permeability, which could explain the enhanced extracellular protein secretion.IMPORTANCE Cell surface structure stabilization is important for extracellular secretion of proteins in Escherichia coli. As the main constituent of the cell wall, peptidoglycan contributes to cell structure robustness and stability. Here, we perturbed the peptidoglycan network by deleting dacA and dacB genes encoding d,d-carboxypeptidase enzymes to improve extracellular protein secretion. This new strategy could enhance the capacity of E. coli as a microbial cell factory for extracellular secretion of proteins and chemicals.
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LANGMUIR
·
2018
·
IF 3.789
Mei Liu,Jiao Li,Baoxin Li
Polyethylenimine (PEI) has antimicrobial activity against Gram-positive (Staphylococcus aureus, S. aureus) and Gram-negative (Escherichia coli, E. coli), bacteria but is highly cytotoxic, and the selective antimicrobial activity against S. aureus is obviously better than that against E. coli. To reduce the cytotoxicity and improve the antibacterial activity against E. coli, we modified PEI with d-mannose through nucleophilic addition between primary amine and aldehyde groups to get mannose-modified polyethylenimine copolymer particles (Man-PEI CPs). The use of mannose may provide good targeting ability toward E. coli pili. The antibacterial activity of Man-PEI CPs was investigated. Man-PEI CPs shows specific and very strong killing capability against E. coli at a concentration of 10 μg/mL, which is the highest antimicrobial efficiency compared to that of unmodified PEI (220 μg/mL). The antibacterial mechanism demonstrated that the enhancement in antibacterial activity is due to specific recognition of the mannose and destroying the cell wall of the bacteria by PEIs. Importantly, the Man-PEI CPs show less cytotoxicity and excellent biocompatibility. The results indicate that Man-PEI CPs have great potential as novel antimicrobial materials to prevent bacterial infections and provide specific applications for killing E. coli.
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Fermentation-Basel
·
2024
·Vol. 10
·期 4
·202
·
IF 3.3
Chong Zhang,Jinglin Liao,Yuancong Li,Shuli Liu,Mengting Li,Di Zhang,Zhenyu Wang,Dong Liu,Hanjie Ying
Human epidermal growth factor (hEGF) holds significant importance in the fields of medicine and cosmetics. Therefore, it becomes imperative to develop a highly efficient fermentation system for hEGF production. In this study, a stable hEGF-secreting expression strain was created by integrating the hEGF gene into the genome ofEscherichia coli(E. coli) BL21, and an immobilized fermentation system was developed based on biofilm to facilitate continuous hEGF production. After optimization of fermentation conditions and gene dosage, the production of hEGF was increased from 13.9 mg/L to 52.4 mg/L in free-cell fermentation. Moreover, genetic modifications targeting dgcC, csgD, bcsA, and bcsB proved to enhance biofilm formation. When thebcsBwas overexpressed in BL21-hEGF-C5, the biofilm-forming ability was enhanced by 91.1% and the production of hEGF was increased by 28% in biofilm-immobilized continuous fermentation. In conclusion, this study successfully confirms the feasibility of continuous hEGF production through the biofilm system ofE. coli, providing valuable insights for the development of other proteins in the field of continuous biomanufacturing.
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JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2024
·
IF 5.7
Zhenqiang Zhao,Jiajia You,Xuanping Shi,Rongshuai Zhu,Fengyu Yang,Meijuan Xu,Minglong Shao,Rongzhen Zhang,Youxi Zhao,Zhiming Rao
Exploring effective remodeling strategies to further improve the productivity of high-yield strains is the goal of biomanufacturing. However, the lack of insight into host-specific metabolic networks prevents timely identification of useful engineering targets. Here, multidimensional engineering strategies were implemented to optimize the global metabolic network for improving l-threonine production. First, the metabolic bottleneck for l-threonine synthesis was eliminated by synergistic utilization of NADH and an enhanced ATP supply. Carbon fluxes were redistributed into the TCA cycle by rationally regulating the GltA activity. Subsequently, the stress global response regulator UspA was identified to enhance l-threonine production by a transcriptomic analysis. Then, l-threonine productivity was improved by enhancing the host’s stress resistance and releasing the inhibitory reaction of glucose utilization. Eventually, the l-threonine yield of THRH16 reached 170.3 g/L and 3.78 g/L/h in a 5 L bioreactor, which is the highest production index reported. This study provides rational guidance for increasing the productivity of other chemicals.
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INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
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2012
·
IF 2.453
Jianying Huang,Hengjun Jiang,Miao Qiu,Xiaodong Geng,Ronghua Yang,Jianrong Li,Chaohua Zhang
Water-soluble quaternary chitin/chitosan derivatives, O -[(2-hydroxy-3-trimethylammonium)propyl chitin (OHT-chitin), N -[(2-hydroxy-3-trimethylammonium)propyl chitosan (NHT-chitosan), and N , N , N -trimethylchitosan (TMC), having identical molecular weight and same anion, were prepared, and their antibacterial activities against Escherichia coli and Staphylococcus aureus were evaluated. Their minimum inhibitory concentration (MIC) values varied from 0.04 mg/mL to 20.48 mg/mL, and the minimum bactericidal concentration (MBC) values varied from 0.16 mg/mL to 40.96 mg/mL against E. coli and S. aureus , respectively. Transmission electron microscopy (TEM) clearly showed that there was serious damage on the bacterial walls, whereas there was some dissimilarity between TMC-treated E. coli and S. aureus .
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ACS Omega
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2024
·
IF 3.7
Xiaofan Wu,Yupo He,Jiaqi Fu,Yanli Zhao
Evaluating the bacterial activity effectively is critical to addressing the challenges posed by bacterial infections. Electrochemistry offers significant advantages in accuracy and cost efficiency compared with methods that are more time-consuming or require expensive instrumentation. This study initially established an electrochemical method for detecting bacterial activity using heat treatment as the pretreatment step. Subsequent optimization of the heat treatment and detection conditions further enhanced the method efficiency. The detection results of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) under different heating conditions showed that the peak current values of E. coli and S. aureus were the highest after heating at 50 °C for 15 min. The dynamic changes in the electrochemical signals from the heat-treated bacteria with alteration in heating conditions were indicated to be related to the effects of heating on the external structure and purine metabolism of the bacterial cells. Although heat-treated E. coli and S. aureus exhibited similar peak potentials, different substances were observed to have contributed to these potentials. A positive linear correlation was observed between the peak current and bacterial concentration. Compared to the plate counting method, the electrochemical method detected the activity changes in E. coli and S. aureus from the logarithmic to the stationary phase earlier. The findings support the broad application of electrochemical methods in microbial detection and provide valuable insights into the purine metabolism of heat-stressed bacteria.
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ANALYTICAL AND BIOANALYTICAL CHEMISTRY
·
2025
·
IF 3.8
Yao Pengnan,Wang Zhengjun,Ma Kefeng,Kong Jinming,Zhang Xueji
Foodborne pathogens, including bacteria, viruses, fungi, and parasites, commonly cause disease outbreaks through contaminated food or water. Escherichia coli ( E. coli ) tends to induce severe illnesses like hemorrhagic colitis and hemolytic uremic syndrome. To address detection challenges, lectins, natural glycoproteins isolated from plants, animals, or microorganisms, demonstrate unique advantages through their specific binding to carbohydrate moieties on pathogen surfaces. This biomolecular recognition mechanism establishes lectins as ideal bioreceptors for developing biosensing platforms targeting foodborne pathogens. In this study, a fluorescent sensor that mediates atom transfer radical polymerization (ATRP) through Concanavalin A (ConA) was developed. This sensor utilizes ConA’s ability to capture pathogens and E. coli ’s ability to catalyze ATRP to identify E. coli . First, ConA is modified onto magnetic beads for initial capture, followed by modification of 2-bromoisobutyryl bromide (BIBB) onto the surface of captured E. coli for subsequent ATRP. If the pathogen captured by ConA is not E. coli , the subsequent ATRP cannot proceed normally, and the sensor cannot generate a fluorescent signal. Under optimal conditions, this sensor demonstrated good linearity from 1.00 × 10 2 to 1.00 × 10 7 CFU/mL. The limit of detection has gone down to 8 CFU/mL. This solution has the advantages of high sensitivity, low cost, and easy operation compared to traditional solutions for bacteria detection, which provides an efficient, stable, and sensitive solution for the detection of E. coli. Graphical In this study, a fluorescent sensor that mediates atom transfer radical polymerization (ATRP) through Concanavalin A (ConA) was developed. The sensor works by modifying ConA on magnetic beads, which allows the capture of pathogens through the recognition of E. coli surface polysaccharides by lectin. Next, 2-bromoisobutyryl bromide (BIBB) was applied to the E. coli surface in preparation for the subsequent ATRP. This solution has the advantages of high sensitivity, low cost, and easy operation compared to traditional solutions for bacteria detection, which provides an efficient, stable, and sensitive solution for the detection of E. coli.
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INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
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2025
·
IF 8.5
Wei Xu,Mi Pan,Lu Liang,Jiayi Li,Le Yang,Junpei Zhou,Wei Xu,Junmei Ding
Phthalate acid esters (PAEs), extensively employed as plasticizers, have emerged as persistent environmental pollutants that pose serious threats to ecosystems and human health. Effective and sustainable strategies for their biodegradation are urgently needed. In this study, a whole-cell biocatalyst was developed by engineering Escherichia coli BL21(DE3) to surface-display the carboxylesterase Ces17 derived from Bacillus licheniformis DBP-4. The ces17 gene was fused to the N-terminal domain of the ice nucleation protein (INPN) from Pseudomonas syringae to facilitate membrane anchoring. Successful surface localization of Ces17 was verified by SDS-PAGE analysis and fluorescence microscopy. The displayed enzyme exhibited optimal catalytic activity at 40 °C and pH 9.0, and demonstrated enhanced storage stability, retaining approximately 71 % of its initial activity after 35 days at 4 °C, compared to only 34 % for its purified counterpart. Biodegradation assays revealed that 1 U of the surface-displayed Ces17 efficiently degraded 81.5 % of dimethyl phthalate, 62.0 % of diethyl phthalate, and 87.0 % of diisobutyl phthalate within 8 h, exhibiting superior performance in both efficiency and operational stability. These findings highlight the advantages of enzyme surface display for improving catalytic properties and durability, and propose a robust whole-cell biocatalytic system with significant potential for PAEs remediation in environmental applications.
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