BIOCHEMICAL ENGINEERING JOURNAL
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2026
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IF 3.6
Xiangcao Cui,Xiaosen Liu,Huiling Zhang,Kun Zhou,Cunzhuo Tang,Chen Deng,Xu Li,Yongjun Qiu,Liqiang Fan,Liming Zhao
A Whole-cell method co-displaying ACT and PPK2 enables low-cost butyrolactam biosynthesis. • Co-display of ACT and PPK2 increases butyrolactam productivity by 46.4% against ACT display alone. • Fed-batch fermentation achieves 39.13 g/L butyrolactam with 94.7% yield, showing industrial potential. Butyrolactam is a high value-added monomer and an important intermediate for various physiologically active substances, and has been widely used in the pharmaceutical, pesticide, and chemical industries. Though the biosynthesis of butyrolactam is gradually advancing, challenges such as the requirement for expensive cofactor adenosine triphosphate (ATP) and the barrier of substrate transmembrane transport in whole-cell synthesis hinder its large-scale industrial application. In this study, an integrated whole-cell catalytic strategy combining ice nucleation protein (INP)-mediated surface display and ATP regeneration was developed. Compared with the butyrolactam productivity of cells intracellularly expressing β-alanine CoA transferase (ACT), co-expressing polyphosphate kinase 2 (PPK2) to regenerate ATP and display ACT on the cell surface only increased it by 23.1% and 25.0%, respectively. Co-displaying ACT and PPK2 further increased the butyrolactam productivity, which was 46.4% more than that of only ACT displayed cells. Using fed-batch fermentation strategy, a final butyrolactam titer of 39.13 g/L and a yield of 94.7% were achieved in the strain co-displaying ACT and PPK2. Our findings demonstrate a potentially attractive strategy for the industrial butyrolactam production.
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BIORESOURCE TECHNOLOGY
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2026
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IF 8.2
Yuan Gao,Shibo Guo,Jun Wu,Jingyan Wang,Huan Liu,Jimin Guo,Xinxiao Sun,Jia Wang,Xiaolin Shen,Qipeng Yuan
Cb SAMT is the most efficient methyltransferase for methyl salicylate biosynthesis. • The engineered 7 × His- Cb SAMT variant increased catalytic efficiency by 12-fold. • SAM regeneration and two-phase fermentation yielded 5.12 g/L methyl salicylate. Methyl salicylate (MeSA) is a volatile methylated aromatic ester widely used in agriculture, food, and pharmaceuticals. Microbial biosynthesis offers a sustainable alternative to plant extraction and petrochemical synthesis; however, efficient MeSA production remains limited by the low catalytic efficiency of carboxyl methyltransferases, insufficient intracellular supply of S-adenosylmethionine (SAM), and severe product volatilization during fermentation. In this study, we systematically screened salicylate carboxymethyltransferase and identified Cb SAMT as a relatively efficient catalyst for salicylic acid (SA). To further improve catalytic performance, we engineered Cb SAMT through fusion-protein design. Remarkably, the catalytic efficiency of 7 × His- Cb SAMT was 12-fold higher than that of the wild-type. Molecular dynamics simulations and computational analyses revealed that the polyhistidine tag reshaped the catalytic microenvironment by remodeling the hydrogen-bonding network and reducing the distance between SA and key substrate-positioning residues, thereby enhancing substrate binding and catalytic turnover. To further enhance MeSA biosynthesis, we reinforced intracellular SAM regeneration and established an in situ two-phase fermentation system using n-dodecane as the extractant to alleviate product volatilization. Finally, fed-batch fermentation achieved a MeSA titer of 5.12 g/L, representing the highest production level reported to date. Collectively, this study highlights the potential of polyhistidine tags in enzyme engineering and provides an efficient platform for the biosynthesis of volatile methylated natural products. Download: Download high-res image (145KB) Download: Download full-size image
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Virulence
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2026
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IF 6.8
Mingyu Cao,Yuhang Li,Guangfu Han,Hanyu Liu,Shanshan Xiong,Xiaona Liu,Menghan Zhao,Yuefan Su,Yanhua Li,Chunliu Dong
The persistent escalation of antibiotic resistance poses a severe threat to global public health. As a common opportunistic pathogen, the rapid development of resistance in Escherichia coli (E. coli) poses severe challenges to clinical anti-infective therapy. Against this backdrop, the combined use of amino acids and antibiotics is considered an effective strategy against multidrug-resistant bacterial infections. This study investigated the antibacterial efficacy and mechanism of action of the combination of arginine (Arg) and florfenicol (FFC) against resistant E. coli. Results indicate that Arg significantly enhances the bactericidal activity of FFC against resistant E. coli both in vivo and in vitro, successfully reversing the resistance phenotype. Mechanistic studies reveal that Arg disrupts fatty acid β-oxidation by inhibiting the arginine succinyl-transferase (AST) pathway, leading to excessive free fatty acid (FFA) accumulation. This triggers membrane structural damage, energy metabolism disorders, and oxidative stress. Concurrently, Arg inhibits the expression and function of multidrug efflux pumps, such as AcrAB-TolC and EmrAB-TolC, reducing FFC efflux and increasing its intracellular concentration. This study systematically reveals a novel pathway whereby Arg synergistically enhances the antibacterial activity of FFC through dual mechanisms of metabolic reprogramming and efflux pump inhibition, providing theoretical support for the development and application of amino acid compounds as antibiotic adjuvants.
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BIORESOURCE TECHNOLOGY
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2026
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IF 8.2
Hai-Fei Zhang,Cheng-Hai Yan,Fang-Hui Chen,Ding Wang,Min Tan,Yan Xu,Richard Ansah Herman,Jun Wang
A new strategy of overexpressing E. coli adhesion gene for biofilm catalysis was proposed. • Adhesin gene overexpression enhances cell adhesion, promotes biofilms formation, and reduces planktonic cell number. • Overexpression of ecp A in E. coli enhances biofilm biomass by 563.88% compared with CK. • The conversion of rutin reached 97.81% using ecpA-overexpressing E. coli biofilms as catalysts. • Overexpressing adhesion genes and carrier adsorption enhance biofilm catalytic performance. Biofilms catalysis has broad application prospects in the biocatalysis due to its high cellular stability, strong reusability, and operational simplicity. However, the naturally limited biofilm-forming ability of Escherichia coli restricts its performance in catalytic processes. In this study, we developed a rational engineering strategy to enhance E. coli -based biofilms catalysis by overexpressing adhesion-related genes in combination with carrier materials. Specifically, adhesin gene overexpression enhances cell adhesion and biofilm formation while decreasing planktonic cell number. Furthermore, biofilm biomass in the ecp A- and pro Q-overexpressing strains increased by 563.88% and 550.36%, respectively ( p < 0.001). In addition, after the addition of hydrophilic polyurethane fiber carriers, rutin conversion rate of 97.81% and isoquercitrin yield of 90.11% were achieved using biofilms catalysts formed by the ecp A-overexpressing strain ( p < 0.05). These findings demonstrate that adhesin gene overexpression combined with hydrophilic polyurethane fibers as carriers is an effective strategy for constructing robust E. coli biofilms catalysts and enhancing biofilm-mediated biocatalysis. Download: Download high-res image (80KB) Download: Download full-size image
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BIOTECHNOLOGY LETTERS
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2022
·Vol. 44
·期 12
·1401-1414
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IF 2.716
Liu Kun,Wang Feng-Qing,Zhao Ming,Gao Bei,Xu Hong,Wei Dongzhi
Human epidermal growth factor (hEGF) has multiple biological functions, such as promoting cell proliferation, differentiation, and migration. In addition, it is a very expensive polypeptide with attractive market prospects. However, the production of hEGF needs for high cost to manufacture polypeptide demands reinvestigations of process conditions so as to enhance economic benefits. Improving the expression of soluble hEGF is the fundamental method to reduce the cost. In this study, a non-extracellular engineered strain of expressed hEGF was constructed, using plasmid pET-22b(+) in Escherichia coli . Preliminary fermentation and high cell density cultivation were carried out in shake flasks and in a 5 L bioreactor, respectively. A high yield of 98 ± 10 mg/L of soluble hEGF and a dry cell weight (DCW) of 6.98 ± 0.3 g/L were achieved in shake flasks. Then, fermentation conditions were optimized for large-scale production, while taking into consideration the expensive equipment required for cooling and conforming to industrial standards. A yield of 285 ± 10 mg/L of soluble hEGF, a final cell density of 57.4 ± 2 g/L DCW (OD 600 141.1 ± 4.9), and hEGF productivity of 14.3 mg/L/h were obtained using a bioreactor at 32 °C for 20 h. The production method developed in this study for the biosynthesis of soluble hEGF is efficient and inexpensive.
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INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
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2022
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IF 8.025
Kunfeng Zhang,Yuzhe Huang,Qianzhen Wu,Wenhua Guo,Hao Chen,Wenna Zhang,Yong Li,Yongming Lu,Qingxi Wu,Wenjuan Pan,Lei Chen,Yan Chen
The objectives of this study were (1) to prepare Armillariella tabescens mycelia polysaccharides (PAT) with remarkably growth inhibitory effect on typical food-borne pathogenic bacteria using a green and efficient polyamide method and (2) to explore the antibacterial mechanism of PAT for use as a natural antibacterial agent. The sugar and uronic acid contents of PAT were 93.41% and 12.24%, respectively. PAT could inhibit the growth of Escherichia coli , Proteus vulgaris , Bacillus subtilis , and Staphylococcus aureus cells, with minimum inhibitory concentrations of 0.5, 1.0, 4.0, and 4.0 mg/mL, respectively. Ultra-high-resolution field emission scanning electron microscopy and high-resolution transmission electron microscopy analysis revealed cell wall and membrane rupture of E. coli treated with PAT. Further, 0.5–4.0 mg/mL PAT was found to significantly ( P < 0.01) and concentration-dependently increase the conductivity of the broth, exudation of the intracellular protein, and alkaline phosphatase and β-galactosidase activities. Confocal laser scanning microscopy revealed morphological changes in E. coli DNA after PAT treatment and intracellular reactive oxygen species accumulation; flow cytometry revealed E. coli cell apoptosis. Our findings provide a theoretical basis and technical support for the development of PAT as a natural antibacterial product.
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ENZYME AND MICROBIAL TECHNOLOGY
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2022
·
IF 3.705
Jinbin Liu,Kaipeng Wang,Mian Wang,Huaxiang Deng,Xiaodong Chen,Yueling Shang,Xiaochen Liu,Xiaohong Yu
An engineered Escherichia coli was constructed by co-expressing L -amino acid deaminase, α-keto acid decarboxylase, alcohol dehydrogenase , and glucose dehydrogenase through two plasmids for tyrosol production. The activity of the rate-limiting enzyme L -amino acid deaminase from Cosenzaea myxofaciens (CmAAD) toward tyrosine was improved by structure-guided modification. The enzyme activity of triple mutant CmAAD V438G/K147V/R151E toward tyrosine was ~5.12-fold higher than that of the wild-type CmAAD. Secondly, the plasmid copy numbers and the gene orders were optimized to improve the titer of tyrosol. Finally, the recombinant strain CS-6 transformed 10 mM tyrosine into 9.56 ± 0.64 mM tyrosol at 45 ℃, and the space-time yield reached 0.478 mM·L −1 ·h −1 . This study proposes a novel idea for the efficient and natural production of tyrosol, which has great potential for industrial application.
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ACS Synthetic Biology
·
2022
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IF 5.249
Zhongshi Huang,Ning Li,Shiqin Yu,Weiping Zhang,Tianmeng Zhang,Jingwen Zhou
Research studies on NAD+ have proven its crucial role in aging and disease. Nicotinamide mononucleotide (NMN), as the key intermediate of NAD+, plays a significant role in supplying and maintaining NAD+ levels. In the present study, a biocatalytic method for the efficient synthesis of NMN was established. First, Escherichia coli was systematically modified to make it more conducive to the biosynthesis and accumulation of NMN. Next, the performance of nicotinamide phosphoribosyltransferase from Vibrio bacteriophage KVP40 (VpNadV) was determined, which has the best catalytic activity to produce NMN from nicotinamide. The accumulation of extracellular NMN was further increased after the introduction of an NMN transporter. Fine-tuning of gene expression and copy number led to the synthesis of NMN at the yield of 2.6 g/L at the shake flask level. The introduction of a nicotinamide transporter, BcniaP, could not obviously increase the production of NMN at the shake flask level, but it decreased the production of NMN at the bioreactor level. Finally, the titer of NMN reached 16.2 g/L with a conversion ratio of 97.0% from nicotinamide, both of which are highest according to currently available reports. The fed-batch fermentation with direct supplementation of nicotinamide could facilitate the industrial-scale production of NMN compared to that achieved by the whole-cell catalysis process. These results also represent the highest reported yield of NMN synthesized from nicotinamide in E. coli.
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Microbial Cell Factories
·
2021
·Vol. 20
·期 1
·1-13
·
IF 5.328
Zhang Yanfeng,He Yongzhi,Zhang Nan,Gan JiaJia,Zhang Shan,Dong Zhiyang
Background Melatonin has attracted substantial attention because of its excellent prospects for both medical applications and crop improvement. The microbial production of melatonin is a safer and more promising alternative to chemical synthesis approaches. Researchers have failed to produce high yields of melatonin in common heterologous hosts due to either the insolubility or low enzyme activity of proteins encoded by gene clusters related to melatonin biosynthesis. Results Here, a combinatorial gene pathway for melatonin production was successfully established in Escherichia coli by combining the physostigmine biosynthetic genes from Streptomyces albulus and gene encoding phenylalanine 4-hydroxylase (P4H) from Xanthomonas campestris and caffeic acid 3- O -methyltransferase (COMT) from Oryza sativa . A threefold improvement of melatonin production was achieved by balancing the expression of heterologous proteins and adding 3% glycerol. Further protein engineering and metabolic engineering were conducted to improve the conversion of N -acetylserotonin (NAS) to melatonin. Construction of COMT variant containing C303F and V321T mutations increased the production of melatonin by fivefold. Moreover, the deletion of speD gene increased the supply of S-adenosylmethionine (SAM), an indispensable cofactor of COMT, which doubled the yield of melatonin. In the final engineered strain EcMEL8, the production of NAS and melatonin reached 879.38 ± 71.42 mg/L and 136.17 ± 1.33 mg/L in a shake flask. Finally, in a 2-L bioreactor, EcMEL8 produced 1.06 ± 0.07 g/L NAS and 0.65 ± 0.11 g/L melatonin with tryptophan supplementation. Conclusions This study established a novel combinatorial pathway for melatonin biosynthesis in E. coli and provided alternative strategies for improvement of melatonin production.
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Catalysts
·
2020
·Vol. 10
·期 9
·1057
·
IF 3.52
Jing Wang,Sheng Xu,Yang Pang,Xin Wang,Kequan Chen,Pingkai Ouyang
To achieve efficient bio-production of phospholipase D (PLD), PLDs from different organisms were expressed inE.coli. An efficient secretory expression system was thereby developed for PLD. First, PLDs fromStreptomycesPMF andStreptomyces racemochromogeneswere separately over-expressed inE.colito compare their transphosphatidylation activity based on the synthesis of phosphatidylserine (PS), and PLDPMFwas determined to have higher activity. To further improve PLDPMFsynthesis, a secretory expression system suitable for PLDPMFwas constructed and optimized with different signal peptides. The highest secretory efficiency was observed when the PLD * (PLDPMFwith the native signal peptide Nat removed) was expressed fused with the fusion signal peptide PelB-Nat inE. coli. The fermentation conditions were also investigated to increase the production of recombinant PLD and 10.5 U/mL PLD was ultimately obtained under the optimized conditions. For the application of recombinant PLD to PS synthesis, the PLD properties were characterized and 30.2 g/L of PS was produced after 24 h of bioconversion when 50 g/L phosphatidylcholine (PC) was added.Keywords:phospholipase D;phosphatidylserine;secretory expression;enzymatic catalysis
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BIOCHEMICAL ENGINEERING JOURNAL
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2020
·
IF 3.475
Nan Jiang,Xuanwei Ding,Yuan Lu
Since the cell-free protein synthesis system is not limited by the cell growth, all the substrates are used to produce the protein of interest, and the reaction environment can be flexibly controlled. All the advantages allow it to synthesize toxic proteins, membrane proteins, and unnatural proteins that are difficult to make in vivo . However, one typical reason why the cell-free system has not been widely accepted as a practical alternative, is its expression efficiency problem. The Escherichia coli -based system was chosen in this study, and the model protein deGFP was expressed to explore a more efficient cell-free system. The results showed that Mg 2+ with a concentration of 15 mM in the cell-free system with BL21 Star (DE3) as the extract could better synthesize protein. The smaller the vectors, the lighter the burden, the higher the protein synthesis. Simulating the crowding effect in the cell does not improve the protein expression efficiency of the optimized cell-free protein synthesis system. Based on the optimized system, the cell-free fundamental research platform, primary screening platform, and portable biomolecular synthesis platform were established. This study provides a robust cell-free protein synthesis toolbox with easy extract preparation and high protein yield. It also enables more researchers to reap the benefits from the cell-free biosynthesis platform.
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CHEMOSPHERE
·
2020
·
IF 5.778
Juan Liu,Zhiyan Huang,Bo Ruan,Huimin Wang,Meiqing Chen,Saeed Rehman,Pingxiao Wu
Polymyxin B is increasingly employed all over the world to treat patients who affected by multidrug-resistant Gram-negative bacteria. Although the mechanism of resistance to polymyxin B is well known, the metabolic role of bacteria in stress response to polymyxin B remains an important task and may help to better understand polymyxin B-related stress response. In this study, the proteome changes of Escherichia coli ( E. coli ) continuously induced in concentrations of 1.0 mg/L and 10.0 mg/L polymyxin B were revealed. Compared to E. coli (PMB 0 ), E. coli exposed to polymyxin B at 1.0 mg/L (PMB 1 ) and 10.0 mg/L (PMB 10 ) resulted in 89 and 314 differentially expressed proteins (DEPs), respectively. Such differences related to fatty acid degradation, quorum sensing and two-component regulatory system pathways. Based on absolute quantitative (iTRAQ) proteomics analysis, this study comprehensively studied the changes of E. coli proteome in culture with concentrations of 1.0 mg/L and 10.0 mg/L polymyxin B through confocal laser scanning microscopy observation, cell viability detection and reactive oxygen species analysis. The results showed that E. coli cultured at concentration of 10.0 mg/L polymyxin B increased the expression levels of multidrug-resistant efflux transporters and efflux pump membrane transporters, which might further improve the pathogens of polymyxin B-resistant bacteria lastingness and evolution. It has emerged globally to resist polymyxin B. The reuse of polymyxin B should be aroused public attention to avoid causing more serious environmental pollution . These findings could provide new insights into polymyxin B-related stress.
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危险品化学品经营许可证（带存储）