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
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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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DOI
高影响因子
BIOTECHNOLOGY & BIOTECHNOLOGICAL EQUIPMENT
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2020
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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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DOI
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2023
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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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DOI
PubMed
高影响因子
ACS Catalysis
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2025
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IF 13.1
Lixin Kang,Haoran Li,Kexin Lin,Shen Hu,Song Liu,Yuben Qiao,Ying Wang,Aitao Li
The biocatalytic production of 25-hydroxyvitamin D3 (25(OH)VD3, calcidiol) represents a superior alternative to traditional chemical synthesis. However, the reported VD3 hydroxylases generally exhibit suboptimal catalytic efficiency, limiting their practical applications. In this study, a cytochrome P450 CYP107Pdh from Pseudonocardia dioxanivorans_CB1190, which displays unique C26 hydroxylation activity on VD3, was identified. Then, structure-guided loop engineering combined with binding pocket reshaping was conducted on CYP107Pdh, leading to the generation of the quintuple variant 89_90insIP/T112A/V161L/G186V (M4). This variant shifted the regioselectivity from 91% C26 in the wild type (WT) to 81% C25 for calcidiol production. In addition, variant M4 showed a remarkable enhancement in catalytic activity, achieving a catalytic efficiency (kcat/Km) that is 75-fold higher than that of the WT. Computational analyses revealed that the regioselectivity shift and activity improvement are primarily attributed to a conformational transition in the substrate-binding pocket from an open to a more closed state, which optimizes substrate binding and facilitates efficient 25-hydroxylation of VD3. Finally, a semipreparative biotransformation yielded 2.64 g of crystalline calcidiol (95% purity) from a 1-L reaction, thereby expanding the enzyme library of VD3 hydroxylases and underscoring its potential for industrial-scale production of calcidiol.
研究领域:
生物化学
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酶工程
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药物合成
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DOI
高影响因子
Molecular Catalysis
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2024
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IF 3.9
Yuqiu Wu,Fanjin Meng,Jiaolong Fu,Yuyang Zhou,Huan Fan,Xin Ju,Liangzhi Li
D-xylose, a biomass derivative, is used as a substrate to generate D-xylulose and D-ribulose with high-added value through a cascade reaction. During this process, the thermal stability and catalytic efficiency of rate-limiting enzymes during cascade reactions are important issues that must be addressed in industrial applications. In this study, we prepared and validated PDA/SiO 2 microspheres as highly loaded immobilization carriers by oxidative self-polymerization of polydopamine (PDA) on silica (SiO 2 ) microspheres. D-xylose was successfully catalyzed by the co-immobilized D-5-phosphate ribose isomerase B (RpiB) and D-tagatose-3-epimerase (DTE), denoted PDA/SiO 2 -RpiB-DTE, to produce D-ribulose and D-xylulose. After immobilization, the K m of co-immobilized RpiB and DTE decreased, which suggested increased substrate affinity. The half-life of the co-immobilized enzymes was 3.5–8 fold higher than that of the free enzymes at 50–70 °C, and the inactivation rate constant was reduced, demonstrating that immobilization improved significantly the thermal stability. The conversion of D-xylose at 10 mM was 29.98% under optimal conditions, which contained 9.85% D-ribulose and 20.13% D-xylulose. PDA/SiO 2 -RpiB-DTE showed up to 89.10% initial enzyme activity after 3 recycle runs. The thermal stability and recovery aspects were improved by immobilization, which facilitates the use of biomass to produce high-value-added products and further exploitation.
研究领域:
生物技术
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酶工程
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生化工程
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DOI
高影响因子
Nature Communications
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2025
·Vol. 16
·期 1
·1-19
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IF 15.7
Qi Houbao,Gao Haiting,Li Meihui,Sun Tianyong,Gu Xiufeng,Wei Li,Zhi Mengfan,Li Zixuan,Fu Dachuan,Liu Yiran,Wei Ziyi,Dou Yu,Feng Qiang
Parvimonas micra ( P. micra ), an opportunistic oral pathogen associated with multiple cancers, has limited research on its role in oral squamous cell carcinoma (OSCC). This study shows that P. micra is enriched in OSCC tissues and positively correlated with tumor metastasis and stages. P. micra infection promotes OSCC metastasis by inducing hypoxia/HIF-1α, glycolysis, and autophagy. Mechanistically, P. micra surface protein TmpC binds to CKAP4, a receptor overexpressed in OSCC, facilitating bacterial attachment and invasion. This interaction activates HIF-1α and autophagy via CKAP4-RanBP2 and CKAP4-NBR1 pathways, driving metastasis. Targeting CKAP4 with masitinib or antibodies impairs P. micra attachment and abolishes P. micra -promoted OSCC metastasis in vitro and in vivo. Together, our findings identify P. micra as a pathogen that promotes OSCC metastasis and highlight that TmpC-CKAP4 interaction could be a potential therapeutic target for OSCC.
研究领域:
肿瘤学
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微生物学
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分子生物学
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癌症研究
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DOI
PubMed
高影响因子
BIOMACROMOLECULES
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2024
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IF 5.5
Yidan Chen,Tiantian Feng,Xiaohong Zhu,Yuting Tang,Yao Xiao,Xiuhua Zhang,Sheng-Fu Wang,Dong Wang,Wei Wen,Jichao Liang,Huayu Xiong
Reactive oxygen species (ROS) have emerged as a promising treatment option for antibacterial and biofilm eradication. However, their therapeutic efficacy is significantly hampered by the unique microenvironments of diabetic wounds. In this study, we designed and synthesized porphyrin-based Fe covalent organic frameworks (Fe-COF) through a Schiff base condensation reaction. Subsequently, Fe-COF were encapsulated with hyaluronic acid (HA) through electrostatic adsorption, resulting in a novel formulation named HA-Fe-COF for diabetic wound healing. HA-Fe-COF were engineered to respond to hyaluronidase in the infected wound, leading to the controlled release of Fe-COF. Those released Fe-COF served a dual role as photosensitizers, generating singlet oxygen and localized heating when exposed to dual light sources. Additionally, they acted as peroxidase-like nanozymes, facilitating the production of ROS through enzymatic reactions. This innovative approach enabled a synergistic therapeutic effect combining photodynamic, photothermal, and chemodynamic modalities. Furthermore, the sustained release of HA from HA-Fe-COF promoted angiogenesis, collagen deposition, and re-epithelialization during the diabetic wound healing process. This “all-in-one” strategy offers a novel approach for the development of antimicrobial and biofilm eradication strategies that minimize damage to healthy tissues in vivo.
研究领域:
材料科学
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纳米技术
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生物医学工程
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伤口愈合
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药学
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DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
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2025
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IF 8.5
Jingqi Sun,Jiaxin Li,Yanzhi Xing,Han Leng,Wei Chen,Yanfeng Zhang,Xuwei Chen
Adenine DNA glycosylase (MutY) is a crucial member of DNA glycosylase family, and the abnormal expression of the human MutY homologs is associated with the pathogenesis of various diseases, therefore, convenient and cost-effective assessing the activity of MutY holds significant biological and medical importance. Herein, the precise identification/excision capacity of MutY to mismatched G-A base pair of dsDNA chains and the DNA-template-dependant fluorescence behaviors of copper nano cluster (CuNCs) was exploited for the accurate sensing of active MutY. Hairpin DNA with G-A base mismatch was excised by MutY to produce dsDNA chains with repetitive AAT-TTA base pairs. The newly formed dsDNA provided more active sites for the growth of CuNCs compared to the original hairpin DNA, resulting in the significantly enhanced fluorescence of final CuNCs. MutY was accurately quantified with a detection limit of 9.98 nmol L −1 . The developed sensing protocol exhibited excellent selectivity toward MutY over various ions, neutral biomolecules, and protein species. Most importantly, The sensing system is capable to distinguish the active MutY from other MutY homologs with low activity, e.g., de-[4Fe4S] cluster MutY (DIS-MutY), and the practicality was well demonstrated by detecting active MutY contents in various cell lysates.
研究领域:
生物化学
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分子生物学
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医学诊断
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DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
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2024
·
IF 7.7
Guocong Luo,Zhaolin Huang,Yingying Zhu,Jiajun Chen,Xiaodong Hou,Dawei Ni,Wei Xu,Wenli Zhang,Yijian Rao,Wanmeng Mu
Lacto-N-neotetraose (LNnT), a representative oligosaccharide found in human milk, has been previously examined for its beneficial traits. However, the LNnT titer is limited by the efficient glycosyltransferase pathway, particularly with respect to the catalysis of rate-limiting steps. As data on the crystal structure of the key enzyme required for synthesizing LNnT are lacking, the synthesis of LNnT remains an uncertainty. Here, for the first time we report the three-dimensional structure of a bacterial β-1,4-galactosyltransferase, Aaβ4GalT, and analyze the critical role played by residues in its catalytic efficacy. Guided by structural insights, we engineered this enzyme to enhance its catalytic efficiency using structure-guided tunnel engineering. The mutant enzyme L5 (K155M/H156D/F157W/K185M/Q216V) so produced, showed a 50-fold enhancement in catalytic activity. Crystal structure analysis revealed that the mechanism underlying the improvement in activity was of the swing door type. The closed conformation formed by dense hydrophobic packing with Q216V-K155M widened and permitted substrate entry. Our results show that altering the tunnel conformation helped appropriately accommodate the substrate for catalysis and provide a structural basis for the modification of other glycosyltransferases.
研究领域:
生物化学
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结构生物学
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DOI
PubMed
高影响因子
ACS Synthetic Biology
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2024
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IF 3.7
Qing Wang,Jiajia You,Yichen Li,Jie Zhang,Yi Wang,Meijuan Xu,Zhiming Rao
In vivo targeted mutagenesis technologies are the basis for the continuous directed evolution of specific proteins. Here, an efficient mutagenesis system (CgMutaT7) for continuous evolution of the targeted gene in Corynebacterium glutamicum was developed. First, cytosine deaminase and uracil-DNA glycosylase inhibitor were sequentially fused to T7 RNA polymerase using flexible linkers to build the CgMutaT7 system, which introduces mutations in targeted regions controlled by the T7 promoter. After a series of optimizations, the resulting targeted mutagenesis system (CgMutaT74) can increase the mutant frequency of the target gene by 1.12 × 104-fold, with low off-target mutant frequency. Subsequently, high-throughput sequencing further revealed that the CgMutaT74 system performs efficient and uniform C → T transitions in at least a 1.8 kb DNA region. Finally, the xylose isomerase was successfully continuously evolved by CgMutaT74 to improve the xylose utilization, indicating that the CgMutaT7 system has great potential for applications in the continuous evolution of protein function and expression components.
研究领域:
合成生物学
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分子遗传学
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DOI
PubMed
高影响因子
Journal of Chemical Information and Modeling
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2025
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IF 5.3
Binbin Chen,Jiahui Huang,Yingchun Liu,Lirong Yang,Qi Wang,Haoran Yu
The catalytic mechanism of a pyridoxal 5′-phosphate (PLP)-dependent UstD was herein studied in atomic detail, employing the computational hybrid QM/MM methodology. UstD is a PLP-dependent enzyme that catalyzes the decarboxylative aldol reactions between l-aspartate and aldehyde or ketone derivatives to form γ-hydroxy-α-amino acids. In the reaction catalyzed by UstD, the loss of CO2 renders the C–C bond-forming reaction effectively irreversible, which makes UstD a special case among the enzymes catalyzing the C–C bond-forming reactions. This enzyme is currently seen as the optimal approach for the regioselective synthesis of γ-hydroxy-α-amino acids, which are very difficult to obtain by standard chemical methods. The results obtained herein showed that the catalytic mechanism of UstD might follow two paths to occur in three phases: (1) decarboxylation of substrate l-aspartate, (2) C–C bond formation by addition of aldehyde, and (3) the regeneration of catalytic sites. Although Path A and Path B showed a negligible difference in the energy barrier of the rate-determining step, Path A involves three additional steps in the overall pathway compared with Path B, which makes the reaction proceed more readily through Path B. According to the QM/MM energy profile of Path B, the rate-limiting step of the catalytic process is the decarboxylation of the side chain of l-aspartate, which has a calculated energy barrier of 19.19 kcal/mol. Two crucial residues, H263 and Y257, were identified to interact with the substrate aspartic acid. The knowledge about the transition states, intermediates, key residues, and protein conformational changes along the reaction path will be valuable for engineering UstD to improve the synthesis of γ-hydroxy-α-amino acids that serve as building blocks of various high-value chemicals such as antidiabetics and nutritional supplements.
研究领域:
生物化学
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计算生物学
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产品关联
DOI
PubMed
高影响因子
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