ACS Sustainable Chemistry & Engineering
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2024
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IF 7.1
Yicong Lu,Xiao Zheng,Wan-Qiu Liu,Shengjie Ling,Jian Li
Cell-free protein synthesis (CFPS) has proven invaluable for expressing a wide array of proteins and enzymes, boasting significant advantages, such as facile manipulation, rapid mass transfer, and high productivity. However, traditional cell-free expression systems typically operate in batch format reactions, posing challenges for enzyme reuse in biocatalysis and rendering supplemented gene templates nonrecyclable. To overcome this limitation, we introduce a chitin-functionalized CFPS system designed to immobilize in vitro-expressed enzymes and gene templates for sustainable biocatalysis and protein synthesis. At its core, this system capitalizes on the strong binding affinity between a chitin-binding domain (ChBD) and crystalline chitin nanofibers (ChNFs). Specifically, we engineer ChBD fusion with target proteins and enzymes, allowing for their cell-free expression and in situ immobilization on ChNFs. This facilitates the effortless recycling of enzymes for multiple biocatalytic reactions, while ChNFs-immobilized enzymes can also be deployed in continuous flow biocatalysis setups. Leveraging the ChBD-ChNF pairing, gene templates can likewise be immobilized and recycled for sustained gene expression. Our results demonstrate that the chitin-functionalized cell-free system significantly enhances CFPS performance through immobilization with ChNF materials. This work underscores the flexibility and robustness of cell-free systems, which can seamlessly integrate with advanced techniques from fields such as chemistry and materials science for impactful applications.
研究领域:
生物化学
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合成生物学
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材料科学
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DOI
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
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2024
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IF 7.7
Tengfei Xiong,Mingyue Yu,Mei Li,Christopher A. Reilly,Xinghui Qiu
Capsaicin and dihydrocapsaicin are the principal pungent compounds in hot peppers. The generalist Helicoverpa armigera and the specialist H. assulta are two of the few insects that can feed on hot pepper fruits. To further understand the mechanisms behind physiological adaptation to capsaicinoids in the two agricultural pests, we investigated the transcriptional response and metabolism capacity of CYP6B6 orthologs from the two closely related species to the capsaicinoids. The results showed that the expression of CYP6B6 was either weakly or moderately induced by capsaicin in a tissue-specific and dose-dependent manner. CYP6B6 induction to greater extent was observed in the larval midguts of the cotton bollworms, indicating that inducible defense is more robust in the generalist. The two CYP6B6 enzymes were able to efficiently metabolize both capsaicin and dihydrocapsaicin, and HassCYP6B6 exhibit a slightly higher activity towards capsaicin than HarmCYP6B6, as evaluated by the specific activity and Vmax values. Multiple known and yet to be elucidated metabolites were detected in the reactions catalyzed by CYP6B6s, with dehydrogenation at the terminal carbon followed by N-macrocyclization and aliphatic hydroxylation being the dominant pathways. The data support the hypothesis that these two Helicoverpa species have well adapted to capsaicinoids via their high P450-mediated detoxification ability.
研究领域:
昆虫学
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分子生物学
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植物与昆虫互作
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PubMed
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
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2024
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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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PubMed
高影响因子
Catalysts
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2024
·Vol. 14
·期 12
·911
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IF 3.8
Shen Yu,Xue-Yong Li,Wen-Jie Si,Ye-Wang Zhang
Formolase, a thiamine pyrophosphate (TPP)-dependent enzyme, catalyzes the carboligation of three one-carbon formaldehyde molecules into one three-carbon dihydroxyacetone molecule. It has many important functions in the biosynthesis of carbon-based compounds and utilization of CO2. However, the enzyme has low activity and stability in the catalytic process, resulting in high cost in the applications. To improve the stability, formolase was immobilized onto magnetic nanoparticles, which were designed to have functional epoxy groups for covalently binding the enzyme. In the immobilization, effects of pH, temperature, and cofactor TPP on the immobilization were investigated and optimized. The results showed that the retention activity of immobilized formolase was highly related to TPP. In the presence of TPP, the specific activity of the immobilized formolase was 6.8 times higher than that without TPP. The optimal immobilization conditions were as follows: a temperature of 20 °C, a pH of 7.0, an immobilization time of 8 h, and an enzyme loading of 20 mg/g. Molecular docking was used to analyze the effect of TPP on the stabilization of the enzyme in the immobilization, which indicated that TTP could stabilize the enzyme structure during the immobilization. The stabilization effect of TPP could be a reference in the immobilization of other enzymes with TPP as the cofactor.
研究领域:
生物化学
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酶工程
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高影响因子
CURRENT ISSUES IN MOLECULAR BIOLOGY
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2024
·Vol. 46
·期 9
·9916-9927
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IF 2.8
Miao Tian,Yuan-Yuan Xu,Yang-Nan Li,Shen Yu,Yi-Lin Wang,Xiao-Lai Ma,Ye-Wang Zhang
Dermatan sulfate and chondroitin sulfate are dietary supplements that can be utilized as prophylactics against thrombus formation. Low-molecular-weight dermatan sulfate (LMWDS) is particularly advantageous due to its high absorbability. The enzymatic synthesis of low-molecular-weight dermatan sulfates (LMWDSs) using chondroitin B lyase is a sustainable and environmentally friendly approach to manufacturing. However, the industrial application of chondroitin B lyases is severely hampered by their low catalytic activity. To improve the activity, a semi-rational design strategy of engineering the substrate-binding domain of chondroitin B lyase was performed based on the structure. The binding domain was subjected to screening of critical residues for modification using multiple sequence alignments and molecular docking. A total of thirteen single-point mutants were constructed and analyzed to assess their catalytic characteristics. Out of these, S90T, N103C, H134Y, and R159K exhibited noteworthy enhancements in activity. This study also examined combinatorial mutagenesis and found that the mutant H134Y/R159K exhibited a substantially enhanced catalytic activity of 1266.74 U/mg, which was 3.21-fold that of the wild-type one. Molecular docking revealed that the enhanced activity of the mutant could be attributed to the formation of new hydrogen bonds and hydrophobic interactions with the substrate as well as neighbor residues. The highly active mutant would benefit the utilization of chondroitin B lyase in pharmaceuticals and functional foods.
研究领域:
生物工程
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酶学
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医药生物技术
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PubMed
高影响因子
FOOD CHEMISTRY
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2024
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IF 8.5
Weijun Leng,Ying Li,Xin Liang,Li Yuan,Xiuting Li,Ruichang Gao
Protein glutaminase (PG; EC 3.5.1.44) is a class of food-grade enzyme with the potential to significantly improve protein functionality. However, its low catalytic activity and stability greatly hindered industrial application. In this study, we employed structural-based engineering and computational-aided design strategies to target the engineering of protein glutaminase PG5, which led to the development of a combinatorial mutant, MT8, exhibiting a specific activity of 31.1 U/mg and a half-life of 216.2 min at 55 °C. The results indicated that the flexible region in MT8 shifted from the C-terminus to the N-terminus, with increased N-terminal flexibility positively correlating with its catalytic activity. Additionally, MT8 notably boosted fish myofibrillar proteins (MPs) solubility under the absence of NaCl conditions and enhanced their foaming and emulsifying properties. Key residues like Asp 31 , Ser 72 , Asn 121 , Asp 471 , and Glu 485 were crucial for maintaining PG5-myosin interaction, with Ser 72 and Asn 121 making significant energy contributions.
研究领域:
食品科学
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酶工程
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蛋白质工程
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计算生物学
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PubMed
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
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2024
·Vol. 90
·期 9
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IF 3.9
Yuan Li,Shu-Fang Li,Lin Zhang,Ya-Ping Xue,Yu-Guo Zheng
Hydroxysteroid dehydrogenases (HSDHs) catalyze the oxidation/reduction of hydroxyl/keto groups of steroids with high regio- or stereoselectivity, playing an essential role in producing optically pure chemicals. In this work, a novel approach was developed to simultaneously improve the stability and activity of 7β-hydroxysteroid dehydrogenase (7β-HSDH) by combining B-factor analysis and computer-aided prediction. Several advantageous mutants were identified, and the most promising variant, S51Y/P202Y, exhibited 2.3-fold improvements in catalytic activity, 3.3-fold in half-life at 40°C, and 4.7-fold in catalytic efficiency (kcat/Km), respectively. Structural modeling analysis showed that the shortened reversible oxidation reaction catalytic distance and the strengthened residue interactions compared to the wild type were attributed to the improved stability and activity of the obtained mutants. To synthesize ursodeoxycholic acid cost-effectively by mutant S51Y/P202Y, a NAD-kinase was employed to facilitate the substitution of nicotinamide adenine dinucleotide phosphate (NADP+) with nicotinamide adenine dinucleotide (NAD+) in the whole-cell catalysis system. The substrate 7-ketolithocholic acid (100 mM) was converted completely in 0.5 h, achieving a space-time yield of 1,887.3 g L−1 d−1. This work provided a general target-oriented strategy for obtaining stable and highly active dehydrogenase for efficient biosynthesis.
研究领域:
生物技术
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酶工程
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生物化学
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化学生物学
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DOI
PubMed
高影响因子
Cell Systems
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2024
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IF 9
Runtao Zhu,Jiao Zhang,Lin Wang,Yunfeng Zhang,Yang Zhao,Ying Han,Jing Sun,Xi Zhang,Ying Dou,Huaxiong Yao,Wei Yan,Xiaozhou Luo,Junbiao Dai,Zhuojun Dai
Functionalizing materials with biomacromolecules such as enzymes has broad applications in biotechnology and biomedicine. Here, we introduce a grafting method mediated by living cells to functionalize materials. We use polymeric scaffolds to trap engineered bacteria and micron-sized particles with chemical groups serving as active sites for grafting. The bacteria synthesize the desired protein for grafting and autonomously lyse to release it. The released functional moieties are locally grafted onto the active sites, generating the materials engineered by living grafting (MELGs). MELGs are resilient to perturbations because of both the bonding and the regeneration of functional domains synthesized by living cells. The programmability of the bacteria enables us to fabricate MELGs that can respond to external input, decompose a pollutant, reconstitute synthetic pathways for natural product synthesis, and purify mismatched DNA. Our work establishes a bacteria-assisted grafting strategy to functionalize materials with a broad range of biological activities in an integrated, flexible, and modular manner. A record of this paper’s transparent peer review process is included in the supplemental information .
研究领域:
生物技术
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生物医学工程
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合成生物学
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PubMed
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2024
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IF 5.7
Jingyu Zhao,Huanhuan Sun,Gege Wang,Qi Wang,Yipeng Wang,Qingbin Li,Shuangyu Bi,Qingsheng Qi,Qian Wang
Two-component systems (TCSs) sensing and responding to various stimuli outside and inside cells are valuable resources for developing biosensors with synthetic biology applications. However, the use of TCS-based biosensors suffers from a limited effector spectrum, hypersensitivity, low dynamic range, and unwanted signal crosstalk. Here, we developed a tailor-made Escherichia coli whole-cell γ-aminobutyric acid (GABA) biosensor by engineering a chimeric GABA chemoreceptor PctC and TCS. By testing different TCSs, the chimeric PctC/PhoQ showed the response to GABA. Chimera-directed evolution and introduction of the insulated chimeric pair PctC/PhoQ*PhoP* produced biosensors with up to 3.50-fold dynamic range and good orthogonality. To further enhance the dynamic range and lower the basal leakage, three strategies, engineering of PhoP DNA binding sites, fine-tuning reporter expression by optimizing transcription/translation components, and a tobacco etch virus protease-controlled protein degradation, were integrated. This chimeric biosensor displayed a low basal leakage, a large dynamic range (15.8-fold), and a high threshold level (22.7 g L–1). Finally, the optimized biosensor was successfully applied in the high-throughput microdroplet screening of GABA-overproducing Corynebacterium glutamicum, demonstrating its desired properties for extracellular signal biosensing.
研究领域:
合成生物学
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生物技术
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分子生物学
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微生物学
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DOI
PubMed
高影响因子
Bioresources and Bioprocessing
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2025
·Vol. 12
·期 1
·1-14
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IF 5.1
Ai Yulin,Huang Yusong,Zhao Hongru,Su Bingmei,Lin Juan
5-Hydroxytryptophan (5-HTP) is widely used as a natural remedy for sleep disorders. In terms of biosafety, bio-derived 5-HTP is preferred over chemically synthesized 5-HTP. However, the low titer of 5-HTP in the reported microbiological methods (< 10 g/L) limits the industrialization of 5-HTP biosynthesis. In the present study, a Trp-accumulating E. coli strain TRP1 was constructed by blocking the degradation path (Δ tnaA ), branching paths (Δ pheA , Δ tyrA ) and repression system (Δ trpR , Δ trpL ). Next, the hydroxylation module employing a phenylalanine hydroxylase mutant XcPAH W179F (XC2) coupled with an MH4 regenerating system (CvPCD-EcFolM system) was screened to convert L-Trp into 5-HTP. Protein engineering was performed on hydroxylase XC2 based on the molecular dynamics simulation of the enzyme-substrate complex, and the strain TRP1-XC4 harboring the triple-mutant XcPAH L98I/A129K/W179F (XC4) was able to produce 319.4 mg/L 5-HTP. Genome editing was carried out focused on accelerating product efflux (strengthening YddG) and increasing MH4 supply (strengthening FolM, FolE and FolX), resulting in a strain TRP5-XC4 to produce 13.9 g/L 5-HTP in 5 L fed-batch fermentation with a space-time yield of 0.29 g/L/h, which is the highest production and productivity record for 5-HTP biosynthesis. This study successfully provided an engineered strain and an efficient green method for the industrial synthesis of 5-HTP. Graphical
研究领域:
生物工程
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微生物学
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合成生物学
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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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PubMed
高影响因子
危险品化学品经营许可证（带存储）