PLoS One
·
2019
·Vol. 14
·期 5
·e0216178
·
IF 2.776
Anamya Ajjolli Nagaraja,Nicolas Fontaine,Mathieu Delsaut,Philippe Charton,Cedric Damour,Bernard Offmann,Brigitte Grondin-Perez,Frederic Cadet
The selection of optimal enzyme concentration in multienzyme cascade reactions for the highest product yield in practice is very expensive and time-consuming process. The modelling of biological pathways is a difficult process because of the complexity of the system. The mathematical modelling of the system using an analytical approach depends on the many parameters of enzymes which rely on tedious and expensive experiments. The artificial neural network (ANN) method has been successively applied in different fields of science to perform complex functions. In this study, ANN models were trained to predict the flux for the upper part of glycolysis as inferred by NADH consumption, using four enzyme concentrations i.e., phosphoglucoisomerase, phosphofructokinase, fructose-bisphosphate-aldolase, triose-phosphate-isomerase. Out of three ANN algorithms, the neuralnet package with two activation functions, "logistic" and "tanh" were implemented. The prediction of the flux was very efficient: RMSE and R2 were 0.847, 0.93 and 0.804, 0.94 respectively for logistic and tanh functions using a cross validation procedure. This study showed that a systemic approach such as ANN could be used for accurate prediction of the flux through the metabolic pathway. This could help to save a lot of time and costs, particularly from an industrial perspective. The R-code is available at: https://github.com/DSIMB/ANN-Glycolysis-Flux-Prediction.
研究领域:
生物信息学
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代谢工程
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计算生物学
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酶学
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DOI
PubMed
高影响因子
Frontiers in Chemistry
·
2021
·
IF 5.221
Jia Hao-Yu,Yang Zi-Yue,Chen Qi,Zong Min-Hua,Li Ning
Catalytic promiscuity is a promising starting point for improving the existing enzymes and even creating novel enzymes. In this work, site-directed mutagenesis was performed to improve promiscuous alcohol dehydrogenase activity of reductive aminase from Aspergillus oryzae (AspRedAm). AspRedAm showed the cofactor preference toward NADPH in reductive aminations, while it favored NADH in the reduction reactions. Some key amino acid residues such as N93, I118, M119, and D169 were identified for mutagenesis by molecular docking. Variant N93A showed the optimal pH and temperature of 8 and 30°C, respectively, in the reduction of 5-hydroxymethylfurfural (HMF). The thermostability was enhanced upon mutation of N93 to alanine. The catalytic efficiency of variant N93A (kcat/Km, 23.6 mM−1 s−1) was approximately 2-fold higher compared to that of the wild-type (WT) enzyme (13.1 mM−1 s−1). The improved catalytic efficiency of this variant may be attributed to the reduced steric hindrance that stems from the smaller side chain of alanine in the substrate-binding pocket. Both the WT enzyme and variant N93A had broad substrate specificity. Escherichia coli (E. coli) cells harboring plain vector enabled selective reduction of biobased furans to target alcohols, with the conversions of 35–95% and the selectivities of >93%. The introduction of variant N93A to E. coli resulted in improved substrate conversions (>98%) and selectivities (>99%).
研究领域:
生物化学
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酶学
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生物催化
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代谢工程
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产品关联
DOI
PubMed
高影响因子
Antibiotics-Basel
·
2024
·Vol. 13
·期 6
·528
·
IF 4.3
Muchen Zhang,Huangwei Song,Siyuan Yang,Yan Zhang,Yunrui Tian,Yang Wang,Dejun Liu
The emergence of carbapenem-resistant Gram-negative pathogens presents a clinical challenge in infection treatment, prompting the repurposing of existing drugs as an essential strategy to address this crisis. Although the anticancer drug 5-fluorouracil (5-FU) has been recognized for its antibacterial properties, its mechanisms are not fully understood. Here, we found that the minimal inhibitory concentration (MIC) of 5-FU againstEscherichia coliwas 32–64 µg/mL, including strains carryingblaNDM-5, which confers resistance to carbapenems. We further elucidated the antibacterial mechanism of 5-FU againstE. coliby using genetic and biochemical analyses. We revealed that the mutation of uracil phosphoribosyltransferase-encoding geneuppincreased the MIC of 5-FU againstE. coliby 32-fold, indicating the role of theuppgene in 5-FU resistance. Additionally, transcriptomic analysis ofE. colitreated with 5-FU at 8 µg/mL and 32 µg/mL identified 602 and 1082 differentially expressed genes involved in carbon and nucleic acid metabolism, DNA replication, and repair pathways. The biochemical assays showed that 5-FU induced bacterial DNA damage, significantly increased intracellular ATP levels and the NAD+/NADH ratio, and promoted reactive oxygen species (ROS) production. These findings suggested that 5-FU may exert antibacterial effects onE. colithrough multiple pathways, laying the groundwork for its further development as a therapeutic candidate against carbapenem-resistant bacterial infections.
研究领域:
微生物学
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药理学
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遗传学
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生物化学
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传染病学
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产品关联
DOI
PubMed
高影响因子
PROCESS BIOCHEMISTRY
·
2024
·
IF 3.7
Pengpeng Cheng,Zhenyu He,Bo Liu,Jinmei Wang,Chuyue Zhang,Lan Tang,Lihua Du,Yuan Lu,Zhimin Ou
Nano-bienzyme conjugates (nano-BECs) for catalysing one-pot reactions can simplify the reaction process and improve the efficiency of biocatalytic reactions. In this work glucose dehydrogenase (GDH) and Candida parapsilosis secondary alcohol dehydrogenase (CpSADH) were immobilised with polyacrylic acid (PAA) to form two types of PAA-based nano-BECs (2-S, 2-P), which achieved in situ regeneration of NADH for the production of chiral alcohols in oil-water biphasic systems, such as the key chiral pharmaceutical intermediate of ( S )-1-(3-trifluoromethylphenyl)ethanol (( S )-TMPE). At a substrate concentration of 100 mM 3-trifluoromethylacetophenone (TMAP), the conversion catalyzed by nano BEC was 98.2 % in dipentyl phthalate-phosphate buffer biphasic system. In contrast, the conversion was only 39.5 % and 38.9 % in the PB buffer and 8.4 % and 7.7 % in dipentyl phthalate system. The experiments on kinetic constants demonstrated that the catalytic efficiencies (K cat /K m ) of nano-BECs (2-S, 2-P) were enhanced by 6.7-fold and 7-fold, respectively, compared to free CpSADH. Additionally, nano-BECs showed stronger coenzyme regeneration ability. Under optimal reaction conditions, nano-BECs achieved 96.2 % conversion of TMAP to ( S )-TMPE in an organic-buffer system with e.e. > 99.6 % at 300 mM substrate concentration. This research expands the potential applications of polymers and proteins, and offers valuable insights into the synergistic effects of PAA on bienzyme catalytic systems in biocatalysis.
研究领域:
生物催化
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药物化学
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材料科学
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酶学
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产品关联
DOI
高影响因子
ACS Sustainable Chemistry & Engineering
·
2018
·
IF 6.14
Dong Yang,Yishan Zhang,Hongjian Zou,Shaohua Zhang,Yizhou Wu,Ziyi Cai,Jiafu Shi,Zhongyi Jiang
Nicotinamide cofactors (e.g., NADH) are essential hydrogen sources for the majority of enzyme reduction reactions. Currently, highly efficient and in situ cofactor regeneration is urgently required but remains challenging. In this study, red phosphorus quantum dots were decorated onto graphitic carbon nitride hollow tubes to prepare high-performance heterojunction photocatalyst (g-C3N4-HTs@rP-QDs) for visible-light-driven NADH regeneration. Energy band analysis combined with photoelectrochemical measurements demonstrates rP-QDs not only act as additional photosensitizers for charge generation but also constitute a type II heterojunction with g-C3N4-HTs for charge transfer, facilitating enrichment of electrons on the g-C3N4-HTs surface. Such enrichment of electrons for g-C3N4-HTs@rP-QDs offers ∼5 times higher reducing active sites than bulk g-C3N4 to trigger the NADH regeneration reaction under visible-light (LED, 405 nm) illumination, thus acquiring superior NADH regeneration efficiency with an initial reaction rate (2 min) of 0.247 ± 0.012 mmol g–1 min–1. In the concerned experimental range, higher g-C3N4-HTs/rP-QDs mass ratios or light intensities that are beneficial for the electron enrichment could result in enhanced regeneration efficiency. The bioactivity of NADH toward enzyme catalysis is further demonstrated by alcohol dehydrogenase-catalyzed hydrogenation of formaldehyde, which enables the sustainable production of methanol.
研究领域:
材料科学
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化学工程
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光化学
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生物催化
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环境科学
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产品关联
DOI
高影响因子
JOURNAL OF BIOTECHNOLOGY
·
2022
·
IF 3.595
Feng Cheng,Wei-Bang Xie,Xiao-Fan Gao,Rong-Liang Chu,Shen-Yuan Xu,Ya-Jun Wang,Yu-Guo Zheng
( S )-2-chlorophenylglycine (( S )-CPG) is a key chiral intermediate for the synthesis of clopidogrel . Herein, a novel, efficient and environmentally friendly chemo-enzymatic route for the preparation of optically pure ( S )-CPG was developed. A straightforward chemical synthesis of the corresponding prochiral keto acid substrate (2-chlorophenyl)glyoxylic acid (CPGA) was developed with 91.7% yield, which was enantioselectively aminated by leucine dehydrogenase (LeuDH) to ( S )-CPG. Moreover, protein engineering of LeuDH was performed via directed evolution and semi-rational design. A beneficial variant Es LeuDH-F362L with enlarged substrate-binding pocket and increased hydrogen bond between K77 and substrate CPGA was constructed, which exhibited 2.1-fold enhanced specific activity but decreased thermal stability . Coupled with a glucose dehydrogenase from Bacillus megaterium ( Bm GDH) for NADH regeneration, Es LeuDH-F362L completely converted up to 0.5 M CPGA to ( S )-CPG in 8 h at 40 °C.
研究领域:
有机化学
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生物化学
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生物催化
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药物化学
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DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2024
·
IF 7.7
Shijin Wu,Xiaojing Ma,Hongde Yan
The asymmetric reduction of α , β -unsaturated compounds conjugated with electron-withdrawing group by ene-reductases (ERs) is a valuable method for the synthesis of enantiopure chiral compounds. This study introduced an ER from Corynebacterium casei ( Cc ER) which was heterologously expressed in Escherichia coli BL21(DE3), and the purified recombinant Cc ER was characterized for its biocatalytic properties. Cc ER exhibited the highest specific activity at 40 °C and pH 6.5, and showcased appreciable stability below 40 °C over a pH range of 6.0–7.0. The enzyme displayed high resistance to methanol. Cc ER accepted NADH or NADPH as a cofactor and exhibited a broad substrate spectrum towards α , β -unsaturated compounds. It achieved complete conversion of 2-cyclohexen-1-one and good performance for stereoselective reduction of ( R )-carvone (conversion 98 %, diastereoselectivity 96 %). This study highlights the robustness and potential of Cc ER.
研究领域:
生物化学
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生物技术
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酶学
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有机化学
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产品关联
DOI
PubMed
高影响因子
ChemCatChem
·
2020
·Vol. 12
·期 12
·3257-3264
·
IF 4.853
Xue-Ying Zhang,Xin Wang,Nan-Wei Li,Ze-Wang Guo,Min-Hua Zong,Ning Li
Graphical Whole-cell biocatalysis : Cofactor-engineered E. coli cells proved to be versatile catalysts to produce furan carboxylic acids from toxic biobased furans with high productivities. The introduction of NADH oxidase (NOX) into E. coli harboring aldehyde dehydrogenases (ALDHs) significantly promoted intracellular NAD + regeneration, thus enhancing ALDH-catalyzed aldehyde oxidation. A group of aromatic aldehydes was efficiently oxidized to target carboxylic acids. More importantly, these engineered whole-cell biocatalysts displayed high substrate tolerance toward toxic furans. Furan carboxylic acids are useful chemicals in various industries. In this work, biocatalytic production of furan carboxylic acids was reported with high productivities by cofactor-engineered Escherichia coli cells. NADH oxidase (NOX) was introduced into E. coli harboring aldehyde dehydrogenases (ALDHs) to promote intracellular NAD + regeneration, thus significantly enhancing ALDH-catalyzed oxidation. These engineered biocatalysts were capable of efficient aerobic oxidation of a variety of aromatic aldehydes. More importantly, they exhibited high substrate tolerance toward toxic furans. E. coli co-expressing vanillin dehydrogenase and NOX ( E. coli_ CtVDH1_NOX) enabled efficient oxidation of 250 mM of 5-hydroxymethylfurfural (HMF) to 5-hydroxymethyl-2-furancarboxylic acid (HMFCA), providing a productivity of 3.7 g/L h. With E. coli_ CtVDH2_NOX as catalyst, up to 240 mM of furfural and 5-methoxymethylfurfural (MMF) could be smoothly oxidized. 2-Furoic acid (FCA, 227 mM) and 5-methoxymethyl-2-furancarboxylic acid (MMFCA, 287 mM) were produced in fed-batch synthesis, providing the productivities of 2.0 and 5.6 g/L h, respectively.
研究领域:
生物技术
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生物化学工程
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代谢工程
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产品关联
DOI
高影响因子
TALANTA
·
2024
·
IF 5.6
Jianwen Wan,Mengge Wang,Songmin Chen,Xiyao Zhang,Wenhui Xu,Di Wu,Qiong Hu,Li Niu
The individualized administration and pharmacokinetics profiling are integral to the safe use of antibody drugs in immunotherapy. Here, we propose an electrochemical platform for the highly sensitive and selective detection of antibody drugs, taking advantage of the affinity capture by the peptide mimotopes together with the signal amplification by the biologically-driven RAFT polymerization (BDRP). Briefly, the BDRP-based platform involves the capture of antibody drugs by peptide mimotopes, the labeling of multiple reversible addition-fragmentation chain-transfer (RAFT) agents to the glycan chains of antibody drugs, and the BDRP-enabled controlled recruitment of numerous redox labels. The BDRP-based signal amplification relies on the reduction of RAFT agents by NADH coenzymes into the carbon-centered radicals, which can propagate efficiently into long polymer chains by reacting with the ferrocene-derivated monomers, recruiting numerous redox labels to the glycan chains of antibody drugs. The BDRP is conducted at the physiological temperature (i.e., 37 o C) and in the absence of external stimuli or radical sources, holding the advantages of biological compatibility and desirable simplicity over conventional RAFT polymerization approaches. The developed platform is highly selective and the detection limit in the presence of rituximab as the target was determined to be 0.14 ng/mL. Moreover, the applicability of the BDRP-based platform in the sensitive assay of antibody drugs in serum samples has been validated. In view of the biocompatibility, desirable simplicity, and cost-effectiveness, the BDRP-based platform shows great promise in the quantitative assay of antibody drugs.
研究领域:
分析化学
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生物技术
·
药学
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电化学
·
免疫疗法
✓ 已验证
产品关联
DOI
PubMed
高影响因子
CHEMICAL ENGINEERING JOURNAL
·
2024
·
IF 13.3
Song Lin,Zhengyu Tao,Zhenhui Li,Shangsong Li,Xiaoliang Wang,Xiaoman Liu,Xin Huang
The development of biotic-abiotic photosynthetic systems is becoming one of the most promising strategies towards the thirsty demand for sustainable energy production. However, considerable efforts are still required to further optimize the design and avoid the utilization of high-value substances while achieving highly efficient hydrogen production. Here we show a way to construct an Escherichia coli ( E. coli )-quantum dots hybrid system by expressing ferredoxin (Fd), Fd-NAD(P) + reductase (FNR), and [FeFe]-hydrogenase ([FeFe]H 2 ase) as a potential biocatalyst. We reveal that electrons originating from the quantum dots could be redirected through the newly established electron transport chain NADH/NAD + -FNR-Fd to [FeFe]H 2 ase, which contributes a key mechanism to boost the highest H 2 production activity (3149.5 μmol g dcw −1 h −1 ) by only using glycerol as the carbon source. Our work sheds light on the advantages of an intracellular biological hybrid system by combining the photocatalytic capacity with the optimized metabolic power to produce biofuels.
研究领域:
生物技术
·
生物能源
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光合作用
·
催化
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产品关联
DOI
高影响因子
TETRAHEDRON-ASYMMETRY
·
2014
·
IF 2.165
Rui-Jie Chen,Gao-Wei Zheng,Yan Ni,Bu-Bing Zeng,Jian-He Xu
A new NADH-dependent carbonyl reductase Rh CR capable of efficiently reducing the ε-ketoester ethyl 8-chloro-6-oxooctanoate (ECOO) to give ethyl ( S )-8-chloro-6-hydroxyoctanoate [( S )-ECHO], an important chiral precursor for the synthesis of ( R )-α-lipoic acid, was identified from Rhodococcus sp. ECU1014. Using recombinant Escherichia coli cells expressing Rh CR and glucose dehydrogenase used for the regeneration of cofactor, 440 g L −1 (2 M) of ECOO were stoichiometrically converted to ( S )-ECHO in a space–time yield of 1580 g L −1 d −1 without the external addition of any expensive cofactor.
研究领域:
生物催化
·
酶工程
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合成化学
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生物技术
✓ 已验证
DOI
高影响因子
ACS ES&T Engineering
·
2024
·
IF 7.4
Zimeng Zhang,Zhiling Li,Xueqi Chen,Jun Nan,Yunxia Zu,Fan Chen,Bin Liang,Aijie Wang
Understanding how nonelectroactive bacteria (non-EAB) perceive and respond to electro-stimulation is pivotal for optimizing electro-biostimulation systems. Here, Escherichia coli, a representative non-EAB, was employed to investigate its electrical response behavior and molecular regulatory mechanism across a spectrum of current densities. Accelerated bacterial growth was observed at current densities ranging from 2 to 10 A m–2 with a maximum growth rate of 1.89 h–1 at 10 A m–2. Moderate electrostimulation (10 A m–2) promoted NADH regeneration and adenosine triphosphate synthesis by modulating intracellular glycolytic flux, tricarboxylic acid (TCA) cycle and electron transport chain (ETC), while cells became inactivated at 20 A m–2 mainly due to the overall inhibition of the TCA cycle and domino collapse of ETC. The presence of reductive stress caused by electro-stimulation not only promoted NADPH and glutamine consumption but also impacted the material exchange fluxes by altering outer membrane proteins (OMPs) from β-fold to β-corner. Additionally, extracellular polymeric substances served as the electron transient medium to sense electro-stimulation. The study revealed that non-EAB possessed approaches different from EET to sense and respond to electro-stimulation. The improved comprehension of regulatory mechanisms governing catabolic pathways under electro-stimulation holds promise for developing more efficient electro-biostimulation systems, with implications for environmental biotechnology applications.
研究领域:
微生物学
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环境生物技术
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代谢工程
✓ 已验证
产品关联
DOI
高影响因子
危险品化学品经营许可证（带存储）