ACS Sustainable Chemistry & Engineering
·
2023
·
IF 8.4
Zhihao Zhang,Jiayu Xue,Zewen Sun,Jiale Wang,Guochao Xu,Ye Ni
Hydrogen-borrowing redox-neutral cascade reactions are promising for the synthesis of lactones from the perspective of sustainable chemistry. In this study, a novel NADPH-dependent Baeyer–Villiger monooxygenase AjBVMO was identified from Acinetobacter johnsonii with a specific activity of 0.96 U·mg–1 in the oxidation of cyclohexanone into ε-caprolactone. To achieve cofactor matching, NADP+-dependent KpADHT215V and CgKR1 with high oxidative activities toward cyclohexanol and 1,6-hexanediol were constructed for developing “substrate-supplied” and “product-convergent” hydrogen-borrowing redox-neutral cascade reactions. The effects of buffer systems on the activities and spontaneous hydrolysis of ε-caprolactone and the ratios of ADHs and BVMO on the conversion rates were explored. In the “substrate-supplied” system employing KpADHT215V and AjBVMO, as high as 68.6 mM ε-caprolactone could be obtained from 100 mM cyclohexanol, with a maximum TOF of 7.2 min–1 and a TTN of 337. In the “product-convergent” system employing CgKR1 and AjBVMO, 115.1 mM ε-caprolactone was produced from 100 mM cyclohexanone, with a maximum TOF of 5.7 min–1 and a TTN of 757. The product-convergent system is more efficient than the substrate-supplied system. Moreover, these two redox-neutral cascade systems could also be applied in the synthesis of lactones with different chain lengths such as γ-butyrolactone and δ-valerolactone. This study established efficient redox-neutral cascade systems for the synthesis of lactones featuring a high atomic economy with only H2O as a byproduct.
研究领域:
生物化学
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可持续化学
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酶工程
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高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
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2025
·
IF 8.5
Li Qiao,Bianqin Ma,Yongjian Jiang,Xiaoting Pan,Zhili Mao,Yi Zhang,Roger A. Sheldon,Anming Wang
Keto reductases are crucial NAD( P )H-dependent enzymes used for the enantioselective synthesis of alcohols from prochiral ketones. Typically, the NADPH cofactor is regenerated through a second enzyme and/or substrate. However, photocatalytic cofactor regeneration using water as a sacrificial electron and hydrogen donor presents a promising alternative, albeit a challenging one. Herein we fabricated several nitrogen-doped carbon dots (CDs) with visible light absorption properties, good water solubility and biocompatibility for photocatalytic regeneration of NADPH. The CD with a smaller size and suitable redox potential gave the highest NADPH yield (55.7 %). Based on this, NADPH-dependent aldo-keto reductase crosslinked aggregates (AKR-CLEs) were initially applied as a stable biocatalyst to reduce the prochiral ketone. ( S )-1-(2-Chlorophenyl) ethanol, an intermediate for LPA1R antagonists, was obtained in 65.3 % yield and 99.99 % enantiomeric excess (ee) under visible light irradiation. The isotope tracer experiment confirmed that water is the hydrogen donor in this light-driven, photo-enzymatic asymmetric hydrogenation system. This method is useful for the sustainable synthesis of chiral alcohols. Moreover, the general principle of utilizing water as the sacrificial hydrogen and electron donor holds potential for application in other redox cofactor regeneration systems.
研究领域:
生物化学
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催化化学
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酶工程
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PubMed
高影响因子
ACS Omega
·
2025
·
IF 4.3
Tianfu Wu,Wei Song,Jing Wu,Jian Wen,Wanqing Wei,Liming Liu
Chiral amino alcohols are vital in many biologically active molecules and natural products and are crucial building blocks for the pharmaceutical industry. However, the chemosynthesis of chiral amino alcohols, particularly bichiral amino alcohols, faces challenges related to chemoselectivity and enantioselectivity. Here, a dual-enzyme cascade pathway for the synthesis of bichiral amino alcohols through difunctionalization, exhibiting both high chemo- and stereoselectivity, was proposed for the first time. Among them, NADPH-dependent meso-diaminopyrimidate dehydrogenase from Proteus vulgaris (PvDAPDH) exhibited (R)-selective reductive amination activity, converting ketones into chiral amines; natural glucose dehydrogenase from Bacillus megaterium (BmGDH) not only facilitated NADPH regeneration but also catalyzed the reductive hydroxylation of ketones to chiral alcohols. Second, the rational design of the rate-limiting enzyme PvDAPDH generated the mutant M4 with increased specific activity and catalytic efficiency (kcat/Km) for 16a up to 3.2- and 10.3-fold, respectively, compared to the wild type. Additionally, the yield of mutant M4 for 1a–16a increased by 1.1- to 37-fold compared to the wild type. Finally, two high-value bichiral amino alcohols (14b and 16b) were asymmetrically synthesized from the corresponding bietone via a one-pot dual-enzyme cascade, exhibiting excellent stereoselectivity (dr ≥ 98:2). These findings provide a potential biosynthetic pathway for the green synthesis of bichiral amino alcohols and complement existing synthetic methods.
研究领域:
生物化学
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合成生物学
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制药工程
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PubMed
高影响因子
Plants-Basel
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2022
·Vol. 11
·期 19
·2487
·
IF 4.658
Yifei Zhang,Yishan Sun,Weiqing Li,Jiayu Li,Rongqiong Xu,Jiarui Du,Zesong Li,Guibin Li,Kejun Yang
To explore the effects of iminodisuccinic acid (a chelating agent) on maize (Zea maysL.) seed germination under lead (Pb) stress, we comparatively analyzed the effects of applying different concentrations of iminodisuccinic acid (0, 5, 20, and 100 mmol·dm−3) and combined an addition of exogenous substances regulating reactive oxygen species production on maize seed germination, seedling growth, H2O2content, NADPH oxidase activity, and antioxidant enzyme activities under Pb-stressed and Pb-free conditions. Iminodisuccinic acid (100 mmol·dm−3) significantly delayed seed germination under normal germination conditions and alleviated the inhibitory effects of Pb stress (20 mmol·dm−3) on seed germination. Under normal conditions (without Pb stress), the iminodisuccinic acid-induced inhibition of seed germination was enhanced by treatment with dimethylthiourea (a specific scavenger of reactive oxygen species) or diphenyleneiodonium chloride (a specific inhibitor of NADPH oxidase), but diminished by treatment with H2O2, CaCl2, diethyldithiocarbamic acid (a specific inhibitor of superoxide dismutase), or aminotriazole (a specific inhibitor of catalase). Under Pb stress, iminodisuccinic acid partially eliminated the excessive H2O2accumulation, improved superoxide dismutase and catalase activity, and weakened the high NADPH oxidase activity. In addition, Ca2+chelation may be essential for maintaining the reactive oxygen species’ balance and improving seed germination and seedling growth by iminodisuccinic acid supplementation in maize under Pb stress. The proposed iminodisuccinic acid supplementation-based method improved maize seed germination in Pb-polluted soil.Keywords:maize;chelator;lead stress;ROS;hydrogen peroxide;NADPH oxidase;seed germination
研究领域:
植物学
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环境科学
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PubMed
高影响因子
CHINESE JOURNAL OF CATALYSIS
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2025
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IF 17.7
Jiaxu Liu,Jiaxin Chen,Xiaoyan Zhang,Daidi Fan,Yunpeng Bai
Enzyme catalysis is a promising way to produce chiral products in a green and sustainable way. However, the high cost of cofactors and their relatively low recycling efficiency hinder the widespread application of enzyme catalysis in industry. In contrast, cofactor regeneration and recycling in cells is highly efficient, mainly due to physical effects caused by the ordered spatial organization of enzymes in vivo . The construction of similar catalytic systems with high cofactor recycling in vitro remains challenging. Here, we present a facile method to generate dual enzyme condensates in vitro based on intrinsically disordered region-mediated liquid-liquid phase separation. Typically, a carbonyl reductase from Serratia marcescens ( Sm CR V4 ) and a glucose dehydrogenase from Bacillus megaterium ( Bm GDH) were co-localized in the condensates. This resulted in an up to 20-fold increase in cofactor recycling efficiency (substrate converted per cofactor per unit time), and a 3.4-fold increase in space-time yield compared to the free enzyme system. The reaction enhancement was shown to be highly correlated with the degree of condensation of the dual enzymes. Fluorescence confocal microscopy showed that the cofactor, nicotinamide adenine dinucleotide phosphate (NADPH), was enriched between neighboring enzymes during the reaction due to the proximity effect, facilitating its regeneration and recycling within the condensate. In a scaled-up synthesis, the consumption of NADPH was reduced 50-fold compared to industrial biocatalytic standards, while the condensate still maintained efficient product synthesis. Concentrating multiple enzymes in a nano- and micro-condensate to increase the reaction rate may provide a general and inexpensive method for improving cofactor-involved enzymatic reactions.
研究领域:
生物技术
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酶学
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化学工程
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DOI
高影响因子
BIOTECHNOLOGY LETTERS
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2023
·Vol. 45
·期 8
·993-1000
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IF 2.7
Peng Shuaiying,Chu Zhongmei,Lu Jianfeng,Li Dongxiao,Wang Yonghong,Yang Shengli,Zhang Yi
The self-sufficient cytochrome P450 BM3 mutant (A74G/F87V/D168H/L188Q) can serve as a biocatalyst for whole-cell catalysis process of indigo. Nevertheless, the bioconversion yield of indigo is generally low under normal cultivation conditions (37 °C, 250 rpm). In this study, a recombinant E. coli BL21(DE3) strain was constructed to co-express the P450 BM3 mutant gene and GroEL/ES genes to investigate whether GroEL/ES can promote the indigo bioconversion yield in E. coli . The results revealed that the GroEL/ES system could significantly increase the indigo bioconversion yield, and the indigo bioconversion yield of the strain co-expressing P450 BM3 mutant and GroEL/ES was about 21-fold that of the strain only expressing the P450 BM3 mutant. In addition, the P450 BM3 enzyme content and in vitro indigo bioconversion yield were determined to explore the underlying mechanism for the improvement of indigo bioconversion yield. The results revealed that GroEL/ES did not increase indigo bioconversion yield by increasing the content of P450 BM3 enzyme and its enzymatic transformation efficiency. Moreover, GroEL/ES could improve the intracellular nicotinamide adenine dinucleotide phosphate (NADPH)/NADP + ratio. Given that NADPH is an important coenzyme in the catalytic process of indigo, the underlying mechanism for the improvement of indigo bioconversion yield is probably related to an increase in the intracellular NADPH/NADP + ratio.
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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PubMed
高影响因子
Oxidative Medicine and Cellular Longevity
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2017
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IF 4.593
Shimei Qi,Zunyong Feng,Qiang Li,Zhilin Qi,Yao Zhang
Myricitrin, a naturally occurring polyphenol hydroxy flavonoid, has been reported to possess anti-inflammatory properties. However, the precise molecular mechanism of myricitrin’s effects on LPS-induced inflammation is unclear. In the present study, myricitrin significantly alleviated acute lung injury in mice. Myricitrin also markedly suppressed the production of NO, TNF-α, IL-6, and MCP-1 in RAW264.7 macrophage cells. The inhibition of NO was concomitant with a decrease in the protein and mRNA levels of iNOS. The phosphorylation of JAKs and STAT-1 was abrogated by myricitrin. Furthermore, myricitrin inhibited the nuclear transfer and DNA binding activity of STAT1. The JAK-specific inhibitor ruxolitinib simulated the anti-inflammatory effect of myricitrin. However, myricitrin had no impact on the MAPK signalling pathway. Myricitrin attenuated the generation of intracellular ROS by inhibiting the assembly of components of the gp91phox and p47phox. Suppression of ROS generation using NAC or apocynin or by silencing gp91phox and p47phox all demonstrated that decreasing the level of ROS inhibited the LPS-induced inflammatory response. Collectively, these results confirmed that myricitrin exhibited anti-inflammatory activity by blocking the activation of JAKs and the downstream transcription factor STAT1, which may result from the downregulation of NOX2-dependent ROS production mediated by myricitrin.
研究领域:
药理学
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免疫学
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分子生物学
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PubMed
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2026
·
IF 6.7
Wenjing Fan,Changgeng Li,Runze Zhao,Tang’en Shi,Liangwen Li,Zhichao Chen,Zichen Yu,Ling Ma,Siyu Zhao,Qingyang Xu
The demand for β-thymidine primarily stems from its role as a raw material and intermediate in antiviral drug production. To address this, β-thymidine was synthesized de novo using Escherichia coli. We engineered a high-yielding β-thymidine strain based on the orotate high-yielding strain Ora14–4. First, we introduced the pyrFE gene cluster from Bacillus subtilis to direct the metabolic flux toward β-thymidine synthesis. Genes involved in β-thymidine catabolism and precursor branching were knocked out, and key enzymes in the β-thymidine biosynthetic pathway were optimized. Enhancing the folate cycle and glycine cleavage system provided sufficient one-carbon donors for deoxythymidine monophosphate synthesis. The Entner-Doudoroff pathway from Zymomonas mobiliz was introduced to enable NADPH production. Last, a growth-adaptive promoter regulated tmk gene expression to minimize carbon loss. The final β-thymidine titer was 25.4 g/L, with a conversion rate of 0.079 g/g glucose, which is, to the best of our knowledge, the highest titer reported to date for de novo microbial production of β-thymidine.
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PubMed
高影响因子
npj Science of Food
·
2026
·
IF 9.7
Yu Peng,Liu Jianing,Xu Weiyin,Peng Lu,Li Yanqi,Shao Simeng,Wang Yiru,Qiu Zhidong,Yang Hongmei
Synaptic loss and aberrant protein S-nitrosylation (SNO) are strongly linked to cognitive decline in both patients and animal models of Alzheimer’s disease (AD). Our recent work in an AD mouse model has shed light on the role of oligosaccharides extracted from black ginseng (OSBG) in ameliorating cognitive impairment. However, the precise molecular mechanisms responsible for therapeutic efficacy of OSBG in AD are not well understood. In the present study, we employed an innovative SNOTRAP-based proteomic approach to quantify SNO proteins in the brain of APP/PS1 mice following OSBG intervention. The results revealed that differentially expressed SNO proteins, such as SNO-NOX1 and SNO- NOX5, confirmed by Western blot (WB), are significantly enriched in pathways related to oxidative stress, such as “Oxidative_stress activation of NADPH oxidase” and “Synaptic target recognition”. OSBG treatment significantly alleviated oxidative stress via inhibition of NADPH oxidase activity in APP/PS1 mouse and PC12 cells by WB immunofluorescence (IF) assays. More importantly, upregulation of PSD-95 and SYN1 was detected in the hippocampal tissue of APP/PS1 mice after OSBG intervention, which was further validated by the corresponding mRNA expression levels. Consistently, histopathological analysis revealed the restoration of hippocampal cellular structure. Overall, our findings highlight the synapse-protective effect of OSBG in an AD model through regulating protein SNO levels and inhibiting NADPH oxidase activity, revealing a novel mechanism by which OSBG alleviated oxidative stress injury.
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高影响因子
BIOTECHNOLOGY AND BIOENGINEERING
·
2024
·Vol. 122
·期 3
·724-735
·
IF 3.5
Xia Ke,Hong-Duo Dong,Xi-Man Zhao,Xin-Xin Wang,Zhi-Qiang Liu,Yu-Guo Zheng
C14-functionalized steroids enabled diverse biological activities in anti-gonadotropin and anticancer therapy. However, access to C14-functionalized steroids was impeded by the deficiency of chemical synthetic methods. Recently, several membrane-bound fungal cytochrome P450s (CYPs) have been identified with steroid C14α-hydroxylation activity. However, the lack of efficient heterologous overexpression strategy hampered their further characterization and molecular engineering. In the present study, sequences of fungi-derived CYP genes encoding putative 14α-hydroxylase were selected and bioinformatically analyzed. Substitution of the N-terminal hydrophobic helix by a soluble maltose binding protein tag significantly enhanced the soluble expression level in Escherichia coli . A novel CYP originated from Bipolaris oryzae was discovered with high steroidal C14α-hydroxylation activity when coupled with the redox partner CPR lun . A catalytically self-sufficient chimeric CYP-CPR was built by intramolecular fusion, and the electronic transfer rate was improved. A coenzyme NADPH regeneration system was finally constructed by the co-expression of glucose dehydrogenase. The developed soluble multi-enzyme cascade biotransformation system supported the selective C14α-hydroxylation toward progesterone with a final titer of 34.54 mg/L, the highest level achieved in E. coli -based heterologous expression system. This study provides insightful ideas on the functional expression of fungi-derived CYPs and promises an efficient C14α-hydroxylation system for steroidal drugs through protein engineering.
研究领域:
生物化学
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分子生物学
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药物开发
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高影响因子
JOURNAL OF BIOTECHNOLOGY
·
2018
·
IF 2.533
Jiapao Wang,Pengfei Cheng,Yifeng Wu,Anming Wang,Fangming Liu,Xiaolin Pei
( R )-Pantolactone (PL) is a key chiral intermediate for the synthesis of calcium ( R )-pantothenate and ( R )-panthenol used as food additives. The commercial production of ( R )-pantothenate is performed by the resolution of racemic pantothenate, which is synthesized through an aldol condensation and a cyanation reaction. In this study, we investigated another synthetic method of ( R )-pantothenate through the stereoselective reduction of ketopantoyl lactone (KPL) by aldo-keto reductase (AKR). A series of conjugated polyketone reductases (CPRs) were discovered from GenBank database by genome mining approach. The putative CPR gene from Candida orthopsilosis Co 90–125 ( Cor CPR) was cloned and functionally expressed in Escherichia coli BL21 (DE3). The optimum pH and temperature of recombinant Cor CPR were 6.0–7.0 and 40 ℃, respectively. The K m and v max toward KPL were1.3 mM and 227.3 μ mol/min/mg protein, respectively. The conserved sequences suggest that Cor CPR belongs to AKR3C family of AKR superfamily. Furthermore, a catalytic tetrad was proposed, and the detailed mechanism was clarified by molecular docking. In a batch reaction, 50 mM KPL was reduced to ( R )-PL with 99% conversion and > 99% enantiomeric excess within 5 h. The recombinant Cor CPR from C. orthopsilosis shows potential application in the asymmetric synthesis of ( R )-pantothenate preparation.
研究领域:
生物化学
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分子生物学
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工业微生物学
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PubMed
高影响因子
Cancers
·
2022
·Vol. 14
·期 2
·345
·
IF 6.575
Hui Chen,Han Zhang,Lixing Cao,Jinling Cui,Xuan Ma,Chong Zhao,Shutao Yin,Hongbo Hu
Simple SummarySelenite, a representative inorganic form of selenium, is preferentially accumulated in tumors. The therapeutic potential of sodium selenite in tumors has received significant attention. However, the effect of sodium selenite in the treatment of established tumors is hampered by its systemic toxicities. In this study, we found selenite exerted a stronger lethality to the cancer cells under the condition of glucose limitation in vitro and an enhanced inhibitory effect on tumor growth when combined with intermittent fasting in vivo. In addition, this treatment showed no obvious toxicity to normal cells and mice. The findings of the present study provide an effective and practical approach for increasing the therapeutic window of selenite and imply that combination of selenite and fasting holds promising potential to be developed a clinically useful regimen for treating certain types of cancer.AbstractCombination of intermittent fasting and chemotherapy has been drawn an increasing attention because of the encouraging efficacy. In this study, we evaluated the anti-cancer effect of combination of glucose limitation and selenite (Se), a representative inorganic form of selenium, that is preferentially accumulated in tumors. Results showed that cytotoxic effect of selenite on cancer cells, but not on normal cells, was significantly enhanced in response to the combination of selenite and glucose limitation. Furthermore, in vivo therapeutic efficacy of combining selenite with fasting was dramatically improved in xenograft models of lung and colon cancer. Mechanistically, we found that SLC7A11 expression in cancer cells was up-regulated by selenite both in vitro and in vivo. The elevated SLC7A11 led to cystine accumulation, NADPH depletion and the conversion of cystine to cysteine inhibition, which in turn boosted selenite-mediated reactive oxygen species (ROS), followed by enhancement of selenite-mediated cytotoxic effect. The findings of the present study provide an effective and practical approach for increasing the therapeutic window of selenite and imply that combination of selenite and fasting holds promising potential to be developed a clinically useful regimen for treating certain types of cancer.Keywords:selenite;glucose limitation;SLC7A11;NADPH;ROS
研究领域:
肿瘤学
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药理学
·
细胞生物学
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PubMed
高影响因子
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