INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
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2024
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IF 7.7
Li Qiao,Jing Zhang,Yongjian Jiang,Bianqin Ma,Haomin Chen,Peng Gao,Pengfei Zhang,Anming Wang,Roger A. Sheldon
Effective photolytic regeneration of the NAD( P )H cofactor in enzymatic reductions is an important and elusive goal in biocatalysis. It can, in principle, be achieved using a near-infrared light (NIR) driven artificial photosynthesis system employing H 2 O as the sacrificial reductant. To this end we utilized TiO 2 /reduced graphene quantum dots (r-GQDs), combined with a novel rhodium electron mediator, to continuously supply NADPH in situ for aldo-keto reductase (AKR) mediated asymmetric reductions under NIR irradiation. This upconversion system, in which the Ti-O-C bonds formed between r-GQDs and TiO 2 enabled efficient interfacial charge transfer, was able to regenerate NADPH efficiently in 64 % yield in 105 min. Based on this, the pharmaceutical intermediate ( R )-1-(3,5-bis(trifluoromethyl)phenyl)ethan-1-ol was obtained, in 84 % yield and 99.98 % ee, by reduction of the corresponding ketone. The photo-enzymatic system is recyclable with a polymeric electron mediator, which maintained 66 % of its original catalytic efficiency and excellent enantioselectivity (99.9 % ee) after 6 cycles.
研究领域:
生物化学
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光催化
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合成生物学
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DOI
PubMed
高影响因子
ACS Sustainable Chemistry & Engineering
·
2019
·
IF 6.97
Guochao Xu,Cheng Zhu,Aitao Li,Yan Ni,Ruizhi Han,Jieyu Zhou,Ye Ni
Cofactor regeneration using diols as “smart cosubstrates” is one of the most promising approaches, due to the thermodynamic preference and 0.5-equiv requirement. In order to establish an efficient NADPH regeneration system with 1,4-butanediol (1,4-BD), a NADP+-dependent alcohol dehydrogenase from Kluyveromyces polysporus (KpADH) was engineered to solve the kinetic imbalance. Several hotspots were identified through molecular dynamic simulation and subjected to saturation and combinatorial mutagenesis. Variant KpADHV84I/Y127M exhibited a lower KM of 15.1 mM and a higher kcat of 30.1 min–1 than WTKpADH. The oxidation of 1,4-BD to 4-hydroxybutanal was found to be the rate-limiting step, for which the kcat/KM value of double mutant KpADHV84I/Y127M was 2.00 min–1·mM–1, 11.6-fold higher than that of WTKpADH. KpADHV84I/Y127M preferred diols with a longer chain length (C5–C6). The ratio of kcat/KM toward 2-hydroxytetrahydrofuran (2-HTHF), in comparison to 1,4-BD, in KpADHV84I/Y127M was dramatically reduced by almost 100-fold compared to WTKpADH, which was advantageous for NADPH regeneration. As much as 100 mM phenylpyruvic acid could be reduced into d-phenylalanine with 99.2% conversion in 6 h using merely 0.5 equiv of 1,4-BD. Both the improved catalytic efficiency toward 1,4-BD and the balanced kcat/KM between 1,4-BD and 2-HTHF contributed to the higher NADPH regeneration efficiency. This study provides guidance for engineering alcohol dehydrogenases for cosubstrate specificity toward diols and its application in NADPH regeneration for the preparation of chiral compounds of pharmaceutical relevance.
研究领域:
生物化学
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代谢工程
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合成生物学
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DOI
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
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2025
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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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DOI
PubMed
高影响因子
ACS Synthetic Biology
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2025
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IF 3.9
Jiaying Ai,Ziyang Yin,Jikai Gao,Wenjing Wang,Fuping Lu,Hui-Min Qin,Shuhong Mao
P450 enzymes are promising biocatalysts and play an important role in the field of drug synthesis due to their high catalytic activity and stereoselectivity. CYP109E1 from Bacillus megaterium was used to convert VD3 for the production of 25(OH)VD3. However, the industrial production was still limited due to the low catalytic performance of CYP109E1. To overcome this, we constructed an engineered strain containing a modified CYP109E1 coupled with an efficient electron transfer chain and NADPH regeneration system. First, Adx4–108T69E-Fpr was identified as the most compatible redox partner for the enzyme based on in-silico analysis. Then, targeted mutations were introduced at the substrate channel of CYP109E1, resulting in higher production efficiency. Next, the production of 25(OH)VD3 was increased by 13.1% after introducing a double Adx4–108T69E expression cassette. Finally, an NADPH regeneration system was introduced by overexpressing zwf, which increased the yield of 25(OH)VD3 48.7%. These results demonstrate that recombinant Escherichia coli BL21 (DE3) coexpressing CYP109E1_R70A-ZWF and 2Adx4–108T69Es-Fpr is an efficient whole-cell biocatalyst for the synthesis of 25(OH)VD3, illustrating an attractive strategy for improving the catalytic efficiency of P450 enzymes.
研究领域:
生物化学
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药物合成
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酶工程
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DOI
PubMed
高影响因子
ACS Omega
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2025
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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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DOI
PubMed
高影响因子
BIOTECHNOLOGY AND BIOENGINEERING
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2024
·Vol. 122
·期 3
·724-735
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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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DOI
高影响因子
JOURNAL OF CATALYSIS
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2024
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IF 6.5
Chun-Xian Cai,Zi-Wen Zhou,Chun-Xiu Liu,Yun-Jie Wei,Xiao-Qi Yu,Li-Hong Zhou,Na Wang
Photo-enzyme catalysis featuring sustainability and selectivity is attractive, but remains challenges from efficiency and enzyme inactivation. This work provides a compatible and efficient photocatalyst-enzyme hybrid system for chiral alcohol synthesis. A covalent organic framework (COF) possessing broad visible light absorption edge to 554 nm was used for photocatalytic NADPH regeneration, achieving 82 % yield in 90 min. Then the influence of photocatalysis on coupled enzyme Lb ADH was unveiled by fluorescence spectra and circular dichroism. Further a facile and mild strategy of entrapping Lb ADH into Ca 2+ /Mn 2+ doped alginate gel was carried out. Impressively, enzyme immobilized strategy not only enhances 3.2 times activity, but also protects Lb ADH from diverse harsh environments, thereby improving photo-enzyme catalytic yield from 26 % to 82 %. To our delight, this one-pot photo-enzyme catalytic system achieves highly yield (up to 99 %) with excellent enantioselectivity (90–99 ee %) for various chiral alcohols, and fulfills semipreparative scale preparation of ( R )-1-[3,5-Bis(trifluoromethyl)phenyl] ethanol, prochiral intermediate of Aprepitant. This work sheds light on the new synthetic route of chiral compounds and development of photo-enzyme catalytic field.
研究领域:
生物化学
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催化化学
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产品关联
DOI
高影响因子
ENZYME AND MICROBIAL TECHNOLOGY
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2025
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IF 3.7
Yingying Zhu,Dawei Ni,Zeyu Li,Zhebin Hao,Liang Wang,Wanmeng Mu
Indigo is an important blue pigment widely used in textile, food, and medicine industries. Biological production of indigo attracts increasing attention recently. Cell factory production of indigo encounters the problem of the toxicity of the precursor indole. Enzymatic production is the alternative biological approach, however, NADPH regeneration should be solved. In this study, flavin-containing monooxygenase from Methylophaga aminisulfidivorans was used for enzymatic production of indigo from indole and formate dehydrogenase from Pseudomonas sp. 101 was co-expressed for NADPH regeneration. Indigo production was enhanced by combination of molecular modification, promoter engineering, and translation initiation region engineering. Finally, 0.183 g/L of indigo was produced from 0.5 g/L of indole and 0.5 mM of sodium formate, with the conversion ratio of 32.5 %. This study demonstrates a feasible and effective strategy for enzymatic production of indigo.
研究领域:
生物催化
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酶促合成
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代谢工程
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工业微生物学
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应用生物化学
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产品关联
DOI
PubMed
高影响因子
Bioresources and Bioprocessing
·
2022
·Vol. 9
·期 1
·1-14
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IF 4.983
Liu Kun,Wang Feng-Qing,Liu Ke,Zhao Yunqiu,Gao Bei,Tao Xinyi,Wei Dongzhi
Progesterone is one of the classical hormone drugs used in medicine for maintaining pregnancy. However, its manufacturing process, coupled with organic reagents and poisonous catalysts, causes irreversible environmental pollution. Recent advances in synthetic biology have demonstrated that the microbial biosynthesis of natural products, especially difficult-to-synthesize compounds, from building blocks is a promising strategy. Herein, overcoming the heterologous cytochrome P450 enzyme interdependency in Mycolicibacterium neoaurum successfully constructed the CYP11A1 running module to realize metabolic conversion from waste phytosterols to progesterone. Subsequently, progesterone yield was improved through strategies involving electron transfer and NADPH regeneration. Mutant CYP11A1 (mCYP11A1) and adrenodoxin reductase (ADR) were connected by a flexible linker (L) to form the chimera mCYP11A1-L-ADR to enhance electron transfer. The chimera mCYP11A1-L-ADR, adrenodoxin (ADX), and ADR-related homolog ARH1 were expressed in M. neoaurum, showed positive activity and produced 45 mg/L progesterone. This electron transfer strategy increased progesterone production by 3.95-fold compared with M. neoaurum expressing mCYP11A1, ADR, and ADX. Significantly, a novel inorganic–biological hybrid system was assembled by combining engineered M. neoaurum and InP nanoparticles to regenerate NADPH, which was increased 84-fold from the initial progesterone titer to 235 ± 50 mg/L. In summary, this work highlights the green and sustainable potential of obtaining synthetic progesterone from sterols in M. neoaurum.
研究领域:
合成生物学
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生物技术
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环境科学
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药学
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微生物学
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产品关联
DOI
PubMed
高影响因子
PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY
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2019
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IF 1.117
Chuhong Zong,Xu Zhang,Fei Yang,Yafeng Zhou,Nan Chen,Zuisu Yang,Guofang Ding,Fangmiao Yu,Yunping Tang
(S)-1-(2, 6-dichloro-3-fluorophenyl) ethanol, the key chiral intermediate of crizotinib, was prepared from 1-(2, 6-dichloro-3-fluorophenyl) ethanone using the alcohol dehydrogenases from Lactobacillus kefir (ADH-LK) with a tetrad mutant (ADH-LKM, F147L/Y190P/V196L/A202W), coupled with glucose dehydrogenase (GDH). In the present study, ADH-LKM and GDH were successfully heterologous expressed in recombinant Escherichia coli. During the regeneration of NADPH with GDH, 150 g/L substrate was totally transformed into target chiral alcohol with an enantiomeric excess value of 99.9% after 12 h at 30 °C (pH 7.0). Our study demonstrates the potential for industrial green production of the key chiral intermediate of crizotinib.
研究领域:
生物催化
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药物化学
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酶工程
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产品关联
DOI
PubMed
高影响因子
BIOCHEMICAL ENGINEERING JOURNAL
·
2024
·
IF 3.7
Shu-Ping Zou,Wei Ding,Yu-Yue Han,Kun Niu,Ya-Ping Xue,Yu-Guo Zheng
L-Phosphinothricin (L-PPT) is the active compound of a broad-spectrum herbicide. In this study, we biosynthesized L-PPT via the asymmetric reduction of 2-carboxy-4-(hydroxymethylphosphoryl) butyric acid (PPO) using a whole-cell biocatalyst. Glutamate dehydrogenase from Pseudomonas moorei ( Pm GluDH), Glucose dehydrogenase from Exiguobacterium sibiricum ( Es GDH) and NAD kinase from Corynebacterium glutamicum ( Cg NADK) were coexpressed in E. coli , significantly increasing the catalytic efficiency by regenerating more NADPH to promote PPO reduction. Following the optimization of balancing the co-expression of the three enzymes in pETDuet-1 and pCDFDuet-1, the coexpressing strain increases in the intracellular NADP(H) concentration to 54.9 μmol/g DCW, which was 3.8-fold higher than the control. The co-expression system exhibited optimal activity at a temperature of 35°C, pH 7.5, and the glucose to PPO molar ratio of 1.4:1. In a 30 L bioconversion system the final concentration and productivity of L-PPT reached 79.8 g/L (>99% ee ) and 1440 g/(L·d), respectively. These results will contribute to the industrial biological production of L-PPT.
研究领域:
生物技术
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生化工程
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代谢工程
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产品关联
DOI
高影响因子
Molecular Catalysis
·
2024
·
IF 3.9
Yuqinxin Xie,Dongzhi Wei,Jinping Lin
(4 S )-3-[(5 S )-5-(4-fluorophenyl)-5‑hydroxy-valyl]-4-phenyl-1,3-oxazacyclopentane-2-one (( S )-Fop alcohol) is a key chiral intermediate for the synthesis of ezetimibe, and could be synthesized via asymmetric reduction of ( S )-4-phenyl-3-[5-(4-fluorophenyl)-5-oxopentanoyl]-2-oxazolidione (Fop dione). However, discovering and engineering of ketoreductases toward bulky-bulky (diaryl) ketones is still challenging. Previously, we identified an alcohol dehydrogenase Gox0525 from Gluconobacter oxydans DSM2343 which possessed strict diastereoselectivity ( d.e. value > 99%) but low activity toward Fop dione. In this study, a semi-rational design based on the focused rational iterative site-specific mutagenesis (FRISM) based on site-directed saturation mutagenesis was performed to improve the catalytic efficiency of Gox0525. The variant M4 (Y92G/P93M/Y94P/L151V) shows a 64-fold enhanced catalytic efficiency ( K cat / K m ) and 47-fold in specific activity compared with the wild type Gox0525. Engineered Escherichia coli cells co-expressing the variant M4 and glucose dehydrogenase from Bacillus subtilis (BsGDH) for NADPH regeneration were employed as biocatalysts for gram-scale reaction of Fop dione. As a result,95 mM (33.76 g/L) Fop dione was completely transformed within 4 h, affording ( S )-Fop alcohol with > 99% d.e. value, the yield of 96%, and the space-time yield of 195.6 g/L/d.
研究领域:
生物技术
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生物化学
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药物化学
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酶学
·
化工
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产品关联
DOI
高影响因子
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