APPLIED AND ENVIRONMENTAL MICROBIOLOGY
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2023
·Vol. 89
·期 3
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IF 4.4
Ling Wang,Xiaowei Wu,Chenghua Gao,Lingrui Wei,Qian Li,Aitao Li
In this study, a new cytochrome P450 enzyme, namely, CYP68J5_Fusarium graminearum (CYP68J5_fg), was identified from Fusarium graminearum via a combination of transcriptome sequencing and heterologous expression in Saccharomyces cerevisiae. The biotransformation of progesterone by whole-cells of S. cerevisiae expressing CYP68J5_fg revealed that the CYP68J5_fg possessed steroidal 12β- and 15α-hydroxylase activities. To the best of our knowledge, this is the first time that a fungal P450 enzyme with 12β-hydroxylase activity has been identified. This advance offers opportunities to boost the efficiency and selectivity of the CYP68J5_fg hydroxylating system and thus shows great potential for further applications of this enzyme for the synthesis of steroid drugs.IMPORTANCE Regioselective and stereoselective hydroxylation is of vital importance in the functionalization of steroids, which remains challenging in organic synthesis. In particular, the C12-hydroxy steroids play a significant role in the synthesis of many important steroidal drugs. In this study, a novel fungal P450 enzyme with 12β-hydroxylation activity was identified, and it shows different substrate specificity and regioselectivity, compared to the bacterial and fungal steroidal hydroxylases that are known to date. This lays the foundation for the creation of effective biocatalysts for the process of 12β-hydroxylation, although further understanding of the molecular structural basis of this fungal P450 is needed to facilitate the engineering of this enzyme for industrial applications.
研究领域:
生物化学
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酶学
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药学
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微生物学
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DOI
PubMed
高影响因子
INTERNATIONAL BIODETERIORATION & BIODEGRADATION
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2023
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IF 4.8
Ni-Hong Yao,Xi Liu,Chenyang Luo,Lei Zou,Jinhua Li,Jing Chen,Dazhong Yan,Hong-Jun Chao
The compound m -cresol ( meta -cresol) is an important representative of alkylphenol pollutants with high toxicity and potential carcinogenicity . The genes and enzymes associated with the m -cresol pathway have not yet been well elucidated. In this study, Comamonas thiooxydans CHJ601 was isolated from industrial wastewater and cultured utilizing m -cresol as the sole source of carbon and energy. m -Cresol catabolism begins with the oxidation of its methyl group in strain CHJ601. Genome sequencing and heterologous expression of key genes revealed its detailed catabolic pathway. m -Cresol methylhydroxylase (MchAB) catalyzed o -cresol ( ortho -cresol), m -cresol, and p -cresol ( para -cresol) in the presence of NADH and FAD. The K m value of m -cresol (48.74 ± 5.02 μM) for MchAB was much lower than that of o -cresol (747.80 ± 53.34 μM) and p -cresol (541.90 ± 41.43 μM). Molecular docking analysis showed that the binding affinities of MchAB against m -cresol with two hydrogen bonds are higher than p -cresol methylhydroxylase with one. The optimal substrate of MchAB was m -cresol, which can be catalyzed into 3-hydroxybenzyl alcohol and then oxidized to 3-hydroxybenzaldehyde. The mchC gene encoded 3-hydroxybenzaldehyde dehydrogenase , which catalyzes the oxidation of 3-hydroxybenzaldehyde to 3-hydroxybenzoate. The results indicated that MchAB and MchC are the first characterized enzymes to catalyze the oxidation of the meta -methyl group of m -cresol in the strain CHJ601.
研究领域:
环境微生物学
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分子生物学
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生物化学
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DOI
高影响因子
Frontiers in Microbiology
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2018
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IF 4.019
Chen Xin,Tang Hongzhi,Liu Yongdi,Xu Ping,Xue Yong,Lin Kuangfei,Cui Changzheng
3-Hydroxybenzoate 6-hydroxylase, an NADH-dependent flavoprotein, can convert 3-hydroxybenzoate which is an important intermediate in the biodegradation of many aromatic hydrocarbons. 3-Hydroxybenzoate is metabolized by entering the TCA cycle through the gentisate pathway. We found a putative 3HB6H gene from a cluster that potentially encodes for gentisic acid degradation from a halophilic Martelella sp. strain AD-3. The corresponding protein was expressed with an N-terminal His-tag and purified by Ni2+-nitrilotriacetic acid affinity chromatography. The protein showed an overexpressed band of about 46 kDa by SDS–PAGE, and it was also proven that the enzyme contains FAD by absorption spectroscopy and HPLC analysis. The optimal activity of 3HB6H from strain AD-3 was observed in phosphate buffer (pH 8.0) at 37∘C without salinity (NaCl) and metal salts. The Km values of 3-hydroxybenzoate 6-hydroxylase were determined to be 72.6 ± 10.1 μM and 104.1 ± 18.2 μM for 400 μM NADH and 3-hydroxybenzoate, respectively. Site-directed mutagenesis showed that residues 305, 306 and 308 are important for FAD binding. In addition, we found that Tyr221 and Gln305 of 3HB6H from strain AD-3 are involved in substrate binding.
研究领域:
微生物学
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酶学
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DOI
PubMed
高影响因子
Journal of Materials Chemistry B
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2023
·Vol. 11
·期 47
·11235-11250
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IF 7
Chan Chen,Haisheng Ren,Weikang Tang,Mengqi Han,Qinfei Chen,Hong Zhou,Jiadong Chen,Yuyue Gao,Wenbin Liu
L-3,4-Dihydroxyphenylalanine (L-DOPA) is widely used in Parkinson's disease treatment and is therefore in high demand. Development of an efficient method for the production of L-DOPA is urgently required. Nanozymes emulating tyrosine hydroxylase have attracted enormous attention for biomimetic synthesis of L-DOPA, but suffered from heterogeneity. Herein, a spherical porous iron–nitrogen–carbon nanozyme was developed for production of L-DOPA. Tannic acid chelated with ferrous ions to form a tannin-iron coordination framework as a carbon precursor. Iron and nitrogen co-doped carbon nanospheres were assembled via an evaporation-induced self-assembly process using urea as a nitrogen source, F127 as a soft template, and formaldehyde as a crosslinker. The nanozyme was obtained after carbonization and acid etching. The nanozyme possessed a dispersive iron atom anchored in the Fe–N coordination structure as an active site to mimic the active center of tyrosine hydroxylase. The material showed spherical morphology, uniform size, high specific surface area, a mesoporous structure and easy magnetic separation. The structural properties could promote the density and accessibility of active sites and facilitate mass transport and electron transfer. The nanozyme exhibited high activity to catalyze the hydroxylation of tyrosine to L-DOPA as tyrosine hydroxylase in the presence of ascorbic acid and hydrogen peroxide. The titer of DOPA reached 1.2 mM. The nanozyme showed good reusability and comparable enzyme kinetics to tyrosine hydroxylase with a Michaelis–Menten constant of 2.3 mM. The major active species was the hydroxyl radical. Biomimetic simulation of tyrosine hydroxylase using a nanozyme with a fine structure provided a new route for the efficient production of L-DOPA.
研究领域:
生物化学
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材料科学
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医学
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DOI
PubMed
高影响因子
BRAZILIAN JOURNAL OF MICROBIOLOGY
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2025
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IF 1.9
Lv Jianhua,Gu Xiangnan,Hui Minmin,Luo Yingjie,Luo Wenbo,Zhao Huiting,Yin Shuqin,Feng Yan,Xue Zhiquan
Phenol hydroxylase (PH) plays a central role in phenol biodegradation pathways. In this study, the enzymatic activity of PH in Candida tropicalis GY8 was evaluated following phenol-induced cultivation, and oxidation products were analyzed via high-performance liquid chromatography (HPLC). Two phenol hydroxylase genes, CtPHE1 and CtPHE2 , were identified, and their expression patterns were examined using RT-qPCR. Functional analysis of the corresponding proteins was conducted by cloning and expressing the genes in Escherichia coli BL21(DE3). Enzymatic activity reached its maximum after 15 h of cultivation ( p < 0.01), and phenol conversion to catechol was verified by HPLC. RT-qPCR results indicated peak transcript levels at 10 h, with CtPHE2 showing consistently higher expression than CtPHE1 at 5, 10, and 15 h ( p < 0.01). Both recombinant proteins were capable of phenol degradation, with CtPHE2 exhibiting greater catalytic activity. These findings provide essential molecular information for advancing the understanding of phenol biodegradation mechanisms.
研究领域:
微生物学
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分子生物学
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环境科学
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DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
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2024
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IF 7.7
BingBing Wang,Jian-Zhong Xu,Shuai Liu,Zhi-Ming Rao,Wei-Guo Zhang
L-Tryptophan hydroxylation catalyzed by tryptophan hydroxylase (TPH) presents a promising method for synthesizing 5-hydroxytryptophan (5-HTP), yet the limited activity of wild-type human TPH2 restricts its application. A high-activity mutant, MT10 (H318E/H323E), was developed through semi-rational active site saturation testing (CAST) of wild-type TPH2, exhibiting a 2.85-fold increase in k cat / K m over the wild type, thus enhancing catalytic efficiency . Two biotransformation systems were developed, including an in vitro one-pot system and a Whole-Cell Catalysis System (WCCS). In the WCCS, MT10 achieved a conversion rate of only 31.5 % within 32 h. In the one-pot reaction, MT10 converted 50 mM L-tryptophan to 44.5 mM 5-HTP within 8 h, achieving an 89 % conversion rate, outperforming the M1 (NΔ143/CΔ26) variant. Molecular dynamics simulations indicated enhanced interactions of MT10 with the substrate, suggesting improved binding affinity and system stability. This study offers an effective approach for the efficient production of 5-HTP.
研究领域:
生物化学
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分子生物学
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合成生物学
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PubMed
高影响因子
BIOCHEMISTRY
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2023
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IF 2.9
Zihan Zhang,Longhao Huang,Zhi-Jun Zhang,Jian-He Xu,Hui-Lei Yu
Cytochrome P450 monooxygenases (CYP450s) play an important role in the biosynthesis of natural products by activating inert C–H bonds and inserting hydroxyl groups. However, the activities of most plant-derived CYP450s are extremely low, limiting the heterologous biosynthesis of natural products. Traditional enzyme engineering methods, either rational or screening-based, are not suitable for CYP450s because of the lack of crystal structures and high-throughput screening methods for this class of enzymes. CYP725A4 is the first hydroxylase involved in the biosynthesis pathway of Taxol. Its low activity, promiscuity, and multispecificity make it a bottleneck in Taxol biosynthesis. Here, we identified key amino acids that affect the in vivo activity of CYP725A4 by constructing the ancestral enzymes of CYP725A4. We obtained positive mutants that showed an improved yield of hydroxylated products based on the key amino acids identified, providing guidance for the modification of other CYP450s involved in the biosynthesis of natural products.
研究领域:
生物化学
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分子生物学
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合成生物学
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产品关联
DOI
PubMed
高影响因子
INTERNATIONAL IMMUNOPHARMACOLOGY
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2025
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IF 4.7
Qingqi Zhou,Xun Li,Decai Hou
Background Osteoarthritis (OA) is an age-related progressive joint disease characterized by loss of cartilage and subsequent inflammation. Perillaldehyde is a compound extracted from Perilla , which has multiple pharmacological activities including anti-inflammatory, and anti-apoptosis. However, it still remains unknown whether and how perillaldehyde could regulate chondrocyte apoptosis and inflammation in OA. Methods The interleukin-1beta (IL-1β)-treated chondrocytes and destabilized medial meniscus (DMM) -induced rats were used as in vitro and in vivo models of OA. Cell viability, proliferation, and apoptosis were investigated by cell counting kit-8 (CCK-8), 5-ethynyl-2′-deoxyuridine (EdU) and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays. The mitochondrial membrane potential was analyzed by 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) staining. Light chain 3 (LC3) location, and expression of NOD-like receptor thermal protein domain-associated protein 3 (NLRP3) and apoptosis-associated speck-like protein (ASC) were detected by immunofluorescence staining. Relative protein levels were measured via western blotting. Tumor necrosis factor-alpha (TNF-α), IL-6 and IL-8 levels were measured by enzyme-linked immunosorbent assay (ELISA). The cartilage injury in rats was investigated via hematoxylin-eosin (HE) staining. Results Perillaldehyde attenuated IL-1β-induced inhibition of viability (from 44.45 % to 92.72 %, p < 0.05) and proliferative potential of chondrocytes ( p < 0.05). Perillaldehyde mitigated mitophagy-associated apoptosis of chondrocytes by enhancing mitophagy ( p < 0.05) and reducing cell apoptosis (from 32.36 % to 8.00 %, p < 0.05). Perillaldehyde attenuated NLRP3-mediated inflammation through reducing NLRP3, ASC, TNF-α, IL-6 and IL-8 levels ( p < 0.05). ALOX5 expression was upregulated in OA (fold change: 2.61, p < 0.05), and decreased by perillaldehyde treatment (fold change: 1.34, p < 0.05). Perillaldehyde inhibits p65 nuclear factor kappa B (NF-κB) signaling activation (fold change: 3.19 to 1.33, p < 0.05) by regulating ALOX5 expression. ALOX5 overexpression reversed the effects of perillaldehyde on mitophagy-associated apoptosis and NLRP3-mediated inflammation ( p < 0.05), and these roles were mitigated due to NF-κB inactivation ( p < 0.05). Perillaldehyde attenuated cartilage injury in OA rats ( p < 0.05). Conclusion Perillaldehyde attenuated IL-1β-induced mitophagy-associated apoptosis and NLRP3-mediated inflammation through decreasing ALOX5 and inactivating NF-κB signaling, indicating the potentially protective potential of perillaldehyde in osteoarthritis.
研究领域:
医学
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药理学
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DOI
PubMed
高影响因子
BIOCHEMICAL ENGINEERING JOURNAL
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2025
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IF 3.8
Jiayan Du,Youtong Bao,Jingyuan Zhu,Xueqing Pang,Depeng Ren,Xinjian Yin,Pingping Zhou
Astaxanthin, a potent antioxidant widely applied in health supplements, cosmetics, and animal feed, faces production challenges in microbial synthesis due to inefficient β-carotene hydroxylase activity. This study identified rate-limiting enzymes and demonstrated that the expressing phytoene desaturase BtCrtI from Blakeslea trispora in Saccharomyces cerevisiae increased β-carotene production 2.8-fold compared to XdCrtI from Xanthophyllomyces dendrorhous . Integrating of XdCrtYB, BtCrtI, CrtE03M, and tHMG1 achieved 208.7 mg/L β-carotene. However, introducing β-carotene ketolase and β-carotene hydroxylase into this strain unexpectedly increased lycopene accumulation, likely due to feedback inhibition by the product and enzyme imbalance. To address this, we utilized a moderate β-carotene producer YJYast-02 for astaxanthin production. Truncating the N-terminal 39 amino acids of β-carotene hydroxylase from Haematococcus pluvialis improved astaxanthin titer by 37.7 %. Rational engineering of the hydrophobicity of the substrate-binding pocket in β-carotene hydroxylase, specifically through mutations such as N183A, C191A and T213A, further increased astaxanthin titer by 15.8 %, 30.5 %, 36.8 %, respectively. The final engineered strain, YJYast-03(t5t39 T213A ), expressing t5OBKTM30 and t39OcrtZM1 T213A , achieved 26.0 mg/L (3.6 mg/g DCW) astaxanthin. This work pioneers the use of molecular docking for engineering β-carotene hydroxylase, offering potential to significantly enhance catalytic activity and optimize astaxanthin biosynthesis in yeast.
研究领域:
生物工程
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合成生物学
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微生物学
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DOI
高影响因子
ENZYME AND MICROBIAL TECHNOLOGY
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2023
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IF 3.4
Feifei Song,Tao Gu,Lin Zhang,Jiaxing Zhang,Shengping You,Wei Qi,Rongxin Su
5-Hydroxytryptophan (5-HTP) is a chemical precursor of serotonin, which synthesizes melatonin and serotonin in animals and regulates mood, sleep , and behavior. Tryptophan hydroxylase (TPH) uses tetrahydrobiopterin (BH4) as a cofactor to hydroxylate L -tryptophan ( L -Trp) to 5-HTP, and the low catalytic activity of TPH limits the rate of hydroxylation of L -Trp. In this study, the catalytic mechanism and structural features of L -Trp-TPH1-BH4 were investigated, and the catalytic activity was improved using a rational design strategy. Then the S337A/F318Y beneficial mutation was obtained. Molecular dynamics simulations showed that the S337A/F318Y mutant formed a salt bridge with TPH1 while forming an additional hydrogen bond with the substrate indole ring, stabilizing the indole ring and enhancing the binding affinity of the variant to L -Trp. As a result, the yield of 5-HTP was increased by 2.06-fold, resulting in the production of 0.91 g/L of 5-HTP. The rational design of the TPH structure to improve the hydroxylation efficiency of L -Trp offers the prospect of green production of 5-HTP.
研究领域:
生物化学
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分子生物学
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代谢工程
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DOI
PubMed
高影响因子
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
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2023
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IF 5
Zhang Xiaohan,Zhang Dong,Ding Yan,Li Zilong,Wang Chenchen,Ye Shuhong
Abstract Resveratrol (RES) is a secondary metabolite synthesized by plants in response to environmental stress and pathogen infection, which is of great significance for the industrial production of RES by fermentation culture. In this study, we aimed to explore the biosynthesis pathway of RES and its key enzymes in the Priestia megaterium PH3, which was isolated and screened from peanut fruit. Through Liquid Chromatography-Mass Spectrometry (LC-MS) analysis, we quantified the RES content and distribution in the culture medium and determined that Priestia megaterium PH3 mainly secreted RES extracellularly. Furthermore, the highest production of RES was observed in YPD, yielding an impressive 127.46 ± 6.11 μg/L. By optimizing the fermentation conditions, we achieved a remarkable RES yield of 946.82 ± 24.74 μg/L within just 2 days, which represents the highest reported yield for a natural isolate produced in such a short time frame. Our investigation revealed that the phenylpropane pathway is responsible for RES synthesis in this bacterium, with cinnamate 4-hydroxylase (C4H) identified as the main rate-limiting enzyme. Overall, our findings highlight the robust RES production capabilities of Priestia megaterium PH3, offering novel insights and potential applications for bacterial fermentation in RES production. Key points
• RES synthesized by the bacterium was confirmed through the phenylpropane pathway.
• The key rate-limiting enzyme for biosynthesis-RES is C4H.
• RES reached 946.82 ± 24.74 μg/L after fermentation for 2 days.
Graphical Abstract
研究领域:
生物技术
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微生物学
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药学
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产品关联
DOI
PubMed
高影响因子
BIOTECHNOLOGY AND BIOENGINEERING
·
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
危险品化学品经营许可证（带存储）