PROCESS BIOCHEMISTRY
·
2023
·
IF 4.4
Xinxin Wang,Xia Ke,Ximan Zhao,Qijie Ren,Jiahao Cui,Zhiqiang Liu,Yuguo Zheng
Understanding the mycobacteria degradation of sterol may facilitate strategies for the directed biotransformation of steroidal drug intermediates. Due to the long and complicated catabolic process, several key steps that involved in the C-C bond cleavage of steroidal side-chain remain unknown. In the present study, an aldolase SalA from Mycobacterium neoaurum was identified that was related to the synthesis of 3-oxo-4-pregnene-20-carboxyl aldehyde and 20-hydroxymethyl pregn-4-ene-3-one (4-HBC). As a member of the thiolase superfamily, the aldolase SalA-SalB is composed of a thiolase-like protein (SalA) and a DUF35 domain (SalB). As a result, the gene salA was determined that had special significance for the synthesis of 4-HBC in sterol metabolism by performing knockout and replenishment. Based on insights provided by the docking model and mutants analysis, the hypothesis of the catalytic mechanism was proposed. Furthermore, the deletion of salA absolutely eliminated the by-product 4-HBC and meanwhile the molar yield of ADD was increased by 10 %. The dominant role of SalA in the C24 steroidal side-chain cleavage as confirmed in this study provided a further clarification on the phytosterol degrading pathway in Mycobacterium neoaurum , which is of significant importance for the directed biotransformation between C19- and C22-steroids.
研究领域:
微生物学
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生物化学
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药物生物转化
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产品关联
DOI
高影响因子
Microbial Cell Factories
·
2017
·Vol. 16
·期 1
·1-10
·
IF 3.681
Xiong Liang-Bin,Liu Hao-Hao,Xu Li-Qin,Sun Wan-Ju,Wang Feng-Qing,Wei Dong-Zhi
Background The strategy of modifying the sterol catabolism pathway in mycobacteria has been adopted to produce steroidal pharmaceutical intermediates, such as 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC), which is used to synthesize various steroids in the industry. However, the productivity is not desirable due to some inherent problems, including the unsatisfactory uptake rate and the low metabolic efficiency of sterols. The compact cell envelope of mycobacteria is a main barrier for the uptake of sterols. In this study, a combined strategy of improving the cell envelope permeability as well as the intracellular sterol metabolism efficiency was investigated to increase the productivity of 4-HBC.Results
MmpL3, encoding a transmembrane transporter of trehalose monomycolate, is an important gene influencing the assembly of mycobacterial cell envelope. The disruption of mmpL3 in Mycobacterium neoaurum ATCC 25795 significantly enhanced the cell permeability by 23.4% and the consumption capacity of sterols by 15.6%. Therefore, the inactivation of mmpL3 was performed in a 4-HBC-producing strain derived from the wild type M. neoaurum and the 4-HBC production in the engineered strain was increased by 24.7%. Subsequently, to enhance the metabolic efficiency of sterols, four key genes, choM1, choM2, cyp125, and fadA5, involved in the sterol conversion pathway were individually overexpressed in the engineered mmpL3-deficient strain. The production of 4-HBC displayed the increases of 18.5, 8.9, 14.5, and 12.1%, respectively. Then, the more efficient genes (choM1, cyp125, and fadA5) were co-overexpressed in the engineered mmpL3-deficient strain, and the productivity of 4-HBC was ultimately increased by 20.3% (0.0633 g/L/h, 7.59 g/L 4-HBC from 20 g/L phytosterol) compared with its original productivity (0.0526 g/L/h, 6.31 g/L 4-HBC from 20 g/L phytosterol) in an industrial resting cell bio-transformation system.Conclusion sIncreasing cell permeability combined with the co-overexpression of the key genes (cyp125, choM1, and fadA5) involved in the conversion pathway of sterol to 4-HBC was effective to enhance the productivity of 4-HBC. The strategy might also be useful for the conversion of sterol to other steroidal intermediates by mycobacteria.
研究领域:
生物工程
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微生物学
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制药工程
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DOI
PubMed
高影响因子
JOURNAL OF BIOLOGICAL CHEMISTRY
·
2026
·
IF 4.1
Yunjie Liu,Yue Zhao,Weihan Sun,Nian Li,Yunjun Pan,Li Ma,Shengying Li
Steroids with anti-inflammatory, anti-allergic, endocrine-regulating, and other pharmaceutical activities represent the second most widely used class of drugs worldwide, following antibiotics. Their industrial production primarily relies on Mycobacteria -mediated biotransformation of sterols into key intermediates, followed by chemical or enzymatic modifications. While the sterol metabolic pathways in Mycobacteria have been intensively studied, the identification and functional characterization of key enzymes, particularly cytochrome P450 enzymes (CYPs or P450s) and their cognate redox partners, remain incomplete. Here, we heterologously expressed 24 P450s, 10 ferredoxin reductases (FdRs), and 12 ferredoxins (Fdxs) from Mycobacterium neoaurum ZC-1 in Escherichia coli . In vitro biochemical experiments identified five P450 enzymes (CYP125A76, CYP125A77, CYP125A78, CYP142A12, and CYP124A1) capable of catalyzing sterol side-chain terminal oxidation. Screening 120 redox partner combinations revealed FdR4662/Fdx4443 as the optimal cognate redox partners for all five P450 enzymes. With this redox partner pair, CYP142A12 achieved a conversion ratio of 89 % for 4-cholesten-3-one with NADH as the preferred cofactor. Structural analyses indicate that the electron-transfer efficiency is primarily governed by electrostatic complementarity around the Fe–S cluster, the redox-center distance between the Fe–S cluster and heme-iron, and the FAD-to-cluster distance within the FdR-Fdx complex. These findings highlight the critical role of redox partner selection in enhancing P450 catalytic efficiency and provide a solid foundation for engineering high-efficiency industrial strains to improve steroid biomanufacturing and reduce production costs.
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DOI
PubMed
高影响因子
JOURNAL OF BASIC MICROBIOLOGY
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2019
·Vol. 59
·期 9
·924-935
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IF 1.76
Wan-Ju Sun,Yong-Jun Liu,Hao-Hao Liu,Jie-De Ma,Yu-Hong Ren,Feng-Qing Wang,Dong-Zhi Wei
Some species of mycobacteria have been modified to transform sterols to valuable steroid synthons. The unique cell wall of mycobacteria has been recognized as an important organelle to absorb sterols. Some cell wall inhibitors (e.g., vancomycin and glycine) have been validated to enhance sterol conversion by interfering with transpeptidation in peptidoglycan biosynthesis. Therefore, two transpeptidase genes, pbpA and pbpB , were selected to rationally modify the cell wall to simulate the enhancement effect of vancomycin and glycine on sterol conversion in a 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC) producing strain (WIII). Unexpectedly, the pbpA or pbpB gene augmentation was conducive to the utilization of sterols. The pbpB augmentation strain WIII- pbpB was further investigated for its better performance. Compared to WIII, the morphology of WIII- pbpB was markedly changed from oval to spindle, indicating alterations of the cell wall. Biochemical analysis indicated that the altered cell wall properties of WIII- pbpB might contribute to the positive effect on sterol utilization. The productivity of 4-HBC was enhanced by 28% in the WIII- pbpB strain compared to that of WIII. These results demonstrated that the modification of peptidoglycan synthesis can improve the conversion of sterols to steroid synthons in mycobacteria.
研究领域:
微生物学
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生物化学
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产品关联
DOI
PubMed
高影响因子
Biotechnology Journal
·
2024
·Vol. 19
·期 11
·e202400489
·
IF 3.2
Bo Zhang,Sifang Zhu,Yi Zhu,Xin Sui,Junping Zhou,Zhiqiang Liu,Yuguo Zheng
Boldenone (BD), a protein anabolic hormone, is commonly used to treat muscle damage, osteoporosis, and off-season muscle building in athletes. Traditional BD synthesis methods rely on chemical processes, which are costly and environmentally impactful. Therefore, developing a more sustainable and economical biosynthetic pathway is crucial for BD production. This study aimed to achieve efficient production of BD. Firstly, the catalytic performance of 17β-hydroxysteroid dehydrogenase and 3-ketosteroid-Δ 1 -dehydrogenase was improved through enzyme engineering, and their expression in the new strain of Mycobacterium neoaurum was enhanced using metabolic engineering. These improvements significantly increased BD production to 4.05 g/L, with a significant decrease in by-product generation. To further increase the yield, a multi-enzyme fusion expression system was constructed, and a key cell wall gene kasB was knocked out, resulting in a spatial-time yield of BD reaching 1.02 g/(L·d). Subsequent optimization of the transformation system further increased the BD production to 5.56 g/L, with a spatiotemporal yield of 1.39 g/(L·d). The green biosynthetic route of phytosterol one-step conversion to BD developed in this study lays the foundation for industrial production.
研究领域:
生物化学
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微生物学
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合成生物学
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产品关联
DOI
PubMed
高影响因子
Systems Microbiology and Biomanufacturing
·
2023
·
IF 0
Li Xuemei,Zhang Rui,Li Jianjiong,Feng Jinhui,Wu Qiaqing,Zhu Dunming,Ma Yanhe
Cholesterol and phytosterols can be served as the substrates for Actinobacteria to produce the precursors used in the synthesis of steroidal medicines. Cytochrome P450s from CYP125 and CYP142 families initiates the metabolism of cholesterol and/or phytosterols. In this study, we demonstrate the functional redundancy and substrate preference of five cytochrome CYP125s and CYP142s by the unmarked cyp genes deletion strains of Mycobacterium neoaurum NRRL B-3805. CYP125-3 was found to be responsible for the catalytic hydroxylation of both cholesterol and the C24-branched side-chains of phytosterols, while CYP142-2 can only oxidize cholesterol and/or cholest-4-en-3-one. The strain harboring the CYP125-3 produced 6.47 g l − 1 and 5.13 g l − 1 of 4-androstene-3,17-dione (AD) when 15 g l − 1 cholesterol or 17.5 g l − 1 phytosterols used, respectively. While, the strain harboring the CYP142-2 produced 7.10 g l − 1 AD when 15 g l − 1 cholesterol used. The other three cytochrome P450s have a smaller contribution for the degradation of cholesterol and phytosterols. CYP125-3 has the hydroxylation activity on more substrates than that of CYP142-2. This study provided possible guidance to improve the efficiency of AD and other metabolites production from different kinds of sterols by the genetic manipulation.
研究领域:
微生物学
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生物化学
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分子生物学
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生物技术
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DOI
Microbial Cell Factories
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2020
·Vol. 19
·期 1
·1-11
·
IF 4.187
Xiong Liang-Bin,Liu Hao-Hao,Zhao Ming,Liu Yong-Jun,Song Lu,Xie Zhi-Yong,Xu Yi-Xin,Wang Feng-Qing,Wei Dong-Zhi
Background The bioconversion of phytosterols into high value-added steroidal intermediates, including the 9 α -hydroxy-4-androstene-3,17-dione (9-OHAD) and 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC), is the cornerstone in steroid pharmaceutical industry. However, the low transportation efficiency of hydrophobic substrates into mycobacterial cells severely limits the transformation. In this study, a robust and stable modification of the cell wall in M. neoaurum strain strikingly enhanced the cell permeability for the high production of steroids. Results The deletion of the nonessential kasB , encoding a β-ketoacyl-acyl carrier protein synthase, led to a disturbed proportion of mycolic acids (MAs), which is one of the most important components in the cell wall of Mycobacterium neoaurum ATCC 25795. The determination of cell permeability displayed about two times improvement in the kasB -deficient strain than that of the wild type M. neoaurum . Thus, the deficiency of kasB in the 9-OHAD-producing strain resulted in a significant increase of 137.7% in the yield of 9 α -hydroxy-4-androstene-3,17-dione (9-OHAD). Ultimately, the 9-OHAD productivity in an industrial used resting cell system was reached 0.1135 g/L/h (10.9 g/L 9-OHAD from 20 g/L phytosterol) and the conversion time was shortened by 33%. In addition, a similar self-enhancement effect (34.5%) was realized in the 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC) producing strain. Conclusions The modification of kasB resulted in a meaningful change in the cell wall mycolic acids. Deletion of the kasB gene remarkably improved the cell permeability, leading to a self-enhancement of the steroidal intermediate conversion. The results showed a high efficiency and feasibility of this construction strategy.
研究领域:
生物化学
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微生物学
·
制药工程
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产品关联
DOI
PubMed
高影响因子
METABOLIC ENGINEERING
·
2019
·
IF 7.808
Wan-Ju Sun,Li Wang,Hao-Hao Liu,Yong-Jun Liu,Yu-Hong Ren,Feng-Qing Wang,Dong-Zhi Wei
The conversion of sterols to steroid synthons by engineered mycobacteria comprises one of the basic ways for the production of steroid medications in the pharmaceutical industry. Here, we revealed that high amounts of reactive oxygen species (ROS) generate during the conversion process of sterols, which impairs the cell viability of mycobacterial cells and thus hinders the conversion of sterols to steroid synthons. Accordingly, the endogenous antioxidants for detoxifying ROS in mycobacteria, ROS scavenging enzymes and low molecular weight thiols, were examined. The results revealed that three antioxidants, catalase (CAT), mycothiol (MSH), and ergothioneine (EGT), demonstrated efficacy toward neutralizing the excessive ROS produced during sterol metabolism . CAT overexpression or MSH or EGT augmentation enhanced the conversion of phytosterols to 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC) by 18.9%, 23.8%, and 32.1%, respectively, and also enhanced the cell viability, indicating the benefits of these antioxidants in reducing ROS-induced stress. Further combinatorial augmentation of CAT, MSH, and EGT demonstrated enhanced effects toward intracellular ROS scavenging, resulting in 54.2% greater cell viability and 47.5% enhancement in 4-HBC production. These findings indicated that the excessive ROS induces cell stress, in turn limiting the conversion of sterols, whereas neutralization of the excessive ROS by combined control of CAT, MSH, and EGT serves as an effective strategy to boost the conversion productivity of sterols to steroid synthons.
研究领域:
药学
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微生物学
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生物技术
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生物化学
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产品关联
DOI
PubMed
高影响因子
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