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Mutation breeding of high 4-androstene-3,17-dione-producing mycobacterium neoaurum ZADF-4 by atmospheric and room temperature plasma treatment

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Journal of Zhejiang University-Science B(Biomedicine & Biotechnology) 2015年第4期16卷 286-295页

作者： Chao LIU Xian ZHANG Zhi-ming RAO Ming-long SHAO Le-le ZHANG Dan WU Zheng-hong XU Hui LI Key Laboratory of Industrial Biotechnology Ministry of Education School of Biotechnology Jiangnan University Laboratory of Applied Microorganisms and Metabolic Engineering School of Biotechnology Jiangnan University Laboratory of Pharmaceutical Engineering School of Pharmaceutical Science Jiangnan University

Steroid medication is used extensively in clinical applications and comprises a large and vital part of the pharmaceutical industry. However, the difficulty of separating 4-androstene-3,17-dione （AD） from 1,4-androstadiene- 3,17-dione （ADD） restricts the application of the microbial transformation of phytosterols in the industry. A novel at- mospheric and room temperature plasma （ARTP） treatment, which employs helium as the working gas, was used to generate mycobacterium neoaurum mutants producing large amounts of AD. After treatment of cultures with ARTP, four mutants were selected using a novel screening method with a color assay. Among the mutants, M. neoaurum ZADF-4 was considered the best candidate for industrial application. When the fermentation medium contained 15 g/L phytosterols and was cultivated on a rotary shaker at 160 r/min at 30 ~C for 7 d, （6.28＋0.11） g/L of AD and （0.82＋0.05） g/L of ADD were produced by the ZADF-4 mutant, compared with （4.83＋0.13） g/L of AD and （2.34＋0.06） g/L of ADD by the original strain, Iv~. neoaururn ZAD. Compared with ZAD, the molar yield of AD increased from 48.3% to 60.3% in the ZADF-4 mutant. This result indicates that ZADF-4 may have potential for industrial production of AD.

关键词： mycobacterium neoaurum Atmospheric and room temperature plasma (ARTP) Mutation breeding 4-Androstene-3 17-dione (AD) 1 4-Androstadiene-3 17-dione (ADD)

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A streamlined high throughput screening method for the mycobacterium neoaurum mutants with expected yield of biotransformation derivatives from sterols

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Chinese Chemical Letters 2018年第8期29卷 1251-1253页

作者： Chaoxiong Wu Junqi Xu Jianping Xie Zhenyu Wang College of Biomedical Engineering Chongqing Medical University Institute of Modern Biopharmaceuticals School of Life Sciences Southwest University

Steroidal drugs have wide indications such as anti-inflammation, anti-tumor, endocrine regulation,fertility management. Phytosterol is the main starting materials for the industrial synthesis of steroid drugs. Microbial transformation of phytosterol is a simple and environmentally friendly process. Efficient microbial strains for industrial phytosterol transformation are critical for the commercial success. To this end, a 96-well plate based method was developed to discriminate the mixture of 4-androstene-3,17-dione, androsta-1,4-diene-3,17-dione, and bisnoraldehyde, with different ratio of the 3 components in the mixture, which mimics the sterol bioconversion products by using the mycobacterium neoaurum. The M. neoaurum bioconversed broth test using phytosterol as substrate also found that the spectrum methodology can evaluate the relative content of different compounds. The method is practical, high throughput and can replace the conventional HPLC-based assay for rapid selection of desired microbial strains.

关键词： mycobacterium neoaurum Sterol Biotransformation Mutants 4-Androstene-3,17-dione (AD) Androsta-1,4-diene-3,17-dione (ADD)

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The function of steroid terminal monooxygenase in cholesterol or phytosterol degradation by the mycobacterium neoaurum NRRL B‑3805

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Systems Microbiology and Biomanufacturing 2023年第4期3卷 585-592页

作者： Xuemei Li Rui Zhang Jianjiong Li Jinhui Feng Qiaqing Wu Dunming Zhu Yanhe Ma National Center of Technology Innovation for Synthetic Biology National Engineering Laboratory for Industrial Enzymes and Tianjin Engineering Research Center of Biocatalytic TechnologyTianjin Institute of Industrial BiotechnologyChinese Academy of Sciences32 Xi Qi DaoTianjin Airport Economic AreaTianjin 300308China

Cholesterol and phytosterols can be served as the substrates for Actinobacteria to produce the precursors used in the synthesis of steroidal *** P450s from CYP125 and CYP142 families initiates the metabolism of cholesterol and/or *** 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 ***125-3 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 *** 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,***,the strain harboring the CYP142-2 produced 7.10 g l^(-1)AD when 15 g l^(-1)cholesterol *** other three cytochrome P450s have a smaller contribution for the degradation of cholesterol and ***125-3 has the hydroxylation activity on more substrates than that of *** study provided possible guidance to improve the efficiency of AD and other metabolites production from different kinds of sterols by the genetic manipulation.

关键词： Sterol degradation mycobacterium neoaurum Steroid C26/C27 monooxygenase Cytochrome CYP125s and CYP142s

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高产雄甾-4-烯-3,17-二酮新金色分枝杆菌的选育及发酵条件优化

高产雄甾-4-烯-3,17-二酮新金色分枝杆菌的选育及发酵条件优化

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作者：刘超江南大学

学位级别：硕士

甾体药物是指分子结构中含有甾核结构的药物,是仅次于抗生素的的第二大类药物,在临床上有广泛的应用。目前甾体药物生产中常用的方法有化学合成法和微生物转化法两种,其中微生物转化法有明显的优势。分支杆菌可以降解植物甾醇生成重要... 详细信息

甾体药物是指分子结构中含有甾核结构的药物,是仅次于抗生素的的第二大类药物,在临床上有广泛的应用。目前甾体药物生产中常用的方法有化学合成法和微生物转化法两种,其中微生物转化法有明显的优势。分支杆菌可以降解植物甾醇生成重要的甾体药物中间体雄甾-4-烯-3,17-二酮(androstene-4-end-3,17-dione,AD)和雄甾-1,4-二烯-3,17-二酮(androstene-1,4-end-3,17-dione,ADD),是合成其他甾体药物的前体物质。3-甾酮-△1-脱氢酶(KSDD)是微生物降解植物甾醇途径中的关键酶,它催化AD生成ADD。如果KSDD缺失,则AD的生成量将会提高。本实验室保存有一株高效转化植物甾醇生成AD和ADD的菌株,mycobacterium neoaurum ZAD,为进一步提高AD的产量和比例,对*** ZAD进行诱变,筛选高产AD突变菌株,并对高产AD的变异菌株进行蛋白组学研究。主要内容如下:(1)以*** ZAD为出发菌株,采用常压室温等离子体(ARTP)诱变技术对出发菌株的单细胞悬液进行诱变处理。然后依据H+依懒型脱氢酶缺失型筛选方法进行初筛,通过摇瓶进行复筛,得到一株高产AD的变异菌株*** ZADF-4。变异菌株*** ZADF-4中,AD产量达6.28±0.11 g/L,AD:ADD为8:1,AD的摩尔得率从48.3%提高到了60.3%。(2)在摇瓶水平上,对*** ZADF-4进行发酵培养优化:其最佳碳源为20 g/L葡萄糖,最佳氮源为大豆蛋白胨10 g/L,K2HPO4 2 g/L,Mg SO4 0.3 g/L,最佳底物投加量为15 g/L,助溶剂与底物之间的最佳比例为3:1;接种量为10%(v/v),培养温度为30 oC,摇瓶实验得AD产量为6.34±0.21 g/L。并用5 L发酵罐在优化后的培养基的基础上进行发酵实验,AD的产量提高到6.68±0.12 g/L。(3)利用2-DE体系串联使用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)技术对变异菌株*** ZADF-4在添加植物甾醇与否两种条件下的蛋白表达差异情况进行研究,共鉴定出16个显著差异蛋白。其中,与AD合成途径相关的酶中,乙醇脱氢酶表达量下降,而硫转移酶和α/β水解酶表达量上调。(4)在转录水平上,利用实时荧光定量PCR(q PCR)技术对2-DE实验结果进行验证。q PCR结果表明,除了乙醇脱氢酶的表达量与预测不同外,其他的q PCR结果与2-DE结果相符。以上研究获得了一株高产AD的变异菌株*** ZADF-4;对ZADF-4蛋白组学研究,为其代谢途径调控提供了理论指导。

关键词：雄甾-4-烯-3,17-二酮常压室温等离子体诱变双向电泳 mycobacterium neoaurum

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