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Received: 2014-10-15

Revision Accepted: 2015-03-11

Crosschecked: 2015-03-18

Cited: 5

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Citations:  Bibtex RefMan EndNote GB/T7714


Chao Liu


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Journal of Zhejiang University SCIENCE B 2015 Vol.16 No.4 P.286-295


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

Author(s):  Chao Liu, Xian Zhang, Zhi-ming Rao, Ming-long Shao, Le-le Zhang, Dan Wu, Zheng-hong Xu, Hui Li

Affiliation(s):  Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China; more

Corresponding email(s):   raozhm@jiangnan.edu.cn

Key Words:  Mycobacterium neoaurum, Atmospheric and room temperature plasma (ARTP), Mutation breeding, 4-Androstene-3, 17-dione (AD), 1, 4-Androstadiene-3, 17-dione (ADD)

Chao Liu, Xian Zhang, Zhi-ming Rao, Ming-long Shao, Le-le Zhang, Dan Wu, Zheng-hong Xu, Hui Li. Mutation breeding of high 4-androstene-3,17-dione-producing Mycobacterium neoaurum ZADF-4 by atmospheric and room temperature plasma treatment[J]. Journal of Zhejiang University Science B, 2015, 16(4): 286-295.

@article{title="Mutation breeding of high 4-androstene-3,17-dione-producing Mycobacterium neoaurum ZADF-4 by atmospheric and room temperature plasma treatment",
author="Chao Liu, Xian Zhang, Zhi-ming Rao, Ming-long Shao, Le-le Zhang, Dan Wu, Zheng-hong Xu, Hui Li",
journal="Journal of Zhejiang University Science B",
publisher="Zhejiang University Press & Springer",

%0 Journal Article
%T Mutation breeding of high 4-androstene-3,17-dione-producing Mycobacterium neoaurum ZADF-4 by atmospheric and room temperature plasma treatment
%A Chao Liu
%A Xian Zhang
%A Zhi-ming Rao
%A Ming-long Shao
%A Le-le Zhang
%A Dan Wu
%A Zheng-hong Xu
%A Hui Li
%J Journal of Zhejiang University SCIENCE B
%V 16
%N 4
%P 286-295
%@ 1673-1581
%D 2015
%I Zhejiang University Press & Springer
%DOI 10.1631/jzus.B1400274

T1 - Mutation breeding of high 4-androstene-3,17-dione-producing Mycobacterium neoaurum ZADF-4 by atmospheric and room temperature plasma treatment
A1 - Chao Liu
A1 - Xian Zhang
A1 - Zhi-ming Rao
A1 - Ming-long Shao
A1 - Le-le Zhang
A1 - Dan Wu
A1 - Zheng-hong Xu
A1 - Hui Li
J0 - Journal of Zhejiang University Science B
VL - 16
IS - 4
SP - 286
EP - 295
%@ 1673-1581
Y1 - 2015
PB - Zhejiang University Press & Springer
ER -
DOI - 10.1631/jzus.B1400274

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)%29&ck%5B%5D=abstract&ck%5B%5D=keyword'>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 atmospheric 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 1;0.11) g/L of AD and (0.82 1;0.05) g/L of ADD were produced by the ZADF-4 mutant, compared with (4.83 1;0.13) g/L of AD and (2.34 1;0.06) g/L of ADD by the original strain, M. neoaurum 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.

采用常压室温等离子体诱变技术获得一株高产雄甾-4-烯-3,17-二酮的突变菌株Mycobacterium neoauru ZADF-4

目的:获得一株高产雄甾-4-烯-3,17-二酮(AD)的Mycobacterium neoaurum突变株。
创新点:获得了一株3-甾酮-Δ1-脱氢酶(KSDD)酶活缺陷型的高产AD的诱变菌株Mycobacterium neoaurum ZADF-4,并采用菌落显色法筛选KSDD酶活缺陷型M. neoaurum突变株。
方法:(1)诱变方法:采用常压室温等离子体(ARTP)诱变技术来处理出发菌株M. neoaurum ZAD。ARTP诱变条件如下:功率40 W,气流量 12.5 L/min,辐射距离1 cm,样品体积10 µl,辐射时间为60、90、120、150和180 s;致死率统计优化后,最适辐射时间为150 s,致死率为90%~96%。(2)筛选方法:将ARTP诱变处理后的菌株点种在硝酸纤维滤膜上,30 °C培养2 d,然后将长有菌落的滤膜小心取出并漂浮在4 mg/ml 二氯靛酚(DCPIP)溶液(0.1 mmol/L 磷酸缓冲液pH 7.0),30 °C培养1 d直到全部菌落染成蓝色。然后将该滤膜取出,漂浮在250 mmol/L AD溶液(2%甲醇和50 mmol/L Tris pH 7.0缓冲液),室温放置15 min左右,观察菌落颜色变化。KSDD在底物AD存在时会脱氢产生雄甾-1,4-二烯-3,17-二酮(ADD)和H+,H+可以使被DCPIP染成蓝色的菌株褪色。因此,酶活缺陷型的菌株会仍保持蓝色,而酶活高的菌株会褪色为黄色 (图3)。(3)对获得的潜在的高产AD菌株进行进一步的酶活检测以及产量验证,以期获得最优的突变株。
结论:获得了4株具有潜在的高产AD能力的菌株,其中,最优的突变株ZADF-4的KSDD酶活相较于出发菌株ZAD下降了81.2%(图4),活性胶也证明其KSDD酶活相较于出发菌株下降明显(图5)。薄层色谱法(TLC)和高效液相色谱法(HPLC)实验证明突变株ZADF-4中,AD的产量有了明显的提高(图6和图7),提高到了(6.28±0.11) g/L,AD/ADD提高到8:1,AD的摩尔产率达到60.3%(表1)。对出发菌株ZAD和突变株ZADF-4的ksdd基因进行克隆和序列比对,发现ZADF-4的ksdd序列在5'端缺失9个核苷酸(atgttctac),导致3个氨基酸(MFY)的缺失;还发生了两个点突变,其中一个是无义突变(g.15a>6t),另一个是有义突变(g.413c>404t),并引起了相应位置上的氨基酸变化(p.138S>135L)。上述的基因突变及其引起的氨基酸序列的变化可能是引起M. neoaurum ZADF-4中KSDD酶活降低及AD产量提高的主要原因。

关键词:Mycobacterium neoaurum;常压室温等离子体 (ARTP);诱变育种;雄甾-4-烯-3,17-二酮(AD);雄甾-1,4-二烯-3,17-二酮(ADD)

Darkslateblue:Affiliate; Royal Blue:Author; Turquoise:Article


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