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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2017, Vol. 37 Issue (3): 65-72    DOI: 10.13523/j.cb.20170309
技术与方法     
利用异源正调控基因acyB2构建埃莎霉素I高产菌株
戴剑漉1, 卢智黎1, 林灵1,2, 王以光1, 赫卫清1
1. 中国医学科学院/北京协和医学院 医药生物技术研究所 国家卫计委抗生素生物工程重点实验室 北京 100050;
2. 东北农业大学生命科学学院 哈尔滨 150030
Construction of the Isomycin I High-yield Strain by Introducing a Heterologous Positive Regulatory Gene acyB2
DAI Jian-lu1, LU Zhi-li1, LIN Ling1,2, WANG Yi-guang1, HE Wei-qing1
1. Key Lab of Antibiotic Biotechnology, National Health and Family Planning Commission, Institute of Medicinal Biotechnology Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China;
2. College of Life Science, Northeast Agricultural University, Harbin 150030, China
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摘要：

必特螺旋霉素（bitespiramycin，BT）是以异戊酰螺旋霉素（简称为埃莎霉素）Ⅰ、Ⅱ和Ⅲ为主要成分的多组分抗生素。通过阻断3-O-酰基转移酶基因（sspA），获得了只产埃莎霉素Ⅰ的WSJ-2菌株，但其发酵产物中含有大量螺旋霉素，而埃莎霉素Ⅰ含量较低。为提高4″-异戊酰基转移酶基因（ist）在WSJ-2中的表达水平，从而提高埃莎霉素Ⅰ的产量，首先构建了含有ist基因和其正调控基因acyB2连锁片段的重组质粒pSET152-ia，然后将其导入到埃莎霉素Ⅰ产生菌WSJ-2中，通过具有阿普拉霉素（apramycin）抗性标记的链霉菌整合型载体pSET152整合到WSJ-2的染色体上，获得新的埃莎霉素Ⅰ产生菌WSJ-IA。在不同发酵时间定量检测ist的表达水平，WSJ-IA中ist的表达量要明显高于WSJ-2。WSJ-IA高产菌株的发酵单位从（280±20）μg/ml提高至（1160 ±108）μg/ml，较原始菌株WSJ-2提高了314%，而且在WSJ-IA发酵产物中埃莎霉素Ⅰ与螺旋霉素Ⅰ含量的比值是WSJ-2的2.4倍左右，说明在引入acyB2基因的同时提高了菌株的发酵单位和埃莎霉素Ⅰ的产量。
关键词： 4″  埃莎霉素Ⅰ;  -异戊酰基转移酶基因(ist);  正调控基因acyB2    
Abstract:

Bitespiramycin (BT) is a multi-component antibiotic consisted mainly of 4″-isovalerylspiramycin (isomycin) I, II and III. An isomycin I producing strain WSJ-2 has been obtained by inactivation of the 3-O-acyltransferase gene (sspA) in WSJ-1 which was responsible for the acylation of spiramycin I to II and III.However, the fermentation products of WSJ-2 contained plenty of spiramycin, and a low percentage of isomycin I. In order to improve the production of isomycin I through increasing the expression of 4″-isovaleryltransferase gene (ist), a recombinant plasmid pSET152-ia, carrying ist gene with positive regulatory gene acyB2 in Streptomyces thermotolerans, was constructed and introduced into Streptomyces spiramyceticus WSJ-2 by protoplast transformation. The recombinant plasmid pSET152-ia was integrated into the chromosome of WSJ-2 via Escherichia coli-Streptomyces shuttle vector pSET152, A new isomycin I producing strain, Streptomyces spiramyceticus WSJ-IA, was generated. The expression of ist in different culture period was detected by real-time quantitative PCR, which indicated that the ist expression of WSJ-IA was higher distinctly than that in WSJ-IA. The fermentation titer of high-yield WSJ-IA strains was up to (1160±108)μg/ml, increased by 314% compared to the original strain whose fermentation titer was only (280±20)μg/ml and the content ratio of isomycin I to spiramycin I in WSJ-IA was about 2.4 times of that in WSJ-2. These results demonstrated that the introduction of acyB2 gene can increase both the fermentation titer and isomycin production.
Key words: Isomycin I    Positive regulatory gene acyB2    4″-isovaleryltransferase gene (ist)
收稿日期: 2016-12-19 出版日期: 2017-03-25
ZTFLH:  Q815  
基金资助:

国家自然科学基金（81172972），国家科技重大专项（2014ZX09201003-002）资助项目
通讯作者: 赫卫清     E-mail: heweiqing@imb.pumc.edu.cn
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引用本文:

戴剑漉, 卢智黎, 林灵, 王以光, 赫卫清. 利用异源正调控基因acyB2构建埃莎霉素I高产菌株[J]. 中国生物工程杂志, 2017, 37(3): 65-72.

DAI Jian-lu, LU Zhi-li, LIN Ling, WANG Yi-guang, HE Wei-qing. Construction of the Isomycin I High-yield Strain by Introducing a Heterologous Positive Regulatory Gene acyB2. China Biotechnology, 2017, 37(3): 65-72.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20170309    或    https://manu60.magtech.com.cn/biotech/CN/Y2017/V37/I3/65

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