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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2017, Vol. 37 Issue (3): 65-72    DOI: 10.13523/j.cb.20170309
    
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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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)     
Received: 19 December 2016      Published: 25 March 2017
ZTFLH:  Q815  
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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.

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

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