Establishment of Conjugation System for the Spiramycin Producer <i>Streptomyces spiramyceticus</i>

doi:10.13523/j.cb.2010009

China Biotechnology

2021, Vol. 41

Issue (2/3): 45-52 DOI: 10.13523/j.cb.2010009

Establishment of Conjugation System for the Spiramycin Producer Streptomyces spiramyceticus

WANG You-bei¹,GUO Si-yu¹,CHANG Bi-bo²,YE Rui-fang¹,HUA Qiang^1,^*(

)

1 State Key Laboratory of Bioreactor Engineering, School of Bioengineering, East China University of Science and Technology, Shanghai 200237, China
2 Topfond Pharmaceutical Co. Ltd, Zhumadian 463000, China

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Abstract

Background:Streptomyces spiramyceticus is used to produce spiramycin (SPM) in China. So far, the successful genetic modification has not been reported in S. spiramyceticus.Objective:The appropriate genetic transformation system of S. spiramyceticus was established in order to modify the composition of SPM and reduce the separation costs of SPM based on genetic operation.Methods:The conjugal transfer system was conducted by conjugation with Escherichia coli ET12567/pUZ8002, and the factors that influence the conjugation efficiency, including culture medium, antibiotic coverage time, and donor/recipient ratio, were investigated and optimized.Results:The experimental results showed that S. spiramyceticus spores were not suitable for conjugal transfer and the best medium for conjugal transfer was ISP4. The maximum transconjugants were obtained when the donor/recipient ratio was 10³∶1. The best conjugation efficiency was 1.93×10^-4 and the percentage of SPM changed significantly.Conclusion:The efficient and simple genetic transformation system for producing strain S. spiramyceticus was established. Based on this method, the sspA gene was knocked out and φC31 locus attB was integrated into S. spiramyceticus genome successfully, which laid the foundation for further biosynthetic gene modification in the strain.

Key words： Streptomyces spiramyceticus Mycelium conjugal transfer Spiramycin CRISRP/Cas9

Received: 12 November 2020 Published: 08 April 2021

ZTFLH:

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Corresponding Authors: Qiang HUA E-mail: qhua@ecust.edu.cn

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	You-bei WANG
	Si-yu GUO
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	Rui-fang YE
	Qiang HUA

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WANG You-bei,GUO Si-yu,CHANG Bi-bo,YE Rui-fang,HUA Qiang. Establishment of Conjugation System for the Spiramycin Producer Streptomyces spiramyceticus. China Biotechnology, 2021, 41(2/3): 45-52.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2010009 OR https://manu60.magtech.com.cn/biotech/Y2021/V41/I2/3/45

Table 1 Spore production of S. spiramyceticus

Fig.1 Colony morphology of S. spiramyceticus cultured in spore culture medium at 28℃ (a) Colony morphology changes with culture time (b) Different colony morphology at 13d of culture

Table 2 Susceptibility of S. spiramyceticus to different antibiotics

Table 3 Growth of S. spiramyceticus in different medium

Fig.2 pCRISRP/Cas9 sspA+HA plasmid

Table 4 Conjugation efficiency of different plasmids

Fig.3 Effects of donor/recipient ratios on conjugation efficiency

Fig.4 Sequencing chromatograms for the △sspA∷φC31 attB mutant

Fig.5 Relative contents of SPM I, SPM II and SPM III (a) Wild-type strain (b) Mutant strain

Fig.6 Schematic diagram of conjugation process of S. spiramyceticus


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