文献类型：期刊文献

中文题名：螺旋链霉菌遗传操作系统-接合转移体系的建立

英文题名：Establishment of Conjugation System for the Spiramycin Producer Streptomyces spiramyceticus

作者：王优蓓[1];郭思妤[1];常碧博[2];叶蕊芳[1];花强[1]

机构：[1]华东理工大学生物工程学院生物反应器工程国家重点实验室,上海200237;[2]河南天方药业股份有限公司,驻马店463000

年份：2021

卷号：41

期号：2

起止页码：45

中文期刊名：中国生物工程杂志

外文期刊名：China Biotechnology

收录：CSTPCD;;北大核心:【北大核心2020】；CSCD:【CSCD2021_2022】；

语种：中文

中文关键词：螺旋链霉菌;菌体接合转移;螺旋霉素;CRISRP/Cas9;

外文关键词：Streptomyces spiramyceticus;Mycelium conjugal transfer;Spiramycin;CRISRP/Cas9

摘要：背景:螺旋链霉菌(Streptomyces spiramyceticus)为国内螺旋霉素(spiramycin,SPM)的生产菌,但目前工业生产中螺旋链霉菌利用遗传操作进行基因改造还没有成功报道。目的:建立螺旋链霉菌遗传操作系统,利用遗传改造的方法改变SPM的组分比例,降低SPM的分离成本。方法:以大肠杆菌(Escherichia coli ET12567/pUZ8002)为供体,螺旋链霉菌为受体进行接合转移实验,建立螺旋链霉菌接合转移的遗传操作系统,并对影响接合转移效率的培养基种类、抗生素覆盖时间、供受体比例等条件进行优化。结果:螺旋链霉菌不适合利用孢子进行接合转移,利用菌体进行接合转移时ISP4培养基为接合转移的最适培养基,供受体比例为103∶1得到的接合子数量最多,接合转移效率为1.93×10-4,SPM中三种组分百分含量变化显著。结论:实验首次建立了螺旋链霉菌接合转移的方法,利用菌体进行接合转移操作,简化了实验操作过程,并且利用CRISRP/Cas9基因编辑系统成功阻断3-O-酰基转移酶基因,并在该位置导入φC31整合位点att B,为后续螺旋链霉菌的基因改造奠定了基础。
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/p UZ8002,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 andφC31 locus att B was integrated into S.spiramyceticus genome successfully,which laid the foundation for further biosynthetic gene modification in the strain.