遗传改造刺糖多孢菌菌株生产多杀菌素J和L  

Engineering the spinosyn J and L producing strain by molecular genetic manipulation

作　　者：党福军[1] 王继栋 覃重军[1] 夏海洋[1] Dang Fu-jun;Wang Ji-dong;Qin Zhong-jun;Xia Hai-yang(Key Lab of Synthetic Biology,Institute of Plant Physiology and Ecology,Shanghai Institutes for Biological Sciences,CAS,Shanghai 200032;Zhejiang Key Laboratory of Antifungal Drugs,Zhejiang Hisun Pharmaceutical Co.Ltd.,Taizhou 317000)

机构地区：[1]中国科学院上海生命科学研究院植物生理生态研究所合成生物学重点实验室,上海200032 [2]浙江省抗真菌药物重点实验室浙江海正药业股份有限公司,台州317000

出　　处：《中国抗生素杂志》2019年第1期52-58,共7页Chinese Journal of Antibiotics

基　　金：科技部863项目(No.2012AA022107)

摘　　要：目的发展刺糖多孢菌遗传操作体系,构建生产多杀菌素J和L遗传重组菌株。方法由于刺糖多孢菌是公认的难操作放线菌之一,本文通过λ-Red和FLP重组酶介导的体内重组体系结合黏粒文库,发展了刺糖多孢菌的高效基因操作体系。结果利用该方法实现目标基因高效敲除,测试基因敲除效率可达66%。利用该技术构建了SpnK失活的刺糖多孢菌突变菌株,HPLCMS和NMR分析显示突变株产生的新化合物为多杀菌素J和L,且产量与出发菌株产生的多杀菌素A和D相当。结论该策略可用于刺糖多孢菌的遗传改造,可将多杀菌素高产菌株改造后直接生产多杀菌素J和L。Objective To develop highly efficient genetic manipulation system and construct recombinant strains for producing spinosyn J and L in Saccharopolyspora spinosa.Methods Because S.spinosa was difficult to manipulate, a highly efficient manipulation system for S.spinosa was developed by adapting cosmid library and in vivo recombination with L-Red and FLP-recombinase.Results This method can bring high efficient gene disruption in S.spinosa.The efficiency for the test gene is 66%.The mutant strain with inactive SpnK,which was constmcted by this method,produced two new compounds.They were verified as the spinosyn J and L by HPLC-MS and NMR analysis.In addition,the production of spinosyn J and L of this mutant was similar to that of spinosad in the original stain.Conclusion This strategy can be employed to construct spinosyn J and L producing strain from any industrial spinosad strains directly.

关 键 词：多杀菌素 乙基多杀菌素 氧甲基转移酶 刺糖多孢菌 

分 类 号：R978.1[医药卫生—药品]