一株产环己酰亚胺的链霉菌遗传操作研究  

A study on the genetic manipulation of a cycloheximide producing Streptomyces strain

作　　者：任禛[1,2] 黄鹤平 余磊[1,2] 夏体渊[1,2] 张永福[1,2] 陈丽娟[1,2] 陈泽斌[1,2] 尹敏[3] 

机构地区：[1]昆明学院农学院,云南昆明650214 [2]云南省高校都市型现代农业工程研究中心,云南昆明650214 [3]云南大学医学院,云南昆明650091

出　　处：《云南大学学报（自然科学版）》2015年第1期163-170,共8页Journal of Yunnan University(Natural Sciences Edition)

基　　金：云南省应用基础研究项目(2013FZ099);云南省教育厅科学研究基金(2013Z051,2013Y367);昆明学院人才引进项目(YJL12002);云南省高校都市型现代农业工程研究中心科研项目(DS-C201402);云南大学中青年骨干教师培养计划(XT412003)

摘　　要：对一株高产环己酰亚胺的链霉菌菌株系统进行遗传操作体系的建立研究,并初步探索了相关生物合成的基因.采用了接合转移,电击转化菌丝体和PEG介导的原生质体转化3种方法进行外源基因的导入实验.使用了7种配方差异较大的培养基进行发酵,并使用HPLC对发酵产物进行分析,摸索了合适的发酵及检测条件.最后通过依赖于λ-RED介导的PCR-targeting方法进行了可能参与环己酰亚胺生物合成的PKS基因片段的敲除.通过实验,最终确定了使用优化的接合转移方法并采用新鲜孢子能够有效地导入外源基因,获得了较高的转化效率(10-6),成功确立了遗传操作体系,获得了合适的发酵条件和HPLC检测方法.在建立遗传操作体系的基础上,成功找到了负责该类化合物生物合成的PKS基因片段,为深入研究其生物合成机理并进一步通过遗传工程改良以获得新化合物及提高化合物产量打下基础,并为链霉菌遗传操作研究提供了新的参考.A Streptomyces strain with high production of cycloheximide was chosen for a systematic genetic manipulation study.Conjugation, mycelium electroporation and PEG mediate protoplast transformation were used for the introduction of foreign genes.Seven different kinds of medium were tested to optimize fermentation condi- tion and HPLC were used to analyze the fermentaiton products, k-RED mediate PCR-targeting was used to knockout a possible PKS fragment which is supposed to be a key gene for CHX biosynthesis.As a result, modified conjugation using fresh spores was proved was be a valid method for introducing foreign genes into this strain, with relatively high conversion efficiency （10-6）. Fermentation and HPLC condition were optimized.By using the ge- netic manipulation system we constructed, a PKS gene which is responsible to the biosynthesis of cycloheximide was found.The study provided a good example for exploring the genetic manipulation on streptomyces and made a solid foundation for obtaining novel compounds and improving the compounds production by optimizing genetic engineering of the strain.

关 键 词：环己酰亚胺 链霉菌 遗传操作 

分 类 号：Q933[生物学—微生物学]