安丝菌素产生菌拟诺卡氏菌EGI80425接合转移体系的建立与应用

安丝菌素是一种有强抗肿瘤作用的天然产物,拟诺卡氏菌Nocardiopsis ansamitocini EGI80425作为安丝菌素的产生菌具有重要的研究价值,因此需要建立其遗传操作体系,并实施对该菌株的遗传改造。首先,通过优化菌丝体接合转移相关的培养时间、供受体比例和培养基中Mg2+浓度等,建立了效率为6.6×10^-3的整合型载体p IB139的接合转移方法;然后在此基础上进一步调整相关条件,建立了效率为6.2×10^-4的游离型质粒p JTU1278的接合转移方法;最后,利用游离型质粒对潜在的PKS竞争基因簇ClusterⅠ和ClusterⅦ进行了缺失。结果显示,ClusterⅠ缺失突变株CXY01中安丝菌素的产量为12.6 mg/L,相比野生型提高了95.3%;ClusterⅦ缺失突变株CXY02中安丝菌素产量为7.4 mg/L,相比野生型提高了15.2%。本研究建立了N. ansamitocini EGI80425的遗传操作体系,并在此基础上对该菌进行了基因组片段敲除,有效提升了安丝菌素产量,为利用N. ansamitocini大量产生安丝菌素奠定了基础。

Establishment and Application of Conjugation System for the Ansamitocin Producer Nocardiopsis ansamitocini EGI80425

Ansamitocin P-3(AP-3) is a potent antitumor agent, and Nocardiopsis ansamitocini EGI80425 is a newly isolated AP-3 producer. Prior to the manipulation of this strain for improving ansamitocin production, an efficient genetic system is required. A conjugation system was firstly established with mycelia and integrative plasmid pIB139, and an efficiency of 6.6×10^-3 was achieved by optimizing incubation time for conjugation, the recipient/donor ratio and concentration of Mg2+ in the medium. In addition, a conjugation system using a replicating vector p JTU1278 for gene deletion was established with an efficiency of 6.2×10^-4. Subsequently, two PKS gene clusters(ClusterⅠand ClusterⅦ), competing with AP3 biosynthesis, were deleted using the replicating vector. Compared to the wild-type, AP-3 productions in the ClusterⅠdeleted mutant(CXY01) and ClusterⅦ deleted mutant(CXY02)were increased by 95.3% to 12.6 mg/L and by 15.2% to 7.4 mg/L, respectively. In this study, an efficient genetic manipulation system was established and applied for gene deletion, which resulted in improved ansamitocin productions and paved a way for further titer increase by genetic engineering with N. ansamitocini.