文献类型：期刊文献

中文题名：基于土壤农杆菌转化法高效构建高产产黄青霉tps1和tps2敲除菌株

英文题名：Efficient Knock-out of tps1 and tps2 Genes in Penicillium chrysogenum using Agrobacterium-mediated Transformation Method

作者：赵骏飞[1];王冠[1];吴宝峰[1];储炬[1];张嗣良[1]

机构：[1]华东理工大学生物反应器国家重点实验室,上海200237

年份：2017

卷号：48

期号：9

起止页码：1293

中文期刊名：中国医药工业杂志

外文期刊名：Chinese Journal of Pharmaceuticals

收录：CSTPCD;;北大核心:【北大核心2014】；CSCD:【CSCD_E2017_2018】；

基金：中荷国际合作项目(No.2013DFG32630)给予资助

语种：中文

中文关键词：农杆菌转化法;产黄青霉;海藻糖循环;同源重组

外文关键词：Agrobacterium-mediated transformation; Penicillium chrysogenum; trehalose cycle; homologous recombination

摘要：产黄青霉(Penicillium chrysogenum)是?-内酰胺类抗生素的主要工业生产菌,高效地对其基因进行改造有利于深入研究产黄青霉的代谢机制,进而提高效价、产率和得率。高产产黄青霉基因测序的完成为菌株定向改造提供了分子基础,但是要实现快速的定向菌株改造还需要高效的基因操作系统。本试验采用土壤农杆菌(Agrobacterium)转化法高效构建了海藻糖循环途径tps1、tps2敲除菌株P.chrysogenum-Δtps1和P.chrysogenum-Δtps2。土壤农杆菌为高毒力的根癌农杆菌AGL-1,载体为p Green II-0179,筛选标记为博来霉素,转化对象为产黄青霉孢子。在本试验条件下农杆菌转化法的转化效率可达60%,其中发生同源重组的效率为25%。本研究为后续探究海藻糖循环在高产产黄青霉中的生理学意义提供了有益的参考。
Over the past decades, the market of β-lactam antibiotics has tremendously increased. Classical strain evolution and process improvement cannot satisfy further increase of the titer, rate and yield （T.R.Y） of β-lactam antibiotics. Penicillium chrysogenum is the main cell factory for the production of β-lactam antibiotics, e.g., penicillins and cephalosporins. Efficient genetic manipulation of its genome facilitates the fundamental studies and the increase of T.R.Y. Genome sequencing of a high-yielding Penicillium chrysogenum lays a foundation on a molecular basis, however, a rapid directed strain improvement should be based on an efficient gene targeting system. In this study, we successfully constructed the tpsl and tps2 knockout strains, P. chrsogenum-△tpsl and P. chrsogenum-△tps2 using the Agrobacterium mediated transformation method. Supervirulent Agrobacterium strain AGL-1 has been selected, with the carrier of pGreenII-0179 vector, the selection marker of bleomycin, and the experimental object of fresh P. chrysogenum spores. The transformation efficiency of Agrobacterium mediated transformation was up to 60%, the efficiency of homologous recombination was up to 25 % in these transformants, and the transformation rate was improved dramatically. This study provided a useful reference for the further exploration of the trehalose cycle in high-yield P. chrysogenum.