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

发布时间：2018-10-26 19:23

【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。
[Abstract]:Penicillium luteum (Penicillium chrysogenum) is the main industrial producing strain of lactam antibiotics. It is helpful to study the metabolic mechanism of Penicillium xanthogenes and to improve its titer, yield and yield by modifying its gene efficiently. The completion of gene sequencing of high-yielding Penicillium flavus provides a molecular basis for the directed transformation of the strain, but the efficient gene operating system is needed to realize the rapid transformation of the directed strain. The trehalose cycle pathway tps1,tps2 knockout strains P.chrysogenum- 螖 tps1 and P.chrysogenum- 螖 tps2. were efficiently constructed by Agrobacterium tumefaciens (Agrobacterium) transformation in this experiment. Agrobacterium tumefaciens was a highly virulent Agrobacterium tumefaciens AGL-1, vector with p Green II-0179, screening marker as bleomycin and transformed to Penicillium flavum spores. In this experiment, the transformation efficiency of Agrobacterium tumefaciens transformation method can reach 60%, and the efficiency of homologous recombination is 25%. This study provides a useful reference for further exploring the physiological significance of trehalose cycle in high yield penicillium luteum.
【作者单位】：华东理工大学生物反应器国家重点实验室;
【基金】：中荷国际合作项目(No.2013DFG32630)给予资助
【分类号】：R915

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