State Key Laboratory of Microbial Technology, Shandong University, Jinan Shandong, China.
FEMS Microbiol Lett. 2010 Feb;303(1):26-32. doi: 10.1111/j.1574-6968.2009.01851.x. Epub 2009 Nov 13.
Genetic transformation is an indispensable tool for basic fungal research, as well as a useful technique for directed improvement of industrial strains. Here we describe a simple and reproducible transformation system for the filamentous fungus Hypocrea jecorina. The system is based on hxk1 (encoding hexokinase) as selectable marker, a hexokinase-negative strain and D-mannitol, which is used as selective carbon source and osmotic stabilizer. Following transformation with the hxk1 gene, the obtained transformants were able to grow on D-mannitol as sole carbon source. Transformation efficiency achieved using D-mannitol as carbon source and osmotic stabilizer was roughly five times higher than that using D-sorbitol. The utility of this system was further demonstrated by transformation of H. jecorina with the egfp (encoding the enhanced green fluorescent protein) gene. Fluorescence microscopy revealed EGFP fluorescence in positive transformants. Our results demonstrated the feasibility of exploiting a carbon source metabolic pathway for the development of promising fungal transformation systems, which provides a new molecular toolbox for genetic modifications of the cell factory H. jecorina.
遗传转化是基础真菌研究不可或缺的工具，也是定向改良工业菌株的有用技术。在这里，我们描述了一种用于丝状真菌Hypocrea jecorina 的简单且可重复的转化系统。该系统基于 hxk1（编码己糖激酶）作为选择性标记物、己糖激酶阴性菌株和 D-甘露醇，D-甘露醇用作选择性碳源和渗透稳定剂。在用 hxk1 基因转化后，获得的转化体能够以 D-甘露醇为唯一碳源生长。使用 D-甘露醇作为碳源和渗透稳定剂的转化效率比使用 D-山梨醇的转化效率大约高五倍。通过用 egfp（编码增强型绿色荧光蛋白）基因转化 H. jecorina，进一步证明了该系统的实用性。荧光显微镜显示阳性转化体中有 EGFP 荧光。我们的结果表明，利用碳源代谢途径开发有前途的真菌转化系统是可行的，这为遗传修饰细胞工厂 H. jecorina 提供了新的分子工具包。
