Liu Gang, Zhang Yan, Li Yun, Yu Shao-wen, Xing Miao
Shenzhen Key Laboratory of Microbial Genetic Engineering, College of Life Sciences, Shenzhen University, Shenzhen 518060, China.
Wei Sheng Wu Xue Bao. 2007 Feb;47(1):69-74.
Comprehensive analysis of the mechanism of metabolite synthesis in filamentous fungi is important for optimization of the filamentous fungus related industrial fermentation processes. In this work, the mechanism of cellulase synthesis in Trichoderma reesei was analyzed with red fluorescent protein (DsRed) as the reporting protein. The expression cassette for heterologous protein expression in T. reesei was constructed, through which the DsRed gene was inserted into the chromosomal DNA of T. reesei. The recombinant T. reesei strain, in which expression of DsRed was controlled by the promoter of cellobiohydrolase gene, was designated as T. reesei TR2. Expression of DsRed in T. reesei TR2 under different culture conditions was analyzed by using a fluorescent microscopy, and thereby the mechanism of cellulase gene expression in T. reesei could be interpreted. With induction of lactose, the pattern of change of red fluorescence in T. reesei TR2 was similar to that of the cellulase activity in the cultivation supernatant. As the culture aged, the red fluorescence in the mycelial increased. This was followed by a reduction in the end of the culture period because of death and autolysis of the mycelial. In the spatial aspect, the red fluorescence was distributed uniformly in the whole hypha after induction, indicating that all the three morphology including apical compartment, subapical compartment and hyphal compartment played a same role in cellulase synthesis. When T. reesei TR2 was cultivated without induction, faint red fluorescence appeared after a relative long period of cultivation, indicating that a small amount of cellulase was still synthesized without induction. This result was useful in explaining the mechanism of cellulase induction by insoluble cellulose.
全面分析丝状真菌中代谢物合成的机制对于优化丝状真菌相关的工业发酵过程具有重要意义。在本研究中，以红色荧光蛋白（DsRed）作为报告蛋白，分析了里氏木霉中纤维素酶合成的机制。构建了用于里氏木霉中异源蛋白表达的表达盒，通过该表达盒将DsRed基因插入到里氏木霉的染色体DNA中。将DsRed的表达受纤维二糖水解酶基因启动子控制的重组里氏木霉菌株命名为里氏木霉TR2。通过荧光显微镜分析了里氏木霉TR2在不同培养条件下DsRed的表达情况，从而可以解释里氏木霉中纤维素酶基因的表达机制。在乳糖诱导下，里氏木霉TR2中红色荧光的变化模式与培养上清液中纤维素酶活性的变化模式相似。随着培养时间的延长，菌丝体中的红色荧光增加。由于菌丝体的死亡和自溶，在培养末期荧光随后降低。在空间方面，诱导后红色荧光在整个菌丝中均匀分布，表明顶端区室、亚顶端区室和菌丝区室这三种形态在纤维素酶合成中发挥相同的作用。当里氏木霉TR2在无诱导条件下培养时，经过相对较长时间的培养后出现微弱的红色荧光，表明在无诱导条件下仍能合成少量纤维素酶。该结果有助于解释不溶性纤维素对纤维素酶的诱导机制。
