Regulation of cellulase enzyme expression in Trichoderma

该项目旨在为丝状真菌里氏木霉纤维素酶表达的转录调控提供新的信息。里氏木霉是一种广泛用于商业生产天然(主要是纤维素酶)酶和越来越多的异源蛋白质的真菌。里氏锥虫的基因组序列是可用的(http://genome.jgi-psf.org/Trire2/Trire2.home.html).虽然里氏木霉基因组编码几种纤维素酶的表达，但cbh1基因与其表达相关的研究最广泛，cbh1启动子被用来驱动异源蛋白的表达。这个项目将建立在我们未发表的数据的基础上，这些数据首次表明了染色质在其调节中的重要性，包括在诱导和抑制过程中核小体位置的变化。此外，我们有初步数据显示，在诱导过程中蛋白质与ORF结合，表明ORF除启动子区域外还参与调控。这在丝状真菌中是前所未有的。该项目的总体目标是在丝状真菌中对目标基因的转录控制定义新的调控原则。该项目的具体实验目标是确认chb1启动子和ORF在诱导和阻遏过程中的核小体重排，并明确已知的调控因子(如CRE1、ACE1)在核小体重排中的作用。第二个目标是定义一个监管cbh1的模式。该项目还将探索蛋白质与ORF结合在转录调控中的作用。可用于探索核小体定位的方法学(包括染色质的分离、核酸酶敏感性分析和杂交研究)、诱导和抑制的培养条件(以及中性条件)，以及一些用于探索某些转录因子的作用的突变菌株，都已在诺丁汉或Roal的研究设施中到位。这个项目为定义真菌转录调控的新机制提供了令人兴奋的可能性。学生将接受微生物学、基因组学和分子生物学方面的培训，并将有机会与诺丁汉的数学家交流，以定义一个调控cbh1基因的模型，该模型可以进行测试，并使用基因阵列方法检查其与整个真菌中的基因调控的相关性，该方法最近才适用于里氏曲霉。这一新信息对Roal来说将是重要的，该公司在其业务中开发丝状真菌，包括T.reesei。

The project aims to provide new information on the transcriptional regulation of expression of cellulase in the filamentous fungus Trichoderma reesei. T. reesei is a fungus exploited extensively commercially for production of native (mainly cellulases) enzymes and, increasingly, heterologous proteins. The genome sequence of T. reesei is available (http://genome.jgi-psf.org/Trire2/Trire2.home.html). Although the T. reesei genome encodes for the expression of several cellulases, the cbh1 gene has been the most extensively studied in relation to its expression and the cbh1 promoter is used to drive expression of heterologous proteins. This project will build upon our unpublished data which show, for the first time, the importance of chromatin in its regulation, including changes in nucleosomal positioning during induction and repression. Moreover, we have preliminary data showing protein binding to the ORF during induction, indicating that the ORF is involved in regulation in addition to the promoter region. That is unprecedented in filamentous fungi. The overall Aim of the project is to define new regulatory principles in the transcriptional control of a target gene in a filamentous fungus. The specific experimental Aims of the project are to confirm the nucleosomal rearrangements of the chb1 promoter and ORF during induction and repression and to specify the roles, if any, of known regulators (e.g. Cre1, Ace1) in the nucleosomal rearrangements. A secondary Aim is to define a model for the regulation of cbh1. The project will also explore the role of protein binding to the ORF during transcriptional regulation. The methodologies available for exploration of nucleosomal positioning (involving the isolation of chromatin, nuclease sensitivity assays, and hybridisation studies), culture conditions for induction and repression (and neutral conditions), and some mutant strains for exploration of the roles of some transcription factors are all in place in Nottingham or the research facility of Roal. This project provides an exciting possibility for defining new mechanisms of transcriptional regulation in fungi. The student will receive training in microbiology, genomics and molecular biology and will have the opportunity to interface with mathematicians in Nottingham in order to define a model for the regulation of the cbh1 gene That model can be tested and its relevance to gene regulation in the fungus as a whole examined using gene array methodology, which is only recently available for T. reesei. This new information will be important for Roal, a company that exploits filamentous fungi, including T. reesei, in its business.