乙酰化修饰在里氏木霉纤维素酶合成调控中的功能研究

Trichoderma reesei is widely used in cellulase industry with its high production of secreted proteins. Although many regulators including transcriptional and non-transcriptional factors involved in synthesis of cellulase were elucidated in the past years, the substantial mechanism to induce the cellulase production is still unknown. The reversible acetylation of protein plays an important role in fungal life cycle, and evidences implying that acetylation may take parts in the regulations of cellulase synthesis and secretion in T. reesei have accumulated recently. In this study, we will select the candidates from all the annotated genes of acetyltransferases and deacetylases in T. reesei genome. Mutated strains would be built by deleting or overexpressing the candidate genes in order to characterize their functions in the regulations of cellulase synthesis and secretion in T. reesei. The mutants with significantly different phenotypes (especially for the cellulase production) would be chosen for the digital gene expression profiling analyses to identify the known regulators or function-unknown proteins with significant expression difference. And the correlation between the transcriptional level of the target proteins and the acetylation status would be further testified. Novel regulators or other functional proteins involved in cellulase synthesis and secretion are expected to be obtained based on the above investigations. These results will help us to know the regulating network of cellulase synthesis and secretion in T. reesei comprehensively. The revelation of the novel mechanisms to regulating cellulase synthesis and secretion in T. reesei will provide clues and foundation for further strain engineering for industry applications.

里氏木霉是应用最广泛的纤维素酶工业生产菌种，它具有强大的纤维素酶诱导分泌能力。近年来鉴定了多个纤维素酶合成相关的转录调控因子及非转录因子功能蛋白。但对其纤维素酶合成诱导机制仍缺乏深入的了解。最近的研究表明，蛋白质的可逆乙酰化修饰，在丝状真菌的生命活动中也扮演着重要角色，这类修饰也可能参与了纤维素酶合成诱导调控。本项目中，我们将从里氏木霉基因组中注释为乙酰转移酶或去乙酰化酶的编码基因中确定候选基因，敲除或过表达这些基因，观测突变菌株在不同产酶条件下生长与产酶的表型，确定产酶表型差异显著的突变株，通过表达谱分析确定表达水平变化显著的已知及未知功能蛋白，并确认它们表达水平的差异与乙酰化修饰水平的相关性，找到参与纤维素酶合成诱导的新功能蛋白。这些研究将为全面认识里氏木霉中纤维素酶的合成诱导调控机制及解析乙酰化修饰与蛋白合成分泌的相关性积累基础数据，并为里氏木霉菌种的设计与进一步改造提供理论根据。

真核生物组蛋白的可逆乙酰化修饰是一种普遍存在的蛋白翻译后修饰，其中去乙酰化酶参与催化这一组蛋白质修饰过程中的乙酰基去除。组蛋白的可逆乙酰化修饰参与 调控真核细胞的生长、发育、次级代谢和多种压力响应等生理生化过程。本项目研究首先基于CRISPR-Cas9建立了高效的里氏木霉基因组编辑系统，利用该编辑系统构建了过表达或敲除系列预测为组蛋白去乙酰化酶编码基因的里氏木霉突变菌株并比较分析这些突变菌株的表型。从中发现组蛋白去乙酰化酶基因Tr26的敲除导致里氏木霉在阻遏碳源葡萄糖存在的条件下纤维素酶的活性显著上调，与出发株相比Tr26敲除株中纤维素酶总调控因子Xyr1及主要纤维素酶基因cbh1和eg1具有较高的转录水平。为了进一步解析里氏木霉组蛋白去乙酰化酶Tr26参与纤维素酶基因表达调控的机制，我们考查了Tr26在氧化压力响应过程的调控功能，发现其能通过上调过氧化氢酶基因cat3的转录水平来参与里氏木霉中的氧化压力响应。此外，我们观察到Tr26纤维素诱导时发生从细胞质向细胞核的募集现象。我们进一步使用酵母双杂交分析发现，Tr26与纤维素酶表达负调控因子Cre1-1和Ace1可能存在弱的相互作用。终上所述，我们的研究表明里氏木霉中组蛋白去乙酰化酶Tr26负调控氧化压力响应过程并通过核质转移参与纤维素酶基因的表达调控。这一基因功能的解析将为进一步设计和改造里氏木霉纤维素酶高产菌株提供理论基础。