斜卧青霉中新转录因子PD003430对纤维素酶基因表达调控的研究

Lignocellulosic materials containing large biopolymers cellulose, hemicellulose are the most abundant renewable organic resource on earth. These biopolymers are degraded by many microorganisms, in particular filamentous fungi, with the aid of extracellular degrading enzymes, cellulases, hemicellulases, which are regulated mainly at the transcriptional level. Penicillium decumbens is known to secrete a large number of cellulases and hemicellulases that can degrade biomass to fermentable sugars. The biosynthesis and secretion of the cellulases of P. decumbens are regulated on transcription levels. Using genomics resources available for P. decumbens, our laboratory constructed a near-full transcription factor deletion strain set. We recently discovered a novel transcription factor called PD003430 that is essential for the cellulose degradation. The transcriptional activator PD003430 may be the central regulator that governs cellulolytic and xylanolytic gene expression in P. decumbens. However, relatively little is known about its functions. In this study, knockout and overexpression strains of PD003430 gene will be obtained. The phenotype of these strains will be investigated, including its hyphal growth, protein secretion level, cellulase secretion level. The ⊿PD003430 mutant and wide-type and overexpression strains will also be performed a systems analysis of the transcriptome associated with pure cellulose utilization and complex plant biomass. Furthermore, we will investigate the binding characteristics and specific target for PD003430 by using ChIP-seq (Chromatin immunoprecipitation followed by sequencing), regulation of PD003430 expression may have a significant impact on the ability of P. decumbens to grow on cellulose and to produce the various degrading enzymes. The comprehensive investigation of the mechanism of transcriptional regulation of PD003430 on cellulase encoding genes will provide theoretical guidance for constructing industrial strains with high cellulase production.

丝状真菌纤维素酶表达调控机理的研究将有助于解析纤维素降解中纤维素酶系的协同作用机制。我们在构建的斜卧青霉(Penicillium decumbens)转录调控因子突变株库的基础上，采用高通量表型筛查方法，发现一新的显著影响纤维素降解的转录调控因子PD003430；本研究首先构建⊿PD003430突变株、回补菌株以及组成型过表达菌株，并对所构建菌株的表型及产纤维素酶特性进行测定，在此基础上，利用基因组表达谱技术检测⊿PD003430突变株、野生菌株和过表达菌株mRNA群体间的差异表达，并采用荧光定量PCR方法进行差异基因的验证，然后，通过染色质免疫共沉淀测序技术(ChIP-seq)以及DNase I印迹试验，研究转录因子PD003430与DNA序列的相互作用，本研究内容的实施，将有助于了解转录因子PD003430在斜卧青霉木质纤维素降解中的调控机制，并为构建纤维素酶高产菌株提供新的思路。

草酸青霉（原命名斜卧青霉）是一种高产纤维素酶丝状真菌。基于构建的草酸青霉转录因子单基因突变株库，我们筛选出了20种影响纤维素酶表达的转录因子，其中ClrB（PDE_05999/PD003430）在纤维素酶基因表达过程中起到关键性的正调控作用。我们利用ClrB突变株转录组和分泌组的分析，获取并系统分析了ClrB调控的靶标基因。我们同时系统构建了纤维素酶转录因子ClrB、CreA、XlnR和AmyR基因突变株，研究了这些转录因子调控纤维素酶基因表达的机制，并特别发现了Δbgl2-gpdA(p)::clrB在非纤维素诱导条件下纤维素酶高表达的现象，这为继续深入拓展纤维素酶表达调控机制的研究提供了新的研究靶点。酵母双杂交实验表明ClrB、CreA、XlnR和AmyR之间存在蛋白质相互作用，结合这些转录因子存在调控纤维素酶表达的协同性，确定并构建了以ClrB为核心的草酸青霉转录调控网络模型。提出了草酸青霉纤维素酶系基因表达调控的“跷跷板效应”，以及纤维素酶基因转录调控网络重构（Reconstruction of Expression Regulation Network，RERN)这一概念。基于以上研究结果，设计并构建了草酸青霉纤维素酶高产菌株（RE-10, RE-27和RE-29），这些突变株相比野生出发菌株纤维素酶活提高十倍以上。同时，本项目研究为突破筛选标记对草酸青霉遗传操作的限制，成功构建了β-rec/six系统介导的筛选标记循环利用系统，为多靶点研究草酸青霉纤维素酶表达调控通路，以及构建高产纤维素酶工业菌株建立了遗传操作平台。本项目所取得的研究成果也对其它丝状真菌纤维素酶表达调控机制的研究和高产菌株的理性构建具有指导作用。