Table_2_The G-protein Coupled Receptor GPR8 Regulates Secondary Metabolism in Trichoderma reesei.XLSX

Changing environmental conditions are of utmost importance for regulation of secondary metabolism in fungi. Different environmental cues including the carbon source, light and the presence of a mating partner can lead to altered production of compounds. Thereby, the heterotrimeric G-protein pathway is of major importance for sensing and adjustment of gene regulation. Regulation of secondary metabolism is crucial in the biotechnological workhorse Trichoderma reesei for knowledge-based adjustment in industrial fermentations, but also with respect to the potential use as a host for heterologous compound production. We investigated the function of the class VII G-protein coupled receptor (GPCR) gene gpr8 that is localized in the vicinity of the SOR cluster, which is responsible for biosynthesis of sorbicillinoids. GPR8 positively impacts regulation of the genes in this cluster in darkness. Accordingly, abundance of trichodimerol and dihydrotrichotetronine as well as other secondary metabolites is decreased in the deletion mutant. Transcriptome analysis moreover showed the major role of GPR8 being exerted in darkness with a considerable influence on regulation of secondary metabolism. Genes regulated in Δgpr8 overlap with those regulated directly or indirectly by the transcription factor YPR2, especially concerning genes related to secondary metabolism. The predicted FAD/FMN containing dehydrogenase gene sor7, one of the positive targets of the cascade triggered by GPR8, has a positive effect on secondary metabolite production, but also cellulase gene expression. Hence SOR7 has some overlapping, but also additional functions compared to GPR8. The G-protein coupled receptor GPR8 exerts a light dependent impact on secondary metabolism, which is in part mediated by the transcription factor YPR2 and the function of SOR7. Hence, T. reesei may apply GPR8 to adjust production of secondary metabolites and hence chemical communication to signals from the environment.

环境条件的动态变化对真菌次级代谢的调控至关重要。包括碳源、光照以及交配伴侣存在在内的各类环境信号，均可改变真菌次级代谢产物的合成谱。其中，异源三聚体G蛋白通路（heterotrimeric G-protein pathway）在感知环境信号并调控基因表达方面发挥核心作用。
次级代谢调控对于里氏木霉（Trichoderma reesei）这一生物技术工业模式宿主菌而言至关重要：一方面可支撑工业发酵的理性调控优化，另一方面也可作为异源化合物合成的宿主菌株，具备潜在应用价值。本研究聚焦定位于山梨菌素类（sorbicillinoids）生物合成相关SOR基因簇附近的VII类G蛋白偶联受体（G-protein coupled receptor, GPCR）基因gpr8，对其功能展开探究。
GPR8在黑暗条件下正向调控该基因簇内的基因表达。相应地，在gpr8基因敲除突变株中，木霉二聚醇（trichodimerol）、二氢木霉四素（dihydrotrichotetronine）及其他次级代谢产物的积累量均显著降低。转录组分析进一步证实，GPR8的核心调控功能主要体现在黑暗条件下，对次级代谢调控具有显著影响。Δgpr8突变株中的差异表达基因与转录因子YPR2直接或间接调控的基因存在大量重叠，尤以次级代谢相关基因为甚。
预测的含FAD/FMN结构域的脱氢酶基因sor7是GPR8介导的信号级联反应的正向靶标之一，其不仅可正向调控次级代谢产物合成，同时也对纤维素酶基因的表达具有调控作用。相较于GPR8，SOR7既存在部分功能重叠，同时也具备独特的调控功能。
G蛋白偶联受体GPR8对次级代谢的调控具有光照依赖性，该调控过程部分通过转录因子YPR2及SOR7的功能来实现。由此可见，里氏木霉可通过GPR8感知环境信号，调控次级代谢产物的合成，进而介导环境信号响应的化学通讯。