Abstract：
White-rot fungi differentially express laccases when they encounter aromatic compounds. However, the underlying mechanisms are still being explored. Here, proteomics analysis revealed that in addition to increased laccase activity, proteins involved in sphingolipid metabolism and toluene degradation as well as some cytochrome P450s (CYP450s) were differentially expressed and significantly enriched during 48 h of o-toluidine exposure, in Trametes hirsuta AH28-2. Two Zn2Cys6-type transcription factors (TFs), TH8421 and TH4300, were upregulated. Bioinformatics docking and isothermal titration calorimetry assays showed that each of them could bind directly to o-toluidine and another aromatic monomer, guaiacol. Binding to aromatic compounds promoted the formation of TH8421/TH4300 heterodimers. TH8421 and TH4300 silencing in T. hirsuta AH28-2 led to decreased transcriptional levels and activities of LacA and LacB upon o-toluidine and guaiacol exposure. EMSA and ChIP-qPCR analysis further showed that TH8421 and TH4300 bound directly with the promoter regions of lacA and lacB containing CGG or CCG motifs. Furthermore, the two TFs were involved in direct and positive regulation of the transcription of some CYP450s. Together, TH8421 and TH4300, two key regulators found in T. hirsuta AH28-2, function as heterodimers to simultaneously trigger the expression of downstream laccases and intracellular enzymes. Monomeric aromatic compounds act as ligands to promote heterodimer formation and enhance the transcriptional activities of the two TFs.IMPORTANCEWhite-rot fungi differentially express laccase isoenzymes when exposed to aromatic compounds. Clarification of the molecular mechanisms underlying differential laccase expression is essential to elucidate how white-rot fungi respond to the environment. Our study shows that two Zn2Cys6-type transcription factors form heterodimers, interact with the promoters of laccase genes, and positively regulate laccase transcription in Trametes hirsuta AH28-2. Aromatic monomer addition induces faster heterodimer formation and rate of activity. These findings not only identify two new transcription factors involved in fungal laccase transcription but also deepen our understanding of the mechanisms underlying the response to aromatics exposure in white-rot fungi.
摘要：
白腐真菌在遇到芳香族化合物时会差异表达漆酶。然而,潜在的机制仍在探索中。这里,蛋白质组学分析表明，除了增加漆酶活性外，参与鞘脂代谢和甲苯降解的蛋白质以及一些细胞色素P450（CYP450）在邻甲苯胺暴露的48小时内差异表达并显着富集，在TrameteshirsutaAH28-2。两种Zn2Cys6型转录因子(TFs),TH8421和TH4300上调。生物信息学对接和等温滴定量热分析表明，它们中的每一个都可以直接结合邻甲苯胺和另一种芳香族单体，愈创木酚.与芳香族化合物的结合促进了TH8421/TH4300杂二聚体的形成。T.hirsutaAH28-2中的TH8421和TH4300沉默导致邻甲苯胺和愈创木酚暴露后LacA和LacB的转录水平和活性降低。EMSA和ChIP-qPCR分析进一步显示TH8421和TH4300与含有CGG或CCG基序的lacA和lacB的启动子区域直接结合。此外，这两种TFs参与了一些CYP450转录的直接和正调控。一起,TH8421和TH4300是在T.hirsutaAH28-2中发现的两个关键调节因子，可作为异二聚体同时触发下游漆酶和细胞内酶的表达。单体芳族化合物充当配体以促进异二聚体形成并增强两种TF的转录活性。当暴露于芳香族化合物时，IMPORTANCEWhite-rot真菌差异表达漆酶同工酶。阐明差异漆酶表达的分子机制对于阐明白腐真菌对环境的反应至关重要。我们的研究表明，两个Zn2Cys6型转录因子形成异二聚体，与漆酶基因的启动子相互作用，并正向调节TrameteshirsutaAH28-2中的漆酶转录。芳族单体的添加诱导更快的异二聚体形成和活性速率。这些发现不仅确定了参与真菌漆酶转录的两个新的转录因子，而且加深了我们对白腐真菌对芳香族化合物暴露反应的潜在机制的理解。
