Analysis Of The Metabolic Regulation And Mechanism Of The Transcriptional Regulators HOS2 And PacC In Trichoderma Harzianum

Fungal secondary metabolites(SMs),also known as fungal natural products,are an important source of new drugs and lead compounds.However,due to the development of natural products chemistry,known compounds have been repeatedly separated.The discovery of fungal natural products encounter bottleneck.With the maturity of genome sequencing technology and the improvement of bioinformatics analysis methods,researchers have discovered that there are a large number of biosynthetic gene clusters(BGCs)in fungi.However,under routine conditions in the laboratory,more than 70%of BGCs are in a "silent" state of low or no expression,and they are huge undiscovered treasure for mining.Therefore,the activation of "silent" gene clusters and the exploration of biosynthesis pathways are now hot spots in the study of fungal natural products.In this study,the biocontrol strain Trichoderma harzianum is selected as the research object.Useing epigenetic regulation and global regulation to explore the cryptic SMs.Meanwhile combined with transcriptome data to explore the functions and mechanisms of the transcriptional regulators HOS2 and PacC shed light on understanding the complex metabolic networks in fungi.In this study,the CRISPR/Cas9 gene editing system was used to knock out the epigenetic regulators expressed in T.harzianum 3.9236,and 6 mutant strains were obtained.The OSMAC strategy was used to screen the largest SMs difference and the optimal fermentation medium.The results were the mutant strain TMY3(knockout histone deacetylase Thhos2)and rice medium.After fermentation,the crude extract was separated by reversed-phase silica gel chromatography and semi-preparative HPLC.Based on HRMS and 1D-,2D-NMR datas,the structures of five compounds were identified,of which three were new compounds named TH1,TH2 and TH3.TH1 was also named trichoharzianin A,which has good anti-inflammatory ability and the inhibitory rate of inflammatory factor NO is 32.89%.Trichoderma harzianum global regulator pacC knockout mutant(KO-pacC/TMY1)and overexpression mutant(OE-pacC/TMY6)were successfully constructed,and they were cultured in PDB medium with initial pH 3.0,5.6,7.1,8.6,10.6 or phosphate buffered PDB along with WT.The SMs profiles,extracellular pH and volatile fatty acids(VFAs)were analyzed as well as transcriptome datas.These results showed that PacC play an important role in regulating the synthesis of SMs of T.harzianum and responding to environmental pH.PacC was directly related to homodimericin A and 8-epi-homodimericin A production.PacC also mainly regulated the environmental pH value by affecting the concentration of formic acid,and indirectly regulates the synthesis of SMs,such as harzianic acid(HA)and iso-harzianic acid(iso-HA).HA and iso-HA were two pH-dependent SMs that are produced in the pH 4.6-7.5 range.Furthermore,a certain amount of PacC was vital to its survival under extreme alkaline conditions,highlighting the importance of PacC and the precise regulation network.PacC played an important role in the antifungal activity of T.harzianum to Fusarium fujikuroi,Sclerotinia sclerotiorum and Rhizoctonia solani.This study enriched the research on the use of epigenetic regulation and global regulation to activate the fungal activity SMs.It is expected to provide more novel compounds for the development of new drugs,and is of great significance to understanding the complex metabolic networks in T.harzianum.