The Biosynthesis Pathway And Functional Characterization Of Secondary Metabolites(Azaphilones) In Trichoderma Guizhouense NJAU 4742

Species of the genus Trichoderma are prolific producers of secondary metabolites with versatile biological activities.However,most studies on the secondary metabolites of Trcichoderma focused on the identification of the structures and in vitro functions while the biosynthetic pathway and biological functions remain elusive.Trichoderma guizhouense NJAU 4742(Tg),isolated and identified in our laboratory,is able to antagonize several plant pathogenic fungi and has been widely utilized in agriculture as the functional agent in bioorgainic fertilizers.Studies on the biosynthesis and the ecological functions of the secondary metabolites in Trichoderma will contribute to better applications of Trichoderma spp and their natural products.In this study,we performed genome mining for biosynthetic gene clusters of the secondary metabolites(smBGCs)in Tg and other 11 common Trichoderma strains.And we found a gene cluster was the most upregulated smBGC during the interaction between Tg and Fusarium oxysporum f.sp.cubense 4(Foc4).By using combination of gene disruption,overexpression of the pathway specific transcription factor,HPLC-MS,NMR and other methods,we identified the structures of products synthesized by this smBGC and the biosynthetic pathwasy and chacterized the biological functions of these metabolites.The main results were as follows:1.Trichoderma guizhouense NJAU 4742 has the potential to produce abundant secondary metabolites.The whole genome of Tg has been sequenced and the smBGCs in Tg and other 11 Trichoderma strains were analysized via antiSMASH and local BLAST.We found that the Harzianum/Virens clades including Tg harboured the lagerst number of smBGCs in their genomes.There are 56 smBGCs in the genome of Tg,but to date only 3 smBGCs have been identified including tricholignan,trichoxide and harzinopyridone,most of the structures and corresponding smBGCs remain unknown.Therefore.Tg still has a huge potential for people to mine new secondary metabolites and new biosynthesis patterns.2.The most upregulated secondary metabolites of Trichoderma guizhouense NJAU 4742 during the interaction with Foc4 are trigazaphilones.Based on the previous research in our laboratory,we found the tgaza cluster was the most upregulated smBGC during the interaction between Tg and Foc4.This gene cluster showed certain similarities with the smBGCs responsible for the azaphilones metabolites asperfuranone and chaetoviridin/chaetomugilin.According to the feature of azaphilones metabolites,we found that tgaza cluster could express in the nutrient PDB but kept silent in GSM.Through disruption of the core PKSs tgaza1 and tgaza2 and overexpression of the pathway specific transcription factors tgaza5 and tgaza8,combined with HPLC-MS and NMR,we found that the isolated and identified products of tgaza cluster were trigazaphilones,including harziphilone,T22azaphilone and isomers or derivatives.Besides,we also determined the range of tgaza cluster(tgazal-tgaza10)via RT-PCR analyses and found that the tgaza cluster was regulated by Tgaza5.3.The biosynthetic pathway of trigazaphiloens in Trichoderma guizhouense NJAU 4742 is similar to that of chaetoviridin/chaetomugilin in Chaetomium.By comfirming the structures of harziphilone and T22azaphilone and the molecular weight of other derivatives,we discovered that harziphilone was the intermediate product synthesized by tgaza cluster.Combining the HPLC-MS data of the extracts of Δtgaza1 and tgaza2 mutants,we confirmed that both two PKSs,Tgazal and Tgaza2,were necessary for the biosynthesis of the intermediate product harziphilone.This is similar to the biosynthesis of intermediate prodcuts in asperfuranone or chaetoviridin/chaetomugilin biosynthetic pathway.On the other hand,after deleting the gene encoding O-acyltransferase(Tgazal0)in Tg,the Δtgaza10 mutant could only produce harziphilone but not other trigazaphilones,comfirming the functional similarity of Tgaza10 to CazE in Chaetomium and suggesting the biosynthetic pathway of trigazaphilones is similar to that of chaetoviridin/chaetomugilin.With the similarity between tgaza cluster and caz cluster,the biosynthetic pathway of trigazaphilones could be proposed as follows:Tgazal supplies a relative highly reduced triketide product to Tgaza2 followed by the Tgaza9-catalytic reaction to form the initial intermediate product,the precursor of harziphilone and its isomers.In addition,Tgazal simultaneously supplies another diketide and transferred via Tgazal0 to synthesize T22azaphilone and other derivatives might be formed via the reaction of addion of oxygen and water.4.The main biological function of trigazaphilones in Trichoderma gui-houense NJAU 4742 is to maintain the intracellular redox homeostasis.Previous in vitro studies have shown that T22azaphilone had the obvious antifungal activity.But in this study,no difference among the Tg wild type(WT),the deleting mutants(Δtgazal,Δtgaza2 and Ptgaza5::gfp)and the overexpressing mutant(OE::tgaza5)were found when confronted with different fungal pathogens.At the same time,there was no significant difference in the antagonistic abilities among the extracts between WT and mutants.By using the green fluorescent-labbled mutant Ptgaza5::gfp,we also found that the tgaza cluster was involved in confrontation with different Fusarium species but the fluorescence response was significantly different.Combined with the staining results of reactive oxygen species(ROS)during confrontation,it was found that the expression of tgaza cluster might be positively correlated to the ROS level.Through comparing the difference of the tolarence between Tg WT and its mutants to hydrogen peroxide and qPCR analyses for the expression of the pathway specific transcription factor tgaza5 under different in vivo redox conditions,we confirmed that the expression of tgaza cluster was strictly regulated by the intracellular redox level.According to the previous study in our laboratory,the main role of trigazaphilones in Tg during interaction with Foc4 is not to inhibit the growth of that pathogenic fungi,but to maintain the intracellular redox homeostasis.5.The tgaza cluster in Trichoderma guizhouense NJAU 4742 is highly conserved among Trichoderma harzianum species complex.By comparing the protein sequences of the two core PKS(Tgaza1 and Tgaza2)in the public database,we discovered that the gene cluster is highly conserved in the Trichoderma harzianum complex species,and the similar tgaza cluster is widely distributed in in Sordariomycetes and Eurotiomycetes,mainly in Penicillium,Aspergillus,Metarhizium and Trichoderma.At the same time,we also found that the tgaza cluster might have the horizontal transfer phenomenon during evolution through the analysis of the integrity of this cluster in above fungal genomes.In addition,there are certain similarities between the smBGC of non-azaphilones sorbicillinoids and the tgaza cluster.Via deleting the gene ypr1,which is encoding the pathway specific transcription factor in sorbicillinoids smBGC,we confirmed that sorbicillinoids and trigazaphilones had the same function,that is,free radical scavenging activity,suggesting that sorbicillinoids might exert effects similar to trigazaphiones in the environment.In summary,we identified the biosynthesis of trigazaphilones(including harziphilone and T22azaphilone),the most obvious secondary metabolites secreted by Tg during the interaction with Foc4,and verified the main biological function of these natural products was to maintain the intracellular redox homeostasis,benefiting Tg to adapt the oxidative stresses in the envrionment.