Bioinformatic Analysis Of Trichoderma Biosynthetic Gene Clusters And Their Activation In Trichoderma Reesei

Natural products produced by filamentous fungi are an important source of drugs or drug lead compounds.Genome analysis has revealed that there are a significant number of silent biosynthetic gene clusters(BGCs)in the genomes of filamentous fungi.How to identify BGCs for synthesis of novel natural products from the large amount of genomic information available,and aslo activate the expression of these BGCs,is a major bottleneck in the current stage of natural product discovery.Trichoderma species are widely used in biological control and have been reported to synthesize a variety of natural products with antimicrobial activity,such as peptaibols,gliotoxin,and Tricholignan A.Although the biocontrol ability of Trichoderma has long been of great concern,their BGCs have not yet been systematically analyzed.Although heterologous expression of BGCs is an important approach for natural product discovery,the lack of efficient genetic manipulation systems in many Trichoderma speices and the low efficiency of large BGCs assembly and integration severely impedes the discovery of BGCs.Trichoderma reesei has the potential to be a heterologous chasis strain for the heterologous expression of Trichoderma BGCs of in view of its high protein expression levels,clean genetic background,and robust metabolism.The present thesis first systematically bioinformatically analyzed the BGCs of Trichoderma and activated the expression of one potential new BGC in T.reesei.To promote efficient expression of heterologous BGCs in T.reesei,a expression system for heterologous BGCs of T.reesei based on the autonomous replicating element AMA1 plasmid was constructed.The main results obtained are as follows:1.Bioinformatic Analysis of BGCs in Trichoderma GenomesThe natural product BGCs in 47 Trichoderma genomes from NCBI and JGI databases was analyzed by AntiSMASH,resulting in the identification of 2,187 biosynthetic gene clusters,including 802 NRPS,665 type I PKS,414 Terpene,293 PKS-NRPS Hybrids,11 RiPPs,and 2 other types of biosynthetic gene clusters.Further analysis revealed that 43 out of 47 Trichoderma strains harbor the BGCs for the synthesis of peptaibols,while 16 out of 47 analyzed Trichoderma strains contain biosynthetic gene clusters for the production of gliotoxin,but studies have shown that only Trichoderma virens can produce gliotoxin due to the absence of the key gene GliH in other analyzed Trichoderma strains.To discover BGCs with the potential for novel compound synthesis in Trichoderma genomes,a phylogenetic analysis was performed using PKS and adenylation domains(A)of NRPS as probes,in combination with the corresponding characterized BGCs in MiBIG database.The result showed that 5 PKS and 3 NRPS branches were not related to any characterized BGCs.BiG-SCAPE analysis revealed that one of the PKS gene clusters(Tr3.2),consisting of highly reducing type Ⅰpolyketide synthase encoding gene pks3,serine hydrolase encoding gene sh2,and glycosyltransferase encoding gene ugt1,was present in 43 analyzed Trichoderma.Transcriptional analysis showed that Tr3.2 was silent in T.reesei under conventional culture conditions.2.Tr3.2 gene cluster ActivationThe expression of sh2 and pks3 was activated by promoter swapping strategy using cellulase promoters Pcel7A and Pcel6A,respectively.Transcription analysis showed that both genes were transcriptionally activated,but HPLC analysis did not detect any newly synthesized products.Further activation of ugtl using Vcel5A resulted in the production of two new compounds.Mass spectrometry and NMR analysis revealed that compound 1 was a sterol-like compound with the modification of 3-hydroxyl-leucine,while compound 2 was a previously characterized PKS-NRPS compound ilicicolin H.Genome analysis suggested that the corresponding BGC for ilicicolin H synthesis was indeed present in T.reesei and transcription analysis revealed the continuous expression of ilicicolin H biosynthetic gene in the late growth stage of the Tr3.2 activation strain.The specific structure,synthesis,and regulatory mechanisms of compound 1 require further investigation.To investigate the relationship between the potential compound synthesized by Tr3.2 and the isolated compounds 1 and 2,the core genes pks3 of Tr3.2 and TriliA of ilicicolin H BGC were knocked out.Our results showed that pks3 deletion lead to the disappear of compound 1 and 2 synthesis,while the knockout of TriliA only only lead to the absence of compound 2 synthesis but not compound 1.These results suggest that the products synthesized by Tr3.2 may have led to the synthesis of compounds 1 and 2.3.Construction of a T.reesei BGCs Expression System based on the AMA1 ElementThe construction of natural product BGCs expression systems for filamentous fungi is of great significance for the efficient mining of the large silent BGCs.AMA1 is an autonomously replicating sequence element identified in Aspergillus nidulans,and studies have shown that it can significantly improve the transformation efficiency of filamentous fungi.Using the AMA1 element and three commonly used selection markers,asl1,hph,and pry4 genes,and three strong constitutive promoters,Pcdnal,Ptef1,and Ptcu1,we constructed expression vectors that can rapidly transform T.reesei.Using fluorescent protein genes BFP,mCherry,and EGFP as reporter genes,we found that simultaneous transformation and expression of the three fluorescent proteins can be achieved by a one-step co-transformation using the three expression vectors.Furthermore,LovB and LovA from the lovastatin biosynthetic pathway were assembled onto the AMA1-based expression vector,which was then transformed into a strain containing LovB,LovG,and LovC,resulting in successful synthesis of lovastatin intermediate Monacolin J(MJ).In summary,the present thesis systematically analyzed the BGCs in Trichoderma,and discovered a PKS gene cluster,Tr3.2,consisting of only three genes.By implementing promoter swapping strategy,the BGC was activated and two compounds,1 and 2,were obtained.In addition,this study established a heterologous BGCs expression system in T.reesei,realizing the efficient transformation and expression of the heterologous BGC,laying the foundation for developing T.reesei as a chasis for expressing heterologous BGCs.