Study On Two Transcription Factors And Biosynthetic Geenes Of Thermomyces Dupontii 2155

Fungi contain a remarkable rich and diversity of secondary metabolic pathways.Genes in these pathways are often grouped in clusters which termed as Biosynthetic Gene Clusters?BGCs?.However,a mass of BGCs in fungal genome is silent or low expressed under laborary conditions,suggesting that the secondary metabolic wealth of filamentous fungi is largely untapped.Our understanding of the regulation of BGCs is expanding at a fast pace.Cluster-specific regulators and global transcriptional factors affect secondary metabolites of fungi in both positive and negative ways.However,most studies are focused on over-expressing positive regulatory factors to induce the expression of silent and/or low expressed BGCs of fungi,little attention has been paid to negative regulatory factors.In addition,expressing of the silent gene in the heterologous host is also an effective way to discover and study the secondary metabolites of fungi.Thermomyces dupontii is a thermophilic fungus with the ability to produce a large number of secondary metabolities?SMs?such as indole alkaloids,Talathermophilins,and a novel nematicidal compound-thermolide.Like many other filamentous fungi,most of the biosynthesis genes in T.dupontii remain silent,for example,the expression of Talth1₀04980_t1?TalA?,the only type I PKS-NRPS hybrid gene in T.dupontii,could not be detected under all laborary medium.Upon bioinformation analysis,a putative cluster special transcription factor-Talthlp4₀05393?TF3?of TalA cluster and a putative global regulator gene Talth1p4₀01383?homologous to Aspergillus McrA?were selected in this thesis to study their function on the production of SMs in T.dupontii.CRISPR-Cas9 gene editing system was empolyed for the first time to generate gene disruption mutant in T.dupontii.Arthrobotrys oligospora,an important predator nematode fungus,was selected as a heterologous host for the first time to express fungal biosynthesis genes.The main results are described as follow:1.Four TF3 and five McrA gene disruption mutant strains??TF3-18,TF3-12,TF3-21,?TF3-14 and ?mcrA-11,?mcrA-34,?mcrA-3,?mcrA-15,?mcrA-12?were obtained through CRISPR-Cas9.Compared with WT,all four TF3 mutant strains produce new peaks in LC-MS analysis,suggesting TF3 might be a negative regulator of SMs in T.dupontii.Further gene expression analysis of biosynthetic genes in TalA cluster would help to evaluate whether TF3 is a TalA cluster special regulator.The two McrA mutant strains ?McrA-3 and AMcrA-15 accumulate a new peak compared with WT on LC-MS.2.A successfully CRISPR-Cas9 edited strain of A.oligospora??215g283-Cas9-1?was obtained in this study,and to our knowldge,this is the first genomic editing strains of A.oligospora to date.LC-MS showed that the metabolic profile of ?215g283-Cos9-1 was in accord with that of gene knockout mutant strain A215g283 obtained by homologous recombination,indicating that the CRISPR-Cas9 gene editing is a reliable method in studying the gene function of A.oligospora.3.Five heterologous expression strains of A.oligospora that expressing biosynthetic gene of thermophilic fungus were successfully obtained,namely AO-TalA-1.AO-TalA-2,AO-TalA-ER-3,AO-TalA-ER-7 and AO-TalA-ER-9.New peaks accumulated in these heterologous expression strains in both LC-MS and GC-MS analysis.Further isolation and identification of these new compounds may contribute in revealing the function of the TalA.4.Growth rate.producing of three-dimensional network and the predator nematode ability of all the heterologous expression strains were declined.