Discovery Of Natural Products From Endocalyx Cinctus、 Penicillium Roqueforti And Colletotrichum Higginsianum

Natural products（generally referred to as secondary metabolites）are one of the main sources of small molecule drug development.Fungi can produce structurally diverse natural products,and the discovery and isolation of natural products from fungi is an important research direction.Genes involved in the synthesis of natural products in fungi are generally clustered on chromosomes（biosynthetic gene clusters）.Genes involved in the synthesis of natural products in fungi are generally clustered on chromosomes（biosynthetic gene clusters）.With the development of bioinformatics and the determination of more and more fungal genome sequences,it is found that the number of secondary metabolite biosynthesis gene clusters contained in the fungal genome is much larger than the number of compounds that can be isolated,which shows that through genome mining（genome mining）means may develop more new compounds from fungi.In this paper,through the bioinformatics analysis of the genome sequences of three fungi（Endocalyx cinctus JCM 7946,Penicillium roqueforti CGMCC 3.7068,Colletotrichum higginsianum ACCC 37053）,it was found that they all contained a considerable number of natural product biosynthetic gene clusters,indicating that they have a large synthetic Secondary metabolite potential.The natural products of the three fungi were developed and studied by traditional fermentation culture,transcriptional factor（TF）overexpression and gene cluster heterologous host expression.1.Two known compounds,6-methoxy-8-hydroxy-3-methylisocoumarin and xylariol A,were isolated by solid fermentation of E.cinctus JCM 7946 in rice medium.6-methoxy-8-hydroxy-3-methylisocoumarin has a molecular formula of C11H10O4 and a molecular weight of 206.42.It has a maximum absorption peak at 238 nm.It is an isocoumarin derivative and can be used as an antibiotic to inhibit the biological activity of various animal cells and human cancer cells.;Xylariol A has a molecular formula of C₁₁H₁₄O₄ and a molecular weight of 210.08.The compound is cytotoxic to Hep G2 cells with IC₅₀ values of 22.3 and 21.2 mg/m L,respectively.It is the first time that these two important active natural products have been identified from Endocalyx fungi.2.After bioinformatics analysis,a gene cluster with the unknown function was found in E.cinctus JCM 7946,which contained a gene cluster encoding polyketide synthase-non-ribosomal peptide synthase（PKS-NRPS）,and a heterologous expression strategy was used to construct The expression plasmid containing the gene cluster was heterologously expressed in the host Aspergillus nidulans.LC-MS analysis of the fermentation product showed that a compound with a molecular weight of 495.01 was expressed.The compound was isolated and purified by mass fermentation（5L PDA）,and3.67 mg was isolated.The specific chemical structure is still being resolved.3.When analyzing the genome of P.roqueforti CGMCC 3.7068,a transcriptional regulator TF was found in a possible polyketide biosynthesis gene cluster,and it was speculated that it could regulate the expression of this gene cluster.The TF was ligated with a hygromycin-resistant vector and transformed into the protoplast of P.roqueforti.The fermentation product of the transformant was detected by LC-MS.It was found that a compound with a molecular weight of 319.91 was successfully activated,and its maximum UV The absorption peak is at the wavelength of 253 nm,the compound has low solubility in methanol and acetonitrile and is currently undergoing large-scale fermentation and product separation and purification.4.Activation of a highly reduced polyketide synthase gene cluster of unknown function in the genome of C.higginsianum ACCC 37053.By cloning the TF gene in the gene cluster into an expression plasmid and transforming it into C.higginsianum ACCC37053 protoplasts.,LC-MS analysis of the fermentation product of the transformant showed that the expression of a compound with a molecular weight of 534.12 and a maximum UV absorption value at 218 nm was activated.The compound is currently being isolated,purified and identified.