The Mining Of Natural Products From Two Strains Of Fungi

Natural products have always been an important source of drug development due to their various pharmacological and biological activities.With the emergence and prevalence of drug resistance of pathogenic microorganisms,existing drugs can no longer meet people’s needs.Humans need to find and develop new drugs to deal with the problem of drug resistance.Because fungi can produce secondary metabolites with diverse structures and activities,they have long been one of the important sources of drug development.However,with the long-term development of microbial resources,the traditional activity-oriented separation method of natural products has the problem of too high re-screening rate.With the development of bioinformatics,people have sequenced the fungal genome and found many silent gene clusters that are not expressed under normal conditions,indicating that its secondary metabolite synthesis potential is far from being exploited.By analyzing the gene sequence of fungi,understanding the metabolic regulation mechanism during the synthesis of secondary metabolites,and constructing a suitable genetic system to activate these silent gene clusters to produce new natural products is an important way to solve the current shortage of new drugs.In this paper,through the bioinformatics analysis of the whole genomes of two fungi:C.higginsianum ACCC 37053 and A.heteromorphus CGMCC 3.4327,two silent highly reduced polyketide gene clusters were found.After being activated by conventional fermentation culture,transcriptional factor（TF）overexpression,and gene cluster heterologous host expression,a number of secondary metabolites that cannot be produced in a conventional culture environment are isolated,and antibacterial experiments and anti-tumor experiments are performed on the compounds to find possible organisms active.The main experimental results are as follows:1.Using PDB as the medium for liquid fermentation of anthracnose bacterium C.higginsianum ACCC 37053,two known compounds,Colletorin A and Colletochlorin A,with molecular weights of 322.0 and 356.5,respectively,are separated,and their common structural feature is the isoprenyl side the chain-linked polysubstituted benzene ring.One-H on the benzene ring of Colletorin A is replaced by-Cl to form Colletochlorin A.It has been reported that Colletochlorin A lacks animal toxicity and has obvious phytotoxicity to the invasive species Ambrosia artemisiifolia.It is a potential the natural plant herbicide proves that the mining of natural products of C.higginsianum has high research value.2.After analyzing the genome of C.higginsianum,a positive regulator TF was found near a silent polyketide biosynthesis gene cluster,and it is speculated that it regulates the expression of this gene cluster.The TF sequence was cloned into an expression vector and transformed into C.higginsianum for overexpression.A new compound 1 was obtained with a molecular weight of 308.0 and maximum ultraviolet absorption at 450nm.The biological activity test showed no antibacterial activity and anti-tumor activity.3.Using Aspergillus nidulans as a heterologous host,the polyketide synthase gene in the above gene cluster was heterologously expressed.Compound 1 was detected in the fermentation broth,which proved that TF is responsible for regulating the expression of the polyketide biosynthesis gene cluster.Hypothetical protein and fatty acid transferase do not participate in the modification of the molecular structure during the synthesis of compound 1.4.There is an unknown polyketide biosynthesis gene cluster containing multiple modified genes in the A.heteromorphus genome.Using Aspergillus nidulans as a heterologous host,plasmids were constructed to express the genes in sequence,and new compounds were detected in the fermentation broth 2,3,4,5.The positive ions detected by mass spectrometry were 289,261,291,and 293,respectively.The monomer of compound 3 was obtained by fermentation and purification.The molecular structure of the compound 3 was found to be an unreported new compound by nuclear magnetic analysis.The molecular formula is C₁₆H₂₀O₃,which is a long-chain polyketone containing multiple C-C double bonds.The molecular structure of compounds 2,4,and 5 is unstable.Antibacterial experiments showed that these compounds have different antibacterial activities.