Isolation And Genome Mining Of Natural Products From Two Fungal Strains

Fungal secondary metabolites have diverse structures and activities and are important sources of clinical drugs,pesticides,and food additives.There are a large number of biosynthetic gene clusters(BGCs)of secondary metabolites in fungal genomes.Polyketide Synthase(PKS)and Non-ribosomal Peptide Synthetase(NRPS)are two giant enzymes that can produce Polyketide(PK)and Non-ribosomal Peptide(NRP)with complex structures and diverse functions.Fungal PKs and NRPs are important sources of anti-bacterial,anti-fungal,and anti-tumor drugs,and have been widely used in the discovery of new drugs.At present,the number of PKs and NRPs isolated from fungi is far less than the number of BGCs of PKS and NRPS in fungal genomes,indicating that many BGCs are not expressed or expressed in low levels under conventional laboratory conditions.Overexpression of Transcriptional Factor(TF)and heterologous expression of fungal PKS and NRPS gene clusters have been effective strategies to obtain new natural products for the development of new drugs.In this study,the natural products of Macrophomina phaseolina ACCC 39267 were isolated by traditional isolation strategy;two putative PKS-NRPS BGCs in the genome of Colletotrichum higginsianum ACCC 37053 were studied by heterologous expression.The results are as follows:1.M.phaseolina was fermented on PDA medium,and two natural cyclic peptides were isolated by preparative HPLC.The molecular formulas of the two compounds are C₃₀H₅₅N₅O₅,and their molecular weights are both 565.The compounds have strong UV absorption at 220 nm and have not shown obvious inhibitory effects in antibacterial experiments.2.In analyzing the genome of C.higginsianum,we found a putative PKS-NRPS gene cluster ACCC68.1 containing multiple modification genes.The BGC was expressed using Aspergillus nidulans A1145 as a heterologous host.Compounds 3 and 4were produced in the expression of the PKS-NRPS alone.The molecular weight of compound 3 is 433,and it has strong UV absorption at 213 nm,and the molecular weight of compound 4 is 403,and it has maximal UV absorption at 196 nm.Compound3 showed a strong inhibitory effect on Mycobacterium tuberculosis,while compound 4showed moderate inhibitory effect on M.tuberculosis and Bacillus subtilis;Compound 3and 4 were still the only products when the backbone gene and modification genes were co-expressed in A.nidulans,it showed that the added genes did not work in the heterologous expression.RT-PCR results showed that the added genes were correctly transcripted in A.nidulans expression system.The added genes did not recognize compounds 3 and 4 as substrates for further transformation.The results of co-expression suggested that compounds 3 and 4 are not the correct products of PKS-NRPS,and the real intermediates were modified by A.nidulans.We used Aspergillus oryzae Ri B40 as an alternative heterologous host,however,only compound 4 was produced.For the modification of intermediates by the hosts,we tried to overexpress the Transcriptional Factor within the ACCC68.1 gene cluster,but no new compounds were produced in the study.3.Another unknown PKS-NRPS gene cluster in the genome of C.higginsianum was studied,the gene cluster contained a PKS-NRPS,an Enoyl reductase,and a FAD-dependent oxidase.The results of heterologous expression in A.nidulans showed that the backbone gene of PKS-NRPS and ER can produce compound 5 with a molecular weight of 295 and strong UV absorption at 230 nm.Through 1-D and 2-D NMR spectrum,the structure of compound 5 was characterized.The antibacterial experiments showed that compound 5 had a strong inhibitory effect on B.subtilis and M.tuberculosis.When the FAD-dependent oxidase gene was co-expressed with backbone genes in Aspergillus nidulans,a new compound 6 was produced,which proved that FAD-dependent oxidase was involved in the biosynthetic pathway.The molecular weight of compound 6 is 329,and it has strong UV absorption at 226 nm.In this study,two known natural pentapeptides were isolated from M.phaseolina.and four new compounds with anti-bacterial activities were produced by heterologous expression.It shows the efficiency of Genome Mining in the discovery of fungal new natural products.