| Microorganisms are the source of various natural products(NPs)which have abundant activity.NPs are an important source of novel molecular structure skeletons,and precursors of various clinical drugs that have been widely used to treat human inflammation,cancer,and other diseases.However,the widespread use of antibiotics brings more and more obvious disadvantages.The "evolution" of drug-resistant bacteria is more and more difficult to control,which greatly reduces the effect of antibiotics.Therefore,human beings have been digging for more effective natural drug molecules.To a certain extent,the evolution and feedback of nature promotes research in the field of natural products.The development of bioinformatics has led to the discovery of many gene clusters that are silenced under conventional conditions.It has been proven that these silenced gene clusters are a huge treasure trove of chemical compounds to be explored by researchers.In this paper,the bioinformatics analysis of the genomes of two fungi: Colletotrichum higginsianum ACCC 37053 and Pestalotiopsis sp.JCM 9685.Bioinformation analysis showed there was a putative non-reduced polyketide synthase and a highly-reduced polyketide in Colletotrichum higginsianum ACCC 37053.The secondary metabolites could not be isolated under conventional conditions were separated by large-scale fermentation culture,overexpression of transcriptional factor(TF)and heterologous expression of gene cluster,and some small molecule compounds were excavated.The experimental results are as follows:1.Large-scale fermentation of Pestalotiopsis sp.JCM 9685 was carried out in rice medium and cultured at room temperature for 30 days.A known compound A was isolated with a molecular weight of 286.0.It has been reported that it is a known compound named methyl 3,8-dihydroxy-9-oxo-9H-xanthene-1-carboxylate.It also has a pair of atropisomer B,its molecular weight is 482.0.The maximum ultraviolet absorption wavelength is at 214 nm and 258 nm.There is no antibacterial activity in the biological activity test.2.A positive regulatory activator TF was found near the BGC of one NR-PKS after genome analysis of C.higginsianum.It was predicted that it could activate the expression of the silent gene cluster.The TF was cloned into an expression vector and transformed into C.higginsianum for overexpression.A compound 1 was obtained with a molecular weight of 300.0 It has the largest absorption peaks at 215 nm,283 nm,and 425 nm.The biological activity test has no bacteriostatic activity and has no obvious cytotoxicity on the growth of cancer cells.Two known compounds 2(Colletorin A)and 3(Colletochlorin A)were also isolated,which have molecular weights of 322.0 and 356.0 respectively,were isolated and characterized by polysubstituted benzene rings linked by isoprene side chains.Activation increased the yield of these two known compounds.3.After heterologous expression and purification of an HR-PKS gene cluster from Colletotrichum higginsianum,compound 4 was obtained.According to NMR spectrum analysis,compound 4 is a known compound which is trans-2-decenedioic acid.It is a nematode control compound.Due to the high commercial value and low yield.In this paper,heterologous expression was achieved in Saccharomyces cerevisiae cells,and its yield was increased by 1.5 times compared with that in Aspergillus nidulans through this approach,achieving the purpose of high yield.In this paper,6 small molecular compounds were obtained by using three genome mining strategies of changing the medium conditions、overexpression of transcriptional factor and heterologous expression for these two fungi.The research group will also carry out more in-depth and detailed research on the basis of the current bacterial species,hoping to obtain more novel and active small molecule compounds to improve human life. |
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