The Discovery Of Bioactive Secondary Metabolites Against Drug-resistant Bacteria From Two Strains Of Fungi Under The Guidance Of Genome

The situation of bacterial drug resistance is becoming more and more serious,and it is urgent to find bioactive secondary metabolites against drug-resistant bacteria.The natural products derived from fungi are the main sources of small molecule drugs because of their complex structure,novel skeleton and rich biological activities.Chaetomium is one of the biological control bacteria for plant pathogens,and its antibacterial mechanism is most likely due to the production of metabolites with antibacterial properties.The discovery of bioactive secondary metabolites against drug-resistant bacteria from Chaetomium is an effective way to develop new drugs.In this paper,two strains of Chaetomium brasiliense SD-596 and Chaetomium cochliodes SD-280 with good activity against SA and MRSA were screened from more than 200 strains of Chaetomium fungi from different sources.Under the guidance of genomic information,targeted mining of active substances against drug-resistant bacteria was conducted.First,the whole genome of the two strains was sequenced to predict the biosynthetic gene clusters of secondary metabolites and the possible secondary metabolites.Then,various modern chromatographic techniques such as silica gel column,gel column and semi-preparative high performance liquid phase were used,combined with thin layer chromatography analysis,1D nuclear magnetic resonance(¹H-NMR and¹³C-NMR),2D nuclear magnetic resonance（¹H-¹H COSY,HMQC,HMBC and NOSEY）and mass spectrometry techniques for the separation and purification of natural products and the identification of chemical structures.Genomic information shows that C.brasiliense SD-596 has 28 secondary metabolite biosynthetic gene clusters.These gene clusters contained 9 type I polyketides（T1PKS）,1 type III PKS（T3PKS）,5 non-ribosomal peptides（NRPS）,5 NRPS-like,1 T1PKS-NRPS,1 Indole,4 Terpene,and 1 Siderophore,and other kinds of gene clusters.Five depsidones were systematically separated and purified from the crude fermentation extract of C.brasiliense SD-596.And identified as:three new compounds mollicellin S（A1）,mollicellin T（A2）and mollicellin U（A3）,and two known compounds mollicellin D（A4）and mollicellin H（A5）.In the bioinformatics analysis of C.brasiliense SD-596,the PKS in Cluster 1 contains a C-methyltransferase（CMe T）domain,which is a biosynthetic gene cluster responsible for the synthesis of depsidones.The biosynthetic pathway of compounds A1-A5 was reasonably speculated.The antimicrobial activity of five compounds was tested.A1,A2,A3,and A5 had specific inhibitory effects against both SA and MRSA.The MIC values of compounds A1,A2 and A5 against SA and MRSA were the same,which were 6.25,12.5 and 25.0μg/m L,respectively.According to the structural differences of depsidones A1,A2 and A4 combined with the antibacterial experiment data,the aldehyde group at C-4 and the methoxyl group at C-7 had positive contributions to inhibitory activities against SA and MRSA.And the positive effects of aldehyde and methoxyl groups might be synergetic.Genomic information shows that C.cochliodes SD-280 has 91 secondary metabolite biosynthetic gene clusters.These gene clusters contained 25 T1PKS,10 NRPS,5 T1PKS-NRPS,8 Terpene,37 Cf＿putative,1 Cf＿fatty＿acid,and other kinds of gene clusters.Eight compounds were isolated from Chaetomium cochliodes SD-280,mainly natural products of Epipolythiodioxopiperazines（ETPs）,and identified as:4-hydroxybenzaldehyde（B1）,ergosterol（B2）,methyl 6-（isoprenyl）-1H-indole-3-carboxylate（B3）,chetomin（B4）,chetomin D（B5）,Chaetocochins G（B6）,uracil（B7）,thymine（B8）.The anti-MRSA activity of chemomin was significantly stronger than that of the positive control.After the processing of the original LC-MS² data by GNPS and Cytoscape,A visual molecular network map was constructed.Combined with the analysis of the standard chetomin data,the four chetomin analogs could be accurately predicted.The constituents of fatty acid metabolites from Chaetomium cochliodes SD-280 were analyzed by gas chromatography-mass spectrometry.The fraction contained 21 kinds of fatty acids,including 13 kinds of saturated fatty acids and 8 kinds of unsaturated fatty acids.The fatty acids had scavenging ability on DPPH·and·OH radicals,and showed a dose-effect relationship.Since the isolated compounds were far less than the number of gene clusters contained,in order to further excavate the secondary metabolites,the fungus Chaetomium cochliodes SD-280 was pretested with OSMAC strategy（one strain many compounds）,and different amino acids of precursor substances were added into the culture medium.The color of the fermentation broth of the L-tryptophan experimental group and the blank group was significantly different.According to the HPLC analysis,the L-tryptophan and L-alanine experimental groups had new signal peaks compared with the blank group,and according to the spectrogram,it was highly possible to produce new diketopiperazines（DKPs）compounds.The signal peaks of the secondary metabolites of the L-leucine group are significantly reduced,but the peak area of the chetomin signal peak increases.It is speculated that leucine stimulates chetomin Produced in large quantities.