Isolation And Purification Of Active Ingredients From Fermentation Broth Of Conidiobolus Coronatus

Because of special growth environment entomogenous fungi have unique metabolic pathways and physiological capabilities and produce some metabolites that are not common in terrestrial microorganisms.Their are not only very important biological control materials,but also ideal resource for natural products with novel structures.Although more and more studies have been conducted on the fungi,there are still more than 90% of the strains with no components study.There are more fewer studies on the metabolites of Zygomycetes.Therefore it is necessary to study metabolites of Zygomycetes.Conidiobolus coronatus(Cost.)Batko belongs to the genus Escherichia of the family Pseudomonas,from Entomophthorales,Zygomyeotina,which are distributed worldwide.They are mainly saprophytic in soil,and also parasitic on many insects.They play an important role in the material cycle of the ecosystem.In this study,a variety of chromatographic integration techniques and spectral analysis methods were used to compare metabolites from 18 strains of C.coronatus and 2 strains of Conidioblous thromboides.And a strain of C.coronatus with significant more of metabolites than other strains were seleted and submitted for metabolites isolation and identification.The research content and results of this test are as follows:First,anti-SMASH database was used to analyze the genome of C.coronatus,surmised there are 2 non-ribosomal peptide gene clusters,2 terpene gene clusters and 2 polyketone gene clusters which implies the fungus has potential to produce non-ribosomal peptides,terpenes and polyketones.HPLC-TOF / MS shows high level of non-ribosomal peptides and polyketones in extracts of C.coronatus.Second,metabolites comparison between the fungus on M102 and SDB media shows that C.coronatus grew faster and produced more second metabolites on SDB medium.Metabolites comparison between mycelia and fermentation broth of the fungus shows that most of the sceond metabolites were excreted in the broth.Third,on the basis of HPLC-TOF / MS analysis,the metabolomics method was used to compare metabolites differences between 20 Conidiobolus strains.Results of principal component analysis show that the 20 strains were clustered in 4 groups.Comparison between 4 strains from the 4 groups shows that the strain G4 has the most second metabolites,and the highest level of TR dipeptide,Cyclo(L-4-Hyp-L-Pro)and Cyclo(L-Pro-L-Tyr).Metabolites comparison between different culture time of strain G4 shows that the best culture time is 16 days.Fourth,a large amount of fermentation culture was performed in an SDB shake flask,followed by ethyl acetate extraction,filtration,concentration,and reconstitution procedures to obtain the original crude extract which was submitted to preparative HPLC.for purification and 5 pure compounds were obtained.The structures were determined by ESI-HRMS,UV,1H NMR,13 C NMR spectral data and X-single crystal diffraction analysis as Cyclic dipeptide(CGP),2-ethyloctahydro-11H-pyrido [1,2-a] pyrrolo [1,2-d]pyrazine-5,11(5a H)-dione,L-Phenylalanine and MG(0:0/18: 1n7).Fifth the 4 compounds were submitted for antibacterial and insecticidal activities evaluation.Results show that they have no antibacterial activity and no inhibitory effect on Candida albicans.But when compound CGP was injected with 5μL1000 ppm into the larvae of Galleria mellonella(Lepidoptera),the mortality rate reached 90% while2-ethyloctahydro-11H-pyrido [1,2-a] pyrrolo [1,2-d] pyrazine-5,11(5a H)-dione has a lethality of 80%.There was no significant difference between the feeding group and the blank group.Despite the fact that the four compounds are known compounds,they are the first tume being isolated from C.coronatus.This study provides basic references on metabolic differences between strains of C.coronatus,metabolic surmise,metabolites isolation and identification,and bioactivities of compounds from the fungus.It is helpful to understand the potential role of the small molecule metabolites of C.coronatus in the process of biocontrol,and provide a basis for the development of more effective,environmentally friendly and safer methods for pests control.