Investigation On Bioactive Metabolites Of Three Insect Symbionts

Insects are the most diverse animal group in the world,which abundant diversity means the diversity of insect symbionts.For the moment,many scholars began to research and develop the secondary metabolites of insect symbionts.However,compared with the diversity of insect symbionts,there only are a small percentage of studies of special environmental microorganisms,and even fewer studies on their secondary metabolites.In this dissertation,one symbiotic actinomycete BYF-112,one fungus BYST-1 of Odontotermes formosanus,and one fungus QTYC-77 of Pantala flavescens were studied,respectively.From the fermentation broth of the above three symbionts,22 compounds were isolated using chromatography techniques including silica gel column chromatography,LH-20 dextran gel column chromatography and recrystallization.The structures of these metabolites,including one new natural product and five new compounds,were elucidated by spectral experiments such as 1D-NMR(¹H-NMR,¹³C-NMR,DEPT135),2D-NMR（¹H-¹H COSY,HMQC,HMBC）and HR-ESI-MS.In addition,we further investigated the antimicrobial,herbicidal and anticancer activities of some compounds that have been isolated.The results were as follows.1.Nine alkaloid compounds（1-9）were isolated and identified from Streptomyces tanashiensis（BYF-112）which was symbiotic actinomycete of O.formosanus,including a new natural product（1）,three new compounds（2-4）and five known compounds N-（2-Hydroxy-5-methylphenyl）acetamide（5）,exfoliazone（6）,venezueline H（7）,3-acetylamino-4-hydroxybenzaldehyde（8）,3-acetylamino-4-hydroxybenzonic acid（9）.The secondary metabolites of BYF-112 were further mined by one strain many compounds（OSMAC）strategy.Compound 10（E-3-methylthio-acrylic acid）was further isolated and purified by comparing the differences of secondary metabolites of BYF-112 cultured in different media.It was found that metabolites 1 and 2 showed significant inhibitory effects on the roots growth of Echinochloa crusgalli at the concentration of 100μg/m L,with the inhibition rates of 66.1%and 65.6%,respectively.Compound 1 showed moderate activity against susceptible and resistant Staphylococcus aureus at the concentration of 30μg/filter paper,with the diameter of inhibition zone of 1.55 mm and 1.25 mm,respectively,which were slightly less than those of the positive levofloxacin with the inhibition zone diameter of 2.93 mm and 2.71 mm,respectively.Metabolite 1 also showed a certain inhibitory activity against human ovarian cancer cell SKOV-3 with an IC₅₀value of 22.33±3.78μM,which was less than that of the positive azithromycin(IC₅₀=0.41±0.12μM).2.A symbiotic fungus BYST-1 was isolated from O.formosanus.The strain was identified as Aspergillus chevalieri.The antifungal activity of fermentation broth against four plant pathogenic fungi was determined.The results showed that BYST-1 fermentation broth had inhibitory activities against four plant pathogenic fungi,including Phytophthora tabaci,Botrytis cinerea,Fusarium oxysporum f.sp.cucumerinum and F.graminearum.When the ratio of fermentation broth to culture medium was 3:20,the inhibition rates of BYST-1 on four pathogenic fungi were 29.2%,21.4%,26.5%and 9.0%,respectively.Subsequently,five compounds（11-15）were isolated,purified and identified,including physcion（11）,preechinulin（12）,2,4-Dihydroxy-6-（（R）-4-hydroxy-2-oxopentyl）-3-methylbenzaldehyde（13）,questinol（14）,hexadecane（15）.The physcion content of BYST-1 in nine different media was determined,and it was found that the highest yield was obtained from fermentation broth in PDB medium with 28.0 mg/L.Meanwhile,whole-genome sequencing of BYST-1 revealed that non-reducing polyketide synthase（Phy G）,β-lactamase（Phy F）,and methyltransferase（Phy H and Phy L）in BYST-1may be involved in the physcion biosynthesis by bioinformatics analysis.3.Seven compounds（16-22）were isolated and identified from Trichoderma harzianum QTYC-77,which was symbiotic fungus of P.flavescens.Among them,16（azaphilone D）and 17（azaphilone E）were new metabolites,and 18-22 were known compounds ergosterol（18）,uracil（19）,pachybasin（20）,harzianone（21）,dodecyl（Z）-9-hexadecenoate（22）.The absolute configuration of the new compound 16 was determined by X single crystal diffraction experiments combined with CD spectra.The anticancer bioassay showed that metabolite 20 had weak cytotoxic activity against human gastric cancer cells MDA-MB231 with an IC₅₀value of 99.69±5.96μM,which was less effective than that of the positive azithromycin with the IC₅₀value of 1.57±0.03 m M.The discovery of our study provided addition evidence that insect symbionts,as one kind of special environmental microorganisms with little research,are the important sources of novel active natural products,which are worthy of further study.