Study On The Anti-bacterial Ingredients From Tiam Mutant And One Marine-derived Actinomycete

Microbial natural products are important resources for discovering new antibiotics,playing a vital role in human health and life.However,with the dissemination of muti-drug resistance(MDR)pathogens,the demand of new antibiotics which can inhibit those MDR pathogens is becoming more and more urgent.A large part of active secondary metabolites derived from microbes are produced by actinomycetes,such as streptomycin found in Streptomyces griseus(Nobel Prize in Physiology,1952),which was the second antibiotic applied in clinical after the penicillin,ended the tragic of the patients infected with tuberculosis waiting for death.The main content of this study is focused on isolating tiacumicin analogues from the ?tiaM(encoding a halognease)mutant of Dactylosporangium subsp.NRRL 18085(Tiacumicin B producer).A series of dehalogenated tiacumicin analogues were obtained in the fermentation broth of the ?tiaM mutant and were evaluated for antibacterial activity.The second part of this study described the screening of 19 actinomycetes derived from South China Sea for antibacterial activities,and one of them,Micromonospora sp.SCSIO 07395,was selected for further chemical studies to exploit new bioactive compounds.The first chapter was on the secondary metabolites of the ?tiaM mutant.A series of gene knockout experiments were carried out characterizing biosynthetic gene cluster for tiacumicins B by post-doctoral Yi Xiao,who provided 23 gene deletion mutants.The ?tiaM mutant was cultured in the YMS medium.The fermentation broth was centrifuged and extracted with organic solvents.The extract was purified by silica gel column,preparative-TLC and semi-HPLC chromatography.Structures of 16 purified compounds were elucidated by the spectroscopic data based on IR,UV,MS,NMR.Twelve of them were new compounds,including 11 tiacumicins analogues(2-12)and one new macrolide(16).The others were 3 known tiacumicins analogues(1,14,15),and one indole3-formaldehyde(16).All of the tiacumicin analogs were found to be dechlorinated derivatives on the aromatic ring of Orsellinic acid or Homo-orsellinic acid,which further confirmed the function of TiaM as a halogenase in Tiacumicin B biosynthetic pathway.Comparing with Tiacumicin B,compounds 1 and the new compounds were assayed for their antibacterial activities.The structure-activity relationship between these tiacumicin analogs indicated that chlorine substitutions on the benzene ring are not necessary for antibacterial acitivities as dechlorinated derivatives 1,2,3,5,6,7,8,9,10 still kept antibacterial acitivities.However,the length of aliphatic chains substituted on the benzene ring impacts the antibacterial activity.The hydroxyl group located at C-18 is an indispensable group for antibacterial bioactivity,because the dehydroxylation or an oxidation to ketone at C-18 would cause decreased activity.One the left partial structure of tiacumicin analogs,esterification at C-4??hydroxyl group(6-methyl-D-rhamnose)would increased antibacterial activity,on the right partial structure of tiacumicin analogs,esterification at C-4?hydroxyl group(2-oxomethyl-D-rhamnose)with Orsellinic acid or Homo-orsellinic acid could also increased antibacterial activity.The second chapter mainly focuses on screening 19 marine-derived actinomycetes for antibacterial activity.In order to adapt to the harsh marine environment,such as high pressure,darkness,high salt,high(low)temperature and oligotrophic,marine-derived antinomycetes evolved unique metabolism and physiology to survive.Studies on secondary metabolites of marine-derived actinomycetes would have more chances to discover novel antibacterial compounds.Butanone extract of 19 marine-derived actinomycetes fermentation broth were evaluated for antibacterial activity.The strain SCSIO 07395 was found to exhibit potent antibacterial activity and was selected for further studies.The main content of the third chapter was the isolation of secondary metabolite from marine-derived actinomycete SCSIO 07395.This strain was cultured with No.1 medium in large scale.Butanone extract of its fermentation broth was isolated on silica gel column and reverse-phase chromatography or semi-HPLC.Three known compounds were obtained,including hypoxanthine(17),succinic acid(18)and uridine(19).Succinic acid and uridine were co-existing mixture with the ratio 3.5:1.Bioactivity-guided isolation suggested that the antibacterial active ingredient may be avilamicin analogues,which need to be confirmed by further efforts.The final part was the brief summary on natural products isolated from marine actinomycetes in recent years.