Screening of Marine Streptomyces Producing Anti-complement Agents and Studies on the Bioactive Metabolites
Author:Xu Xiao Na
Supervisor:bai feng wu zhao xin qing
The human complement system plays an important role in host immune defense against infection and in the clearance of antigen-antibody complexes from the bloodstream.However,when it is excessively activated,a variety of diseases such as rheumatoid arthritis,Alzheimer’s disease,and acute respiratory distress syndrome(ARDS)may be induced.The anti-complement agents derived from natural products have the advantages that they can be produced sustainably and can be directly digested and absorbed.Therefore,it is of importance to develop natural products as anti-complementary agents.Due to the ease of cultivation and genetic manipulation,as well as convenience in quality control,anti-complementary compounds from microorganisms have great application potential.However,so far studies on microbial-derived anti-complement agents are still very limited.Marine actinomycetes have the potential to produce complex and diverse secondary metabolites which have numerous biological activities,such as antibacterial,antifungal,antiviral,cytotoxic and antitumor activities.Therefore,marine actinomycetes have become sources of new drugs.However,studies on anti-complement substances from marine actinomycetes are only at the preliminary stage.In this thesis,marine actinomycetes that produce anti-complement agents were screened,and two active strains,namely,Streptomyces xinghaiensis NRRL B24674T(shorted as S187)and Slreptomyces sp.DUT11(shorted as DUT11)were focused.The results obtained are as below:(1)Anti-complement activities of the fermentation broth of 42 marine streptomycetes,which were isolated from the sediment samples about 10 meters below sea level from Dalian Xinghai Bay and Xiaoping Island were tested.It was found that the fermentation broth of seven marine actinomycetes had good anti-complement activity.At the same time,the 16S rRNA sequence analysis of these strains of actinomycetes showed that these marine actinomycetes all belonged to different species.These results imply that diverse marine streptomycetes strains could produce anti-complement agents.(2)The active metabolites of the strain S187 that shows good potential to produce anti-complement agents were further investigated.Firstly,seven different fermentation mediums were selected to optimize the fermentation conditions of strain S187,and the M12 medium was determined as the best medium.The medium was used as the basic medium to optimize the fermentation conditions.The optimum fermentation conditions were as follows:initial pH was 7.0,temperature was 28 ℃,and culture time was 6 days.Then,the synthesis of the secondary metabolites of the wild-type S187 strain and the mutant strain △sinH-P3 with the disappearance of anti-complement activity was compared.It was found that there were multiple compound peak differences,and combined with HPLC-Mass Spectometry analysis,it was possible to preliminarily identify the anti-complement compounds.Finally,the crude extract of the strain S187 was isolated and purified by anti-complement activity tracking method.The secondary metabolites were purified by using different chromatography techniques including normal and reversed phase silica gel column chromatography,sephadex LH-20 column chromatography,thin layer preparation chromatography and high performance liquid chromatography.Four compounds were purified.On the basis of chemical reaction and spectral data analysis(LC-MS,1H-NMR,13C-NMR)and comparison with related literatures,the chemical compounds 1-4 were seperately elucidated as:2-pyrrolic acid,p-hydroxybenzoic acid,o-hydroxybenzonitrile and anthranilic acid.These four compounds all showed anti-complement activity,and the CH50 was 2.0 ± 0.3,0.8 ± 0.1,2.4 ± 0.5 and 2.5±0.6 mmol/L,respectively.(3)The strain DUT11 which produces relatively high anti-complement activity was further investigated.The draft genome sequences of strain DUT11 were obtained,which were used to guide the analysis of the active metabolites.Tunicamycin gene cluster was detected in the genome of strain DUT11,which promotes examination of anti-complement activity of tunicamycin.When fermentation broth of strain DUT11 was analyzed,tunicamycin was detected in both the mycelia and the fermentation broth.Then the fermentation medium was optimized,and large scale fermentation was performed,and the crude extracts were separated and purified.The active secondary metabolites of the strain DUT11 were isolated by anti-complement activity tracking method.The secondary metabolites were purified by using different chromatography techniques including normal and reversed phase silica gel column chromatography,sephadex LH-20 column chromatography,thin layer preparation chromatography,high performance liquid chromatography and re-crystallization.Finally,nine compounds were purified.On the basis of chemical reaction and spectral data analysis(LC-MS,1H-NMR,13C-NMR)and comparison with related literatures,the chemical compounds 1-9 were seperately elucidated as:3-indolecarboxylic acid,p-hydroxybenzoic acid,2-pyrrolic acid,tunicamycin Ⅰ,Ⅱ,Ⅴ,Ⅶ,Ⅹ and nonactin.In addition to nonactin,the other eight compounds have good anti-complement activity in the anti-complement activity test.Among them,tunicamycin X showed the best anti-complement activity with a CH50 of 0.041 ± 0.03 mg/mL.(4)The optimal conditions for production of anti-complement substances and tunicamycins from strain DUT11 were further studied.The strain DUT11 can grow at a NaCl concentration up to 10%,therefore the strain can be regarded as medium halophilic bacteria.When the NaCl concentration is 3%,the yield of tunicamycin is the highest,which is 30.78 mg/L.In contrast,there was no significant change in the anti-complement activity when the concentration of NaCl was 0-5%.Secondly,seven different media were selected to optimize the culture of tunicamycin in strain DUT11,and M33 medium was used as the basic medium.Finally,optimization of tunicamycin was performed by response surface optimization.The optimal yield of tunicamycin was determined according to the optimization results.The optimized conditions are:soluble starch concentration(A),soybean powder concentration(B)and yeast powder concentration(C)49.97,24.88 and 4.15 g/L,respectively,and the tunicamycin concentration was 57.03 mg/L.On the other hand,the optimal conditions for anti-complement agents were:A,B and C were 47.56,18.00 and 3.92 g/L,respectively.The different optimal conditions suggest that in addition to tunicamycin,other anti-complement agents were also produced by the strain DUT11.The results in this thesis demonstrated that many marine actinomycetes can be used to produce anti-complement agents.The studies on the anti-complementagents of S.xinghaiensis S187 and Streptomyces sp.DUT11 provided basis for further development of anti-complement active substances from microbial sources,as well as efficient and economic production of anti-complement compounds.