Study On The High-yielding Strain Improvement And Culture Condition Optimization For Deacetylmycoepoxydiene

Natural products with "drug-likeness and biological friendliness",are important resources of new drugs and new drug lead compounds,play an irreplaceable role in the drug development process.With the in-depth study,it's more and more difficult to find and develop new drugs from terrestrial organisms,in order to overcome this bottleneck,marine, which is more complicated than terrestrial in the ecology and species,has been the research topic.Marine fungi,as an important member of the marine habitats,during adapting to oceanic ecosystem,has a special pathway,can generate unique structure,novel skeletons and diverse bioactivity,have attrached more and more attention.Mangrove plants as important new resources for potential pharmaceutical because of their ecosystems that straddled the land and the sea,from freshwater to seawater,often with distinct microorganism species,have gradually attracted more and more attention of natural products chemists.Deacetylmycoepoxydiene(DAM),a second metabolic product from the endophytic fungul A123,containing an oxygen-bridged cyclooctadiene,is novel and represents a new class of fungal metabolites.The IC50 of Raji cell line is 3.0μg/mL.But the yield in the wild-type is too low,about only 0.8mg/L, hinder the development DAM.This paper established the antimicrobial spectrum of DAM,based on the apectrum,a bioassay method for quantitative determination of DAM was developed using the strain Bacillus subtillis,as the indicator organismBased on this technique,a convenient,low-cost and time-saving agar-plug method coupled with TLC and HPLC analysis was constructed for screening of mutants.This method was successfully applied in the high throughput screening procedure.Through the protoplast mutation technology and high-throughput screening methods,after four round mutagenesis,an excellent mutant A818 was selected from 4000 mutants,with the yield of DAM increased to 198 mg/ L,nearly 250 times than starting strain A123.This paper successfully solved the problem of inoculation in liquid fermentation,and through the screening of kinds of media and the components of media,settled the difficult that DAM couldn't produce in liqiud fermentation.After single-factor optimization,four key factors with strong impact on DAM, namely,glucose,potato,vitamin B1 and mannitol were selected;centre composition design of response surface method was applied to optimizate the concentration of the four factors.The optimization level of each ingredients were:glucose 87g/L,potato 170 g/L,vitamin B1 5 mg/L and mannitol 14 g/L, and the yield of DAM predicted in liquid mediem reached 221.59 mg/L.After the quadratic regression model equation,the yield of DAM was 201.39 mg/L.