Optimization And Breeding Of Indolocarbazole Alkaloid Producing Strains

This article adopts the method of combination of biological and chemical tostudy the staurosporine-producing bacteria strains, including Strains of thehigh-yield strains, High-yield strains of the optimization of culture conditions andmedium composition optimization, mutagenic breeding of high yield strain,high-yield strain plasmid verified. In our laboratory strains of bacteria depositedlibrary T1-28as the starting strain after UV mutagenesis, UV-LiCl mutagenesis,nitrous repeatedly mutagenesis, with the staurosporine production of highperformance liquid chromatography as an indicator to screen high-yield strains, onthe basis of serially passage five generations, measured staurosporine production,stability testing mutagenic strains. Eventually screened stable genetic characteristicsof two strains of high-yield strains TUV10-5and TN20-12, its production ofstaurosporine improved912.71%and882.31%than the original strain.A study of media regulation were based on the strain L25-1, which wasscreened from the marine Streptomyces YB-24isolated from north pole, we chooseof six different media, found in ISP5medium, the strain produces the greatestamount of staurosporine. Experiments shows that in fermentation of high-yieldL25-1, the product of staurosporine in mycelium was higher than that in the liquid.Then compared the two different medium, the ISP5medium and the improved gauseone medium. We found that the different elements between the two mediums areglycerol and L-asparagines, and found that both of them coud influence theproduction of staurosporine, but not as good as when used in combination high yield. When liquid culture, the medium added looked like no difference with improvedgause one medium, but the medium added L-asparagines has undergone significantchanges, the mycelium shows a light green. There was no obvious differencebetween the three mediums in solid medium.The high-yield strain L25-1was treated by UV mutagenesis, UV LiClmutagenesis, nitrous repeatedly mutagenesis, with the staurosporine production ofhigh performance liquid chromatography as an indicator to screen high-yield strains,screened a high-yielding strain5L-N15-1, its production of staurosporine increasedby91.31%. Optimization of protoplast preparation and regeneration conditions, afterthe orthogonal experiment to determine the optimal combination is: glycine1.0%,sucrose0.3M, enzyme concentration2mg/mL and temperature37℃. The L25-1fusion and actinomycetes UV2m-5, H10-5and T1-28, screened a strain fusionstrains, production increased by75.51%.Treated the strain L25-1with high temperature and acridine orange to eliminateits plasmid, after elimination, after three consecutive Strains stabilized culture,strains were treated Pyricularia test activity and by high performance liquidchromatography staurosporine production testing, the plasmid eliminate strain stillinhibit the activity of Pyricularia oryzae, and the HPLC test results also show thatthere are still fermented products staurosporine generation, thus determined, inProducing Strains L25-1, and with staurosporine not produce plasmid genesinvolved.