Studies On Exploiting Actinomycetic Strain Resources For Drug Screening By A New Approach Using Antibiotic-resistance Mutation Technique

During the new drug screening from microbial strains, absolute majority of pure strains that do not produce bioactive metabolites under a definite condition with given bioassay were destroyed, which make greatly waste. It is very significant how to make good use of the microbial strains that do not produce bioactive metabolites. To discover new bioactive metabolites ,the non-active strains were chosen as target strains for exploiting microbial new strain sources for new drug screening by inducing antibiotic-resistance and chemical mutagens to these strains. we attempted to obtain antibiotic-resistant mutants that can produce anti-tumor active metabolites by using the bioassay。The studies included screening of the wild strains of marine-derived microbes and the anti-tumor activity was assayed for the wild strains, screening of antibiotic-resistant mutants that can produce bioactive metabolites by chemical mutagen combined use of antibiotic-resistance mutation technique, fermentation studies, bioassay-guided fractionation, structural elucidation and preliminary evaluation for anti-tumor activities of pure compounds.1. Isolation of the wild strains of marine-derived microbes and screening of the strains producing metabolites showing anti-tumor activity155 wild strains including 126 actinomycetes and 29 fungi were isolated from the soil samples collected at the tideland of Bohai Bay around Lüjühe in Tanggu district of Tianjin, China and their fermentation were assayed by MTT method using human chronic myelogenous leukaemia K562 cells. The results showed that 20 anti-tumor active strains including 17 actinomycetes and 3 fungi inhibited the proliferation of human chronic myelogenous leukaemia K562 cells with the inhibition rates of more than 20 at the concentration of 100μg/mL. These 20 strains are 15.9% of 155 strains, while samples from 19 actinomycetic strains showed no any inhibitory activity (IR%<6%) even at the concentration of 1000μg/mL. the wild strains obtained that produce metabolites showing anti-tumor activity have provided the microbial strains for further investigation on the bioactive metabolites, at the same time, the microbial strains that do not produce bioactive metabolites might also become of an initial strain resource for the studying on exploiting and using non-active strains to exploit medical strains resource.2. Isolation of antibiotic-resistant mutants and screening of the mutants producing metabolites showing anti-tumor activityThe actinomycetic wild strains L35-1 and L8-5x, the sample from which showed no inhibitory effect on K562 cells with the 2.8% and 4.8% of inhibition rate at 1000μg/mL, was used for the screening of their antibiotic-resistant mutants and 444 mutant strains were obtained. Among the mutant strains obtained, 324 strains of mutation were fermented in liquid media and the crude extracts obtained were subjected to the evaluation of anti-tumor by bioassays using human chronic myelogenous leukaemia K562 cells, samples from 127 strains showed anti-tumor activity with the inhibition rates over 20% at concentration of 1000μg/mL (among them , 14 anti-tumor active mutant strains with the inhibition rates of more than 20% at 1000μg/mL). these active mutant strains showed obvious difference in the morphology and color from their parent strain. The TLC analyses of the fermentation products showed that the secondary metabolites of the active mutant strains were much different from that of the initial strain L35-1 and L8-5x.3. studies on compounds that different from the fermentation of initial strains in the fermentation of active mutantsThe active mutants D2s4-1and csn8-15 were chosen as target strains for obtaining the bioactive metabolites production. the time course experiments for the fermentation of the two strains were carried out. On the basis of the experiments, large-scale fermentation of the two mutants were performed respectively. according the result of TLC, the six compounds that were different from the fermentation of initial strains were isolated from the fermentation of active mutants by the means of modern chromatographic methods , and their structures were elucidated mainly by use of spectroscopic methods. they are 1,9-dicarbomethoxyphenazine (1), N-(2-hydroxyphenyl)-acetamide (2), Pyrrole-2-carboxylic acid (3), Isoflavone daidzein (4), Cyclo-(4-hydroxy-Pro-Leu) (5), Uracil (6). Among them, three compounds showed the anti-tumor activity by MTT method using K562 cells, 1,9-dicarbomethoxyphenazine was a new natural products which was a new anti-tumor agent found occurring in nature for the first timeIn summary, 155 wild strains were isolated from the soil samples collected at the tideland of Bohai Bay and 20 anti-tumor active strains and many non-active strains were obtained by MTT method using human chronic myelogenous leukaemia K562 cells. two non-active strains was used for the screening of their antibiotic-resistant mutants and 444 mutant strains were obtained. Among the mutant strains obtained, 324 strains of mutation were fermented and assayed by bioassays using human chronic myelogenous leukaemia K562 cells, 14 anti-tumor active mutant strains were obtained with the inhibition rates of over 20% at 1000μg/mL.Then we studied metabolites of two active mutation and obtained six compounds, three of which showed anti-tumor activities. These results in our studies mentioned above have been a good demonstration to prove that our new strategy of screening of antibiotic-resistant mutants and mutagen-induced mutations will be practical and successful in exploiting the actinomycete strains that do not produce bioactive metabolites as microbial new strain sources for new drug screening.