Studies On Exploiting Actinomycetic Strain Resources For New Drug Screening By Streptomycin-Resistance Mutation Technigue

When we screen the activity of microorganism, a majority of strains don't show any activity under the available model. The improvement of the productivity of microbial strains is an important field in secondary metabolites from microorganisms. That how to make good use of the non-active microorganisms under the available model is also interesting. It was found that antibiotic productions could be activated dramatically by a certain ribosomal mutation and an idea occurred that bacterial gene expressions may be changed dramatically by modulating the ribosomal proteins or rRNA, which could eventually lead to the activation of inactive (silent) genes. One of the most conventional ways to modulating the ribosome is the introduction of mutations conferring resistance to antibiotics that attack the ribosome. The research focused on exploiting new microbial resources for medicinal uses based on ribosome-engineering.Total 127 microbial strains, including 80 fungi and 47 actinomycetes, were isolated from soil samples collected at the tideland of Bohai Bay around Lüjühe in Tanggu district of Tianjin, China. The anti-tumor activity was assayed by MTT method using K562 cells together with morphological observation of K562 cells under inversed light microscope. Among them, samples from 4 actinomycetic and 4 fungal strains inhibited the proliferation of K562 cells with the inhibition rates over 40% at the 100μg/mL of concentration, while the samples from another 6 actinomycetic strains showed anti-tumor activity with inhibition rates ranging in 20% to 40% at the 100μg/mL concentration. The 80 fungal and 47 actinomycetic strains were isolated from the Bohai marine-environment samples, from which, the 4 fungal and 10 actinomycetic strains that produce metabolites with anti-tumor activity have been obtained. The frequency of obtaining the bioactive metabolite producing strains is obviously higher in actinomycetes than in fungi. These microbial strains including both the bioactive metabolite producing and non-producing strains have provided microbial strain resources for further studies on the bioactive metabolites and on the alteration of metabolic capacity of microbes to produce bioactive metabolites by ribosome-engineering, respectively.A mutation CHS-21101 (streptomycin-resistant) showed stronger cytoclasis bioactivity on K562 cells It was selected to be fermented. The whole fermentation broth was separated by the means of modern chromatographic methods in a bioassay-guided separation manner. From the bioactive part, EtOH extract, tow compounds were isolated by silica gel, Sephadex LH-20 and reverse-phase high performance liquid chromatragraphy. The structures of one compounds were elucidated mainly by use of spectroscopic methods. Among them, two compounds showed the antitumor activities by the use of MTT and flow cytometry using mammalian cancer K562 cells.In summary, Ribosome-engineering technology was stochastically carried out on 2 non-active strains by inducing the antibiotic resistance and 232 mutated strains were obtained. 4 mutated strains, which showed anti-tumor activity with inhibition rates ranging over 40% at the 100μg/mL concentration.This research proves that it is feasible to exploit new microbial resources for medicinal usage by applying the ribosome-engineering technology. This method is economical and simple to be carried out. The silent genes of microorganisms can be activated and more and more metabolites with novel structures can be obtained by using this ribosome-engineering technology.