| Secondary metabolites of microorganisms have been considered to be an important source of new drugs and leading compounds. However, of late, the discovery rate of novel bioactive metabolites through traditional cultivation techniques is extremely low, because most gene clusters are not able to be activated by that way. Meanwhile, after bioactive screening, most inactive fungi can not be directly used in the active products research thus results in the waste of fungal strains and investment. So, how to more simply and efficiently regulate the metabolism of these fungal strains to produce active metabolites has become a key point of our research.The antibiotic resistant screening technique related with ribosome engineering has been extensively used in the metabolism function regulation and strains improvement of prokaryotic microorganism such as bacterium and actinomycete. However, this method has not been applied to fungus due to relatively poor susceptibility to ribosomes-targeting drugs and shielding effect of the fungal cell wall. Our approach, using the solvent DMSO to improve the permeability of aminoglycoside antibiotics entering into cell and active the biosynthesis system, can make the fungus to produce newly active metabolites. The obtained mutant strains will be tested against tumor cell line and pathogenic fungus, and the products of the active fungal strains will be separated and identified.Penicillium purpurogenum G59, a marine-derived inactive strain, was chosen as the initial strain. The EtOAc extract of its fermentation products did not show any inhibitory activity against human chronic myelogenous leukaemia K562 cells and two pathogenic fungi at 100μg/mL and 1000μg/mL respectively. By the DMSO-mediated gentamicin-resistance mutation technique, total 181 of gentamicin-resistant (gen) mutants were obtained. Among them, the EtOAc extracts of 9 gen mutants showed inhibitory effect against K562 cell line with the inhibition rates over 30% at the concentration of 100μg/ml. 2 gen mutants showed activie against Candida albicans ATCC 10231 with inhibition zone about 10 mm at 1000μg/mL respectively. Compared with the initial strain, these mutants can produce bioactive metabolites. The above results revealed that the secondary metabolism of the inactive wild-type fungal strains can be altered by DMSO-mediated gentamicin-resistance mutation technique.Two antitumor-active gen mutants 5-1-4 and 2-5-3-1 were selected as target strains to be investigated for their antitumor metabolites. The time-effect of fermentation was investigated. Based on the results, large-scale fermentations of the 2 mutants were performed respectively at the same condition. After dealing with the same post-treatment method, the EtOAc extracts of the 2 mutants were obtained. Then, the bioactive metabolites produced by them were isolated by a bioassay-guided separation procedure with various chromatographic methods, combined with direct comparison with the sample of original strain. Structures of the obtained compounds were identified mainly according to spectroscopic data (MS, 1H-NMR, 13C-NMR, DEPT, COSY, HMQC, HMBC). Theantitumor activity of the EtOAc extract samples and the obtained pure compounds were assayed by MTT method on K562 cells. Four bioactive metabolites 1~4 were isolated from the fermentation products of mutant 5-1-4, and identified as janthinone (1), fructigenine A (2), aspterric acid methyl ester (3) and citrinin (4). Compounds 5~8 were obtained from fermentation product of mutant 2-5-3-1, and identified as ergosterol (5), rugulosuvine A (6), emodin (7) and citreorosein (8). Compounds 1~8 inhibited the proliferation of K562 cells with the inhibition rates of 34.6%, 60.8, 31.7%, 67.1%, 52.8%, 41.2%, 84.6% and 37.1% at 100μg/mL, respectively.In summary, 181 mutants were obtained by selecting on PDA plates using a DMSO-mediated gentamicin-resistant screening technique, including 9 mutants inhibiting the proliferation of K562 cells with the inhibition rates over 30% at the 100μg/mL of the crude extracts, and 2 mutants were bioactive on Candida albicans ATCC 10231 with inhibition zone about 10 mm at 1000μg/paper. Two antitumor-active mutants were chosen as target strains for investigating their antitumor metabolites. Compounds 1~8 were isolated and identified. Their antitumor activities were subjected to preliminary assessment by MTT method.The results in our studies mentioned above prove that our new strategy of altering inactive wild-type fungal strains to bioactive mutants will be practical and successful in exploiting the fungal strains that can not produce bioactive metabolites as new strain sources for new drug screening. |
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