| Microbial metabolites are the most important sources of new drugs and their leading compounds,among which the secondary metabolites of fungi received much more attention for the riches in chemical diversity. However, most of the wild fungi reveal no relevant biological activity during the screening for bioactivities, resulting in a waste of natural resources. The study on microbial genomics research indicates that fungal genomic typically possess great potential in the products of secondary metabolism. But on ordinary culture conditions, many biosynthetic pathways of secondary metabolism are silenced. In recent years, our group tried to establish a series of simple and effective technical means to activate the silent secondary metabolic pathway of fungi, including random diethyl sulfate(DES) mutagenesis mediated by dimethyl sulfoxide(DMSO), introduce antibiotics-resistance into fungi by dimethyl sulfoxide(DMSO) and introduce antibiotics-resistance into fungi by ultrasound. Now, our group trys to introduce neomycin-resistance into a marinederived Penicillium purpurogenum G59 strain by DMSO, and we have obtained dozens of neomycin-resistant mutants with anti-tumor activity. In order to test whether the neomycin-resistance has been introduced into the mutants successfully, we picked out 3 mutants for neomycin-resistant verification. Fresh spores of both the mutant and parent G59 were treated the same conditions with neomycin and DMSO, finally we found the growth of G59 had been significantly inhibited, but little effect on the growth of mutant. The result indicated that the mutants have received neomycin-resistance already, which may lead to the different in the growth and the metabolisms compared to the parent G59. In our previous work, we have selected out 8 neomycin-resistant mutants with anti-tumor activity during the introduction of neomycin-resistance to Penicillium purpurogenum G59. Then we carried a secondary active screening on the 8 mutants, and four of which maintained stable activities. The HPLC analysis on the metabolites of the 8 mutants and parent G59 shows that there are obviously differences between the original G59 and the mutants. Through the secondary active screening and the HPLC analysis, mutant 4-30 was picked out for its stability on anti-tumor activity and the significant differences in the metabolites compared with parent G59.Fermentations of mutant 4-30 were taken in both liquid and solid medium. During the separation of the fermentation products, bioassay-guided and chemical detecting analysis was used combined with kinds of chromatographic methods. Then we got 5 newly-produced compounds(1-5) from the liquid medium fermentation and newly-produced 4 compounds(6-9) from the liquid medium fermentation. The structure of compounds 1-9 were identified in used of modern spectrum technology and the modified Mosher’s method. Among them, compound 6 was a new structure which belongs to cyclopentachromones(CPCs) that are very rare in nature. The special structural characteristics of compound 6 were the unique sulfur-containied side chain, and the positions of substituents on the cyclopentene ring. The relative configuration at C-2 and C-3 was determined by the coupling constant of C-1 and H-3. Then the absolute configuration of C-2 and C-3 in compound 6 was determined by ECD calculations. And the modified Mosher’s method was used to determine the absolute configuration of C-17. Since compound 6 possess a special structure compared with other cyclopentachromones that were reported, there should be a unique biosynthetic pathway, and a reasonable estimate of biosynthesis pathway was given.In HPLC-PDAD-UV and HPLC-ESI-MS analysis, compounds 1-9 were all detected in the extracts from the liquid and solid medium fermentations of 4-30, but none of them were detected in the control G59 extracts also from the liquid and solid medium fermentations. These results indicated that all 1-9 were newly-produced by the mutant 4-30, through the activation of silent metabolic pathways in the parent strain G59, including PKS, NRPS, by the introduction of neomycin resistance in the mutant.The antitumor activities of compounds 1-9 were tested against human tumor cell lines including K562, HL-60, BGC-823, HeLa and MCF-7 by MTT method. All of the compounds 1-9 showed some inhibition on the tumor cell lines at 100 ?g/mL, and give the inhibition rates(IR%) ranging from 27%~89%. At the same time, positive control 5-Fluorouracil give inhibition rates between 42%~47%. The new compound 6, exhibit an anti-tumor activity with half inhibitory concentration(IC50) of five human tumor cell lines mentioned above ranging from 16~75 μM.In summary, introduction neomycin-resistance into Penicillium purpurogenum G59 strain by DMSO resulted in the generation of neomycin-resistant mutant 4-30 with anti-tumor activity, which expanded our recognition of active metabolites of fungi, especially for cyclopentachromones. At the same time, it verified the feasibility and effectiveness of our method that introduce neomycin resistance into the mutant to active silent pathways in parent strain for discovering new bioactive compounds. And it provides a very important reference for the subsequent scientific researchers.
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