Abstract
A new approach to activate silent gene clusters for dormant secondary metabolite production has been developed by introducing gentamicin-resistance to an originally inactive, marine-derived fungal strain Penicillium purpurogenum G59. Upon treatment of the G59 spores with a high concentration of gentamicin in aqueous DMSO, a total of 181 mutants were obtained by single colony isolation. In contrast to the strain G59, the EtOAc extracts of nine mutant cultures showed inhibitory effects on K562 cells, indicating that the nine mutants had acquired capability to produce antitumor metabolites. This was evidenced by TLC and HPLC analysis of EtOAc extracts of G59 and the nine mutants. Further isolation and characterization demonstrated that four antitumor secondary metabolites, janthinone (1), fructigenine A (2), aspterric acid methyl ester (3) and citrinin (4), were newly produced by mutant 5-1-4 compared to the parent strain G59, and which were also not found in the secondary metabolites of other Penicillium purpurogenum strains. However, Compounds 1–4 inhibited the proliferation of K562 cells with inhibition rates of 34.6% (1), 60.8% (2), 31.7% (3) and 67.1% (4) at 100 μg/mL, respectively. The present study demonstrated the effectiveness of a simple, yet practical approach to activate the production of dormant fungal secondary metabolites by introducing acquired resistance to aminoglycoside antibiotics, which could be applied to the studies for eliciting dormant metabolic potential of fungi to obtain cryptic secondary metabolites.
Highlights
Natural products are important sources of new drugs and drug leads [1,2,3,4]
The fungus Penicillium purpurogenum G59 is insensitive to gentamicin
The above results have demonstrated that introduction of gentamicin-resistance by a high concentration of gentamicin in aqueous DMSO effectively activates dormant secondary metabolite production in strain G59, which could be useful in fungal metabolite studies as a simple, yet practical approach to elicit the biosynthetic potential of fungi in order to increase chemical diversity for drug screening
Summary
Natural products are important sources of new drugs and drug leads [1,2,3,4]. It has been reported that approximately 50% of new drugs approved from 1981 to 2006 are natural molecules or derived therefrom [4]. The chemical epigenetics approach manipulates epigenetic processes by administering small-molecule epigenetic modifiers to fungal cultures to induce silent biosynthetic pathway transcription to activate the production of cryptic metabolites, including new compounds. Way to activate cryptic biosynthesis pathways of secondary metabolites by introducing drug-resistant mutation in bacteria [26,49] Application of this approach to fungi has not been reported. We have undertaken a research work to develop a similar mutation-based new approach to activate silent gene clusters to awake dormant secondary metabolite production in fungi. Using these strains, we have established a new approach to activate dormant secondary metabolite production by introducing antibiotic-resistance to obtain antitumor secondary metabolites. We report in detail recent results of our research on Penicillium purpurogenum G59 using an aminoglycoside antibiotic, gentamicin
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