Abstract
A new ultrasound-mediated approach has been developed to introduce neomycin-resistance to activate silent pathways for secondary metabolite production in a bio-inactive, deep-sea fungus, Aspergillus versicolor ZBY-3. Upon treatment of the ZBY-3 spores with a high concentration of neomycin by proper ultrasound irradiation, a total of 30 mutants were obtained by single colony isolation. The acquired resistance of the mutants to neomycin was confirmed by a resistance test. In contrast to the ZBY-3 strain, the EtOAc extracts of 22 of the 30 mutants inhibited the human cancer K562 cells, indicating that these mutants acquired a capability to produce antitumor metabolites. HPLC-photodiode array detector (PDAD)-UV and HPLC-electron spray ionization (ESI)-MS analyses of the EtOAc extracts of seven bioactive mutants and the ZBY-3 strain indicated that diverse secondary metabolites have been newly produced in the mutant extracts in contrast to the ZBY-3 extract. The followed isolation and characterization demonstrated that six metabolites, cyclo(d-Pro-d-Phe) (1), cyclo(d-Tyr-d-Pro) (2), phenethyl 5-oxo-l-prolinate (3), cyclo(l-Ile-l-Pro) (4), cyclo(l-Leu-l-Pro) (5) and 3β,5α,9α-trihydroxy-(22E,24R)-ergosta-7,22-dien-6-one (6), were newly produced by the mutant u2n2h3-3 compared to the parent ZBY-3 strain. Compound 3 was a new compound; 2 was isolated from a natural source for the first time, and all of these compounds were also not yet found in the metabolites of other A. versicolor strains. Compounds 1–6 inhibited the K562 cells, with inhibition rates of 54.6% (1), 72.9% (2), 23.5% (3), 29.6% (4), 30.9% (5) and 51.1% (6) at 100 μg/mL, and inhibited also other human cancer HL-60, BGC-823 and HeLa cells, to some extent. The present study demonstrated the effectiveness of the ultrasound-mediated approach to activate silent metabolite production in fungi by introducing acquired resistance to aminoglycosides and its potential for discovering new compounds from silent fungal metabolic pathways. This approach could be applied to elicit the metabolic potentials of other fungal isolates to discover new compounds from cryptic secondary metabolites.
Highlights
Marine microbial natural products, especially those derived from fungi, have come to be rich and promising sources of novel compounds with biological and pharmaceutical properties, providing a number of drug leads and other healthcare ingredients [1,2,3,4,5,6]
According to our previous experience in the treatment of P. purpurogenum G59 spores by gentamicin in aqueous dimethyl sulfoxide (DMSO) at 4 °C to introduce drug resistance [27], and further, in view of the documented effect of ultrasound on the membrane permeability [42,43,44,45,46,47,48,49], we tested the introduction of drug resistance in A. versicolor ZBY-3 by neomycin treatment of ZBY-3 spores at 4 °C for different times after ultrasound irradiation of the spores in the presence of neomycin at different concentrations
200–800 mg/mL neomycin at 4 °C did not inhibit strain growth on potato dextrose agar (PDA) plates at 28 °C
Summary
Especially those derived from fungi, have come to be rich and promising sources of novel compounds with biological and pharmaceutical properties, providing a number of drug leads and other healthcare ingredients [1,2,3,4,5,6]. A great number of novel and bioactive compounds have been increasingly discovered from marine microbial sources [1,2,3,4,5,6,7,8,9,10,11], the great biosynthetic potential of microbial isolates, including the fungi, has so far not been fully elicited, because their major biosynthetic pathways that produce secondary metabolites are silent in laboratory culture conditions [12,13,14,15,16]. Ribosome engineering [22,23] has been able to activate silent pathways by introducing drug-resistance mutations in bacteria to discover new antibacterial agents [24]. This strategy has been recently extended to fungi [27]. In spite of the strategies mentioned, additional simple approaches that are suitable for use by microbial chemists are still needed for research work on secondary metabolites from silent pathways
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