Activation of the silent secondary metabolite production by introducing neomycin-resistance in a marine-derived Penicillium purpurogenum G59.
Introduction of neomycin-resistance into a marine-derived, wild-type Penicillium purpurogenum G59 resulted in activation of silent biosynthetic pathways for the secondary metabolite production. Upon treatment of G59 spores with neomycin and dimethyl sulfoxide (DMSO), a total of 56 mutants were obtained by single colony isolation. The acquired resistance of mutants to neomycin was testified by the resistance test. In contrast to the G59 strain, the EtOAc extracts of 28 mutants inhibited the human cancer K562 cells, indicating that the 28 mutants have acquired the capability to produce bioactive metabolites. HPLC-photodiode array detector (PDAD)-UV and HPLC-electron spray ionization (ESI)-MS analyses further indicated that diverse secondary metabolites have been newly produced in the bioactive mutant extracts. Followed isolation and characterization demonstrated that five bioactive secondary metabolites, curvularin (1), citrinin (2), penicitrinone A (3), erythro-23-O-methylneocyclocitrinol (4) and 22E-7α-methoxy-5α, 6α-epoxyergosta-8(14),22-dien-3β-ol (5), were newly produced by a mutant, 4-30, compared to the G59 strain. All 1-5 were also not yet found in the secondary metabolites of other wild type P. purpurogenum strains. Compounds 1-5 inhibited human cancer K562, HL-60, HeLa and BGC-823 cells to varying extents. Both present bioassays and chemical investigations demonstrated that the introduction of neomycin-resistance into the marine-derived fungal G59 strain could activate silent secondary metabolite production. The present work not only extended the previous DMSO-mediated method for introducing drug-resistance in fungi both in DMSO concentrations and antibiotics, but also additionally exemplified effectiveness of this method for activating silent fungal secondary metabolites. This method could be applied to other fungal isolates to elicit their metabolic potentials to investigate secondary metabolites from silent biosynthetic pathways.
State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China. wucj2009@163.com.|Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China. wucj2009@163.com.
Chang-Jing
CJ
Wu
State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China. yileamms@126.com.
Le
L
Yi
State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China. cuicb@126.com.|Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China. cuicb@126.com.
Cheng-Bin
CB
Cui
State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China. sdrlcw@126.com.
Chang-Wei
CW
Li
State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China. ammswang@126.com.
Nan
N
Wang
State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China. nullah876@sina.com.
Xiao
X
Han
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000903:Antibiotics, Antineoplastic / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D059001:Aquatic Organisms / Q000187:drug effects* / Q000302:isolation & purification / Q000502:physiology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002681:China; D003080:Cold Temperature; D045424:Complex Mixtures / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D055808:Drug Discovery / Q000379:methods*; D025141:Drug Resistance, Fungal / Q000187:drug effects; D005285:Fermentation; D006801:Humans; D015394:Molecular Structure; D009153:Mutagens / Q000494:pharmacology; D009355:Neomycin / Q000494:pharmacology; D010140:Pacific Ocean; D010407:Penicillium / Q000187:drug effects* / Q000302:isolation & purification / Q000502:physiology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D064210:Secondary Metabolism / Q000187:drug effects*; D012988:Soil Microbiology; D013172:Spores, Fungal / Q000187:drug effects / Q000302:isolation & purification / Q000502:physiology; D053833:Wetlands
D000900:Anti-Bacterial Agents; D000903:Antibiotics, Antineoplastic; D045424:Complex Mixtures; D009153:Mutagens; D011500:Protein Synthesis Inhibitors; D009355:Neomycin; D004121:Dimethyl Sulfoxide