title: Production and secretion of naphthoquinones is mediated by the MFS transporter MFS1 in the entomopathogenic fungus Ophiocordyceps sp. BCC1869.
abstract: Naphthoquinones are deep red polyketide pigments produced by the ant-pathogenic fungus Ophiocordyceps sp. BCC1869. In culture, biosynthesis of these naphthoquinones remains at a low level during the first 20 days and reaches its maximum production level at approximately 50 days. The MFS transporter gene MFS1 was previously identified in Ophiocordyceps sp. BCC1869 from a subtractive EST library between the fungus grown under naphthoquinone-inductive and naphthoquinone-repressive conditions. We cloned and sequenced this transporter gene, which has an open reading frame of 1505 bp and three introns (48, 52, and 58 bp). Phylogenetic analysis showed this MFS transporter was tightly clustered with fungal riboflavin transporters. Functional analysis of this gene was performed by overexpression of MFS1 under the control of a strong, constitutive promoter. We successfully transformed the fungus with this overexpression plasmid using PEG-protoplast transformation, which generated nine transformants per µg of plasmid. RT-PCR indicated that the MFS1 expression level in the overexpressing strains increased 3- to 10-fold compared to the wild type. HPLC analysis of crude extracts of mutants and wild type demonstrated that four naphthoquinone derivatives, erythrostominone, epierythrostominol, deoxyerythrostominone, and deoxyerythrostominol, were the major naphthoquinones produced and excreted in staggering quantities (20- to 2300-fold) in 7-day old liquid cultures by the mutant C7, compared to the wild type. High resolution electrospray ionization mass spectrometry verified mass spectra of these purified metabolites. Three other naphthoquinone derivatives, whose structures have not been identified, were also detected in high amount in the mutant liquid cultures.
authors.affiliation: Bioresources Technology Unit, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Thailand Science Park, Thanon Phahonyothin, Tambon Khlong Nueng, Amphoe Khlong Luang, Pathumthani, 12120, Thailand.|Department of Genetics, Faculty of Science, Kasetsart University, Bangkok, 10900, Thailand.
authors.forename: Pratoomporn
authors.initials: P
authors.lastname: Khaokhajorn
authors.affiliation: Department of Genetics, Faculty of Science, Kasetsart University, Bangkok, 10900, Thailand.
authors.forename: Sompid
authors.initials: S
authors.lastname: Samipak
authors.affiliation: Bioresources Technology Unit, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Thailand Science Park, Thanon Phahonyothin, Tambon Khlong Nueng, Amphoe Khlong Luang, Pathumthani, 12120, Thailand.
authors.forename: Sutichai
authors.initials: S
authors.lastname: Nithithanasilp
authors.affiliation: Bioresources Technology Unit, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Thailand Science Park, Thanon Phahonyothin, Tambon Khlong Nueng, Amphoe Khlong Luang, Pathumthani, 12120, Thailand.|School of Bioresources and Technology, King Mongkut's University of Technology Thonburi, Bangkok, 10140, Thailand.
authors.forename: Morakot
authors.initials: M
authors.lastname: Tanticharoen
authors.affiliation: Bioresources Technology Unit, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Thailand Science Park, Thanon Phahonyothin, Tambon Khlong Nueng, Amphoe Khlong Luang, Pathumthani, 12120, Thailand. alongkorn@biotec.or.th.
authors.forename: Alongkorn
authors.initials: A
authors.lastname: Amnuaykanjanasin
mesh_terms: D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D020224:Expressed Sequence Tags; D005820:Genetic Testing; D006999:Hypocreales / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009285:Naphthoquinones / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D016366:Open Reading Frames; D010802:Phylogeny; D010860:Pigments, Biological / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D004271:DNA, Fungal; D026901:Membrane Transport Proteins; D009285:Naphthoquinones; D010860:Pigments, Biological
keywords: Erythrostominone; MFS transporter; Naphthoquinone; Ophiocordyceps; Overexpression; Secretion