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HONDA, Yoichi

KUWAHARA, Masaaki

MOUE, Manabu

WATANABE, Takashi

Kyoto University Research Information Repository

Title<Preliminary>Genetic Transformation of white Rot FungusPleurotus ostreatus to Carboxin Resistance usingElectroporationAuthor(s)MOUE, Manabu; HONDA, Yoichi; WATANABE, Takashi;KUWAHARA, MasaakiCitationWood research : bulletin of the Wood Research Institute KyotoUniversity (2002), 89: 17-18Issue Date2002-09-30URL http://hdl.handle.net/2433/53128RightType Departmental Bulletin PaperTextversionpublisherKyoto UniversityPreliminaryGenetic Transformation of white Rot FungusPleurotus ostreatus to Carboxin Resistanceusing Electroporation*lManabu MOUE*2, Yoichi HONDA*2,2 M k' K *2,3Takashi WATANABE* and asaa I UWAHARA(Received May 31, 2002)Keywords: Pleurotus ostreatus, white rot fungi, transformation, electroporationIntroductionWhite rot basidiomycetes, such as Phanerochaetechrysosporium and Pleurotus ostreatus, are known to be able todegrade plant cell wall lignin extensively. E~tracel.lul~renzymes secreted by these fungi, includmg lIgnInperoxidase (LiP), manganese peroxidase (MnP) andlaccase (Lac) have been demonstrated to be major, I' . b' d d' t 1,2) Thecomponents of the Ignm 10 egra atIOn sys ems .fungal lignin degrading systems are also the focus ofconsiderable research attention because of their ability todegrade a variety of organic pollutants including PCBs anddioxins3,4) .Although expression of genes encoding ligninolyticenzymes has been studied using various host organisms,including Esherichia coli, yeast, Aspergillus spp, and Bacurovirus system, recombinant enzymes obtained with theseheterologous systems had unusual properties such aselevated molecular wei9"h ts, requirements of re-folding andlow specific activities5- ). A homologous gene expressionsystem can provide an enzyme in both active and nativeform, which makes it possible to characterize the enzymeproperties and to elucidate its physiological role in thebiological functions. Recently, we have developed aDNA-mediated transformation system using a drugresistant marker gene to a systemic fungicide, carboxin.And a homologous gene expression of P. ostreatus MnP3•• i" 8)isozyme was achIeved USIng the translormatIOn systemBesides its potential as an effective producer of MnPenzyme, the MnP-overproducing transformants havepossibilities for advantages in mycelial treatment of ligninor aromatic pollutants.. While molecular breeding by genetic transformationitself is a powerful tool in investigation and development ofspecific functions of wood rot fungi, to achieve a g~netargeting or functional cloning, new transformatIOnprotocol wi th even higher transformation efficiency shouldbe developed. Here we reprot on DNA-mediated trans-*' A part of this work was presented at the 5 th Annual meetingof the Japan Society of Mushroom Science andBiotechnology in Kyoto, July, 2001.*2 Laboratory of Biomass Conversion, Wood ResearchInstitute, Kyoto University, Uji, Kyoto 611-0011, Japan.*3 Present address: Institute of Wood Technology, AkitaPrefectural University, Noshiro, Akita 016-0876, Japan.17formation of P. ostreatus using electroporation procedure.Materials and MethodsP. ostreatus dikaryotic strain #261 (ATCC 66376)was usedas a host strain. Protoplasts were prepared by digestingliquid-cultured mycelium with a lysing enzyme solutioncontaining 0.1 % protoplasting enzyme (MushroomExperimental Station, Horst, The Netherlands), 0.5%Zymolyase 20 T (SEIKAGAKU Corp., Tokyo), and 0.2%Chitinase (Sigma St Louis, MO, USA). Protoplasts, , 9) .were mixed with 1-10 pg ofpTMI DNA In SMYM (I %sucrose, I % malt extract, 0.4% yeast extract and 0.5 Mmannitol) liquid medium. Electroporation experimentswere conducted on SMYM-suspended protoplasts usingGene Pulser II (Bio-Rad Laboratories, Richmond, CA,USA). Following delivery of the electric pulse, thesuspension was added to 5 ml of SMYM liquid mediumand incubated at 28°C for 2-5 days prior to plating onSMYM agar plates containing I pg/ml of carboxin(AccuStandard Inc., New Haven, CT, USA). Drugresistant colonies were subcultured individually on PDA(potato-dextrose-agar, Nissui, Tokyo) plate containing Ipg/ml of carboxin for the secondly screening. Detectionof the introduced sequence in the drug resistant strains wascarried out by specific amplification of the CbxR markersequence with PCR. The primers used in the PCR wereMI3 (5'-GTAAAACGACGGCCAGT-3') and SDH7 (5'-CACACA AATCATT GAACC-3'). Each PCR cycleTable 1. Number of the drug-resistant colonies.Exp. Protoplasts Cuvette Voltage r No. of(X 108 ) gap (cm) (V) (msec) coloniesI 1.0 0.4 1,020 6.12- I2 2.0 0.4 1,020 7.74 123 1.6 0.4 1,030 7.74 274 4.0 0.4 1,030 6.46 I5 4.0 0.2 490 2.68 I6 1.5 0.2 490 3.28 27 2.0 0.2 480 3.20 28 1.3 0.4 500 3.98 29 1.2 0.4 500 3.14 210 3.2 0.4 1,030 7.52 6II 3.2 0.4 2,030 5.72 I12 3.2 0.4 1,510 6.40 2r= capacitance X resistance.WOOD RESEARCH No. 89 (2002)Results and discussionconsisted of template denaturation at 98°C for 30 sec,primer annealing at 52°C for I minm and DNA extensionat 72°C for I min. After 25 cycles, an additional extensionstep of 10 min at 72°C was included. All the PCR-amplified fragments were analysed on a 0.8% agarose gel.M :. A-EcoT141 digestFig.' 1. Detection of CbxR sequence in the transformantsby specific amplification with PeR. Lanes are:M, .EcoT14I-digestd lambda phage DNA as amo1ecu1arstanderd; wt, wild-type #261 strain asa negative control; lane 1-6, transformants.Bands wi.th expected size (1.3 kb) were indicatedby an arrow.PDA plates containg the same concentration of the drug.All the isolated colonied showed continuous growth in thepresence of carboxin suggesting that they were nottransient transformants with unstable drug resistance.The presence of the introduced DNA sequence waschecked by PCR using ~rimers which specifically annealsto the CbxR construct) (Fig. I)., No fragments wereamplified when genomic DNA extracted from wild typehost strain was used as a template (lane wt). Whereasfragments of the expected size (1.3 kb) were amplifiedwhen DNA from the drug resistant strains were used as atemple (lane 1-6). These results indicated that the CbxRsequence was introduced and maintained in the drugresistant strains. Preliminary results of Southern blotanalysis suggested that the CbxR 'DNA sequence wasintegrated ectopically into the chromosomal DNA withmultiple copy nurnbers in these drug resistant strains (datanot shown).It was demonstrated that electroporation method is analternative procedure for transformation of P. ostreatus tocarboxin resistant, however, its efficiency is as high as thatobtained by transformation of the PEG/CaCI2 method.References1) M.H. GOLD and M. ALIC: Microbiol.Rev., 57, 605-622(1993).2) D. CULLEN: j. Biotechnol., 53(2':"'3), 273-289 (1997);3) J.A. BUMPUS, M: TIEN, D. WRIGHT and S.D. AUST: ScienCe228, 1434-1436 (1985).4) K. VALLI, H. WARIISHI and M.H. GOLD: J. Bacteriol.,174(7), 2131-2137 (1992).-5) R.E. WHITWAM, I.G. GAZARIAN and M. TIEN: Biochem.Biophys. Res. Commun., 216(3), 1013-1017 (1995).6) P. STEWART, RE. WHITWAM, P.J. KERSTEN, D. CULLEN andM. TIEN: Appl. Environ. Microbio!., 62(3), 860-864 (1996).7) F.J., RUIZ-DUENAS, M.J. MARTINEZ and A.T. MARTINEZ:Appl. Environ. Microbiol., 65(10), 4705-4707 (1999). .8) T. IRIE, Y. HONDA, T. WATANABE and M. KUWAHARA: Appl.Microbiol. Biotechnol., 55(5), 566-570 (2001).9) Y. HONDA, T. MATSUYAMA, T. IRIE, T. WATANABE andM.KUWAHARA: Curro Genet., 37(3), 209-212 (2000).transformants23456M wtElectroporation experiments were conducted on severalconditions summerized in Table I, and in eachexperiments, germinating colonies were observed on thefirst screening plates containing I pg/ml of carboxin.Total 59 drug resistant colonies were obtained and, in thebest condition, 27 drug resistant colonies were obtained atfield strength of 2.58 kV/cm and time constant of 7.74msec. These colonies were isolated and subcultured on- 18-

<Preliminary>Genetic Transformation of white Rot Fungus Pleurotus ostreatus to Carboxin Resistance using Electroporation

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<Preliminary>Genetic Transformation of white Rot Fungus Pleurotus ostreatus to Carboxin Resistance using Electroporation

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