Genetic transformation and mutant isolation in Ganoderma lucidum by ...

FEMS Microbiology Letters 233 (2004) 201–204 www.fems-microbiology.org

Genetic transformation and mutant isolation in Ganoderma lucidum by restriction enzyme-mediated integration Sunkyung Kim a, Jaemahn Song b, Hyoung T. Choi a

a,*

Microbial Physiology Laboratory, Division of Life Sciences, Kangwon National University, Chunchon 200-701, South Korea b CabioTech, Songnae Venture Building B-401, Songnae 2-dong 558, Sosa-Gu, Buchon, South Korea Received 15 January 2004; received in revised form 11 February 2004; accepted 16 February 2004 First published online 27 February 2004

Abstract A white-rot basidiomycete Ganoderma lucidum has long been used as a medicinal mushroom in Asia, and it has an array of enzymes important for wood degrading activity. There have been many reports about the ingredients which show health aiding effects. In order to analyze gene functions and introduce foreign genes into this fungus, genetic transformation is required. We have successfully transformed G. lucidum to geneticin resistance using pJS205-1 which has the antibiotic resistance genes against geneticin and phosphinothricin. Many different mutants have been generated during the transformation by restriction enzyme mediated integration, and the transformation yield was 4–17 transformants (lg plasmid DNA)
1 . The plasmid was integrated stably into the recipient chromosome, which was confirmed by PCR with the plasmid-specific primers. Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Fungal transformation; Geneticin; Ganoderma lucidum

1. Introductizon Ganoderma lucidum is very interesting organism since it has long been used as the health aiding foodstuff, and it is also a white-rot basidiomycete which can degrade many recalcitrant substances. Trametes versicolor, another medicinal mushroom, has been investigated not only for its pharmaceutical effect [1] but for genes related in lignin-degrading enzymes [2,3] and for genetic transformation to hygromycin B resistance gene [4]. However, G. lucidum has been primarily investigated for its pharmaceutical components [5,6], while other basic research has been poorly reported. There are two papers on the biochemistry of G. lucidum; induction and characterization of lignin-modifying enzymes [7], and biochemical characterization of laccase isozymes [8] have been reported. *

Corresponding author. Tel.: +82-33-250-8543; fax: +82-33-2414627. E-mail address: [email protected] (H.T. Choi).

Genetic transformation of basidiomycetes has been reported in Agaricus bisporus [9], Pleurotus ostreatus [10], and Coprinellus ( ¼ Coprinus) congregatus [11]. There are many different kinds of selectable markers for transformants, and antibiotic resistance genes have been widely used. Antibiotics such as hygromycin B [4,12], geneticin [13] and phosphinothricin [11] show good growth inhibition against many fungi. In the transformation of filamentous fungi, the transforming vector plasmid is integrated into the chromosomal DNA of recipient cell usually at random sites. Many mutants can be generated during the genetic transformation by restriction enzyme mediated integration (REMI). Furthermore, REMI is useful in tagging the gene(s) related in the mutant characteristics [14]. Therefore REMI is a very good method to clone genes from various mutants when there are no mutant strains available in an organism. We have constructed a transforming vector which confers resistance against geneticin and phosphinothricin, and used successfully in the transformation of G. lucidum isolated in Korea by the REMI.

0378-1097/$22.00 Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2004.02.010

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2. Materials and methods 2.1. Fungal strains and vector plasmid construction Ganoderma lucidum ASI 7071-9 (monokaryon) was obtained from National Institute of Agricultural Science and Technology (NIAST) in Korea. The monokaryon was used as a recipient cell in the transformation experiment, and this strain was maintained on SGCM agar slant [8]. Transforming vector pJS205-1 was constructed as follows: pBARGPE1 which had phosphi-nothricin resistance gene (bar) was cut with SmaI, and geneticin resistance gene (kan) (PvuII and EcoRI fragment) was inserted after filling the sticky end. This generated pJS205-1 which was about 6.5 kb in length. 2.2. Transformation by REMI Ganoderma lucidum monokaryon was grown in the minimal liquid medium [11] in a shaking incubator at 30 °C for 5 days. The whole cells were ground with a Waring blender and the homogenate was transferred (10% v/v) to fresh minimal medium for 3 days. The above step was repeated once and the fungal cells were grown for one day. Protoplast generation and genetic transformation by REMI were carried out by following LeemÕs protocol [11] except using three different restriction enzymes during the transformation as follows: EcoRV, NotI and XhoI, which had one restriction site, respectively. The transformants were selected on the minimal medium containing geneticin (200 lg ml
1 ) during 3–10 days after regeneration. 2.3. Characterization of transformants Regenerated transformants were transferred to the selective medium which contained higher concentrations of geneticin (600 lg ml
1 ), and also transferred to the same medium with phosphinothricin (200 lg ml
1 ). Some transformants showed different growing patterns such as a fluffy mycelial mat or slower growth. Some transformants which showed decreased lignin degrading activity were selected, using the de-colorizing ability of poly-R medium (red to yellow) by the lignin degrading enzymes [4]. These transformants were selected and their chromosomal DNAs were isolated by the CTAB-proteinase K method [15]. The chromosomal DNAs of the transformants and the recipient strain were used as the templates of the PCR to confirm the integration of the pJS205-1 using the kan-specific primers. The nucleotide sequence of the forward primer was 50 -GAA CAA GAT GGA TTG CAC GC-30 and that of the reverse primer was 50 -GAA GAA CTC GTC AAG AAG GC-30 . The kan-specific DNA fragment in PCR mix containing 0.5

lg of chromosomal DNA, dNTPs and Taq polymerase was amplified as follows: 1 cycle of 5 min at 95 °C, and next 30 cycles of denaturation (1 min at 95 °C), annealing (1 min at 55 °C), polymerization (2 min at 72 °C), then 1 cycle of final extension (7 min at 72 °C). The length of the PCR product should be 786 bp long. Secreted laccase isozymes [8] of transformants and the recipient strain were analyzed by native, non-denaturing PAGE [11].

3. Results and discussion Genetic transformation of protoplasts generated from G. lucidum 7071-9 monokaryon was successfully carried out with the transforming plasmid pJS205-1, and the transformation yield was 4–17 transformants (lg plasmid DNA)
1 . When the REMI was carried out using different restriction enzymes which digested pJS205-1 at a single site such as EcoRV, NotI and XhoI, the mean transformation yield was 3.8  1.1, 6.2  2.1, 16.4  4.7 cells (lg plasmid DNA)
1 , respectively. Transformation of C. congregatus to phosphinothricin resistance with REMI showed a high yield to 550 transformants (lg plasmid DNA)
1 [11]. The transformation frequency of T. versicolor to hygromycin B resistance by REMI was 25–50 transformants (lg plasmid DNA)
1 [4]. In cases of Aspergillus nidulans [16] and A. bisporus [9], transformation frequencies of 5–20 and 1–5 transformants (lg plasmid DNA)
1 were obtained, respectively. In case of Lentinula edodes using its own promoter with hygromycin B resistance gene, the yield was 16 transformants (lg plasmid DNA)
1 [12]. Therefore the transformation yield was in a range of average level when pJS205-1 was used. The chromosomal DNAs of several transformants were used as the templates for the PCR using the kanspecific primers in order to confirm the integration of the plasmid into the host chromosomes. When the products were analyzed by the agarose gel electrophoresis, the expected 786 bp amplified bands appeared (Fig. 1). When the transformants (719n9, 719n27, 719xh1 and 719xh43) which showed changed growth rate or irregular colony margin were transferred to the minimal medium with geneticin (600 lg ml
1 ), they showed good growth while the recipient strain could not (Fig. 2). The transformants also showed good growth on the phosphinothricin plate (data not shown). Since fungal laccase was one of the lignin degrading enzymes, the enzyme activity of the transformants were analyzed. When the transformants on SGCM plate were reacted with o-tolidine to check laccase activity [11], 719n27 strain showed poor activity (Fig. 3). Laccase from culture supernatants grown on SGCM were analyzed by non-denaturing

S. Kim et al. / FEMS Microbiology Letters 233 (2004) 201–204

Fig. 1. Confirmation of integration of pJS205-1 plasmid into transformant chromosomal DNAs by PCR with the kan-specific primers using different DNA sources. Lanes: M, DNA 1 kb ladder; 1, pJS2051; 2, transformant 719n9; 3, transformant 719n27; 4, transformant 719xh1; 5, transformant 719xh43; and 6, recipient strain.

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PAGE, and 719n9, 719n27 and 719xh1 showed different isozyme patterns although 719n27 had very poor activity when compared with the recipient strain (Fig. 4). The major isozyme in the recipient strain was decreased in the three transformants, and they had strong isozyme activities which moved slower. When the laccase isozymes in this fungal strain were purified, three isozymes showed very similar biochemical characteristics and the same N-terminal amino acid sequences. When the carbohydrate moieties of the three isozymes were removed, the de-glycosylated protein showed same electrophoretic mobility [8]. Therefore, there is a possibility that the transformants had some changes in the protein glycosylation. One of the advantages of transformation by REMI is generation of various mutants, which can be used in the cloning of the genes related in the mutant characteristics [17]. A phospholipase D gene fragment was identified from a transformant generated by REMI in C. congregatus by retrieving the chromosomal DNA region including the inserted vector [14]. The transformant of C. congregatus showed much slower growth than the recipient strain, and showed very low laccase activity [11]. In order to identify gene(s) which were implicated in the growing pattern and enzyme modification in G. lucidum, transformants which showed different growing patterns or decreased lignin degrading activity were selected for further experiment. We have got many different kinds of mutants, and these will give information about various genes.

Fig. 2. Growth of several transformants on minimal medium with geneticin (600 lg ml
1 ): recipient strain (7071-9), transformant 719n9, transformant 719n27, transformant 719xh1, and transformant 719xh43 (counterclockwise from the top).

Fig. 3. In situ laccase reaction by o-tolidine flooding on the SGCM culture. Fungal strains were same as in Fig. 2.

Fig. 4. Characterization of laccase isozymes by native PAGE (10% gel). The gel was stained with o-tolidine after the electrophoresis was carried out until the bromophenol blue dye moved out from the gel. Lanes 1, 719n9; 2, 719n27; 3, 719xh1; 4, 719xh43; 5, 7071-9. Solid arrow represented the major laccase isozyme in the recipient strain, and the open arrows represented the slower moving laccase isozymes in the transformants.

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