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Molecular cloning and characterization of a cDNA encoding cellobiose dehydrogenase from the wood-rotting fungus Grifola frondosa
FEMS Microbiology Letters 217 (2002) 225^230
www.fems-microbiology.org
Molecular cloning and characterization of a cDNA encoding cellobiose dehydrogenase from the wood-rotting fungus Grifola frondosa Makoto Yoshida, Tsuyoshi Ohira, Kiyohiko Igarashi, Hiromichi Nagasawa, Masahiro Samejima Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan Received 20 September 2002; accepted 17 October 2002 First published online 6 November 2002
Abstract Cloning of a cDNA encoding cellobiose dehydrogenase (CDH) from the wood-rotting fungus Grifola frondosa, which produces the edible maitake mushroom, was performed using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends. The CDH cDNA consisted of 2469 bp, including an open reading frame encoding the 18-amino acid signal peptide at the N-terminal region and the 750-amino acid mature protein with a predicted molecular mass of 79.6 kDa and a pI value of 4.32. Analysis of the amino acid sequence revealed that it contains a flavin-binding motif, two glucose-methanol-choline oxidoreductase motifs, and two possible residues for heme ligand binding (Met61 and His58). The amino acid sequence of G. frondosa CDH (GfrCDH) has a high degree of identity with three known CDHs from basidiomycetes, but not with two CDHs from ascomycetes. In addition, transcription of the CDH gene in G. frondosa grown on several carbon sources was analyzed by RT-PCR. mRNA of GfrCDH was detected from mycelia grown on cellobiose and cellulose, but not on glucose. Consequently, transcription of the GfrCDH gene seems to be promoted under conditions favoring cellulose degradation, and to be regulated by carbon catabolite repression. : 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Cellobiose dehydrogenase; Grifola frondosa; Cellulose degradation ; Edible maitake mushroom
1. Introduction The fruiting body of the wood-rotting fungus Grifola frondosa is the edible maitake mushroom, for which there is a substantial consumer demand in Japan, China and the USA [1]. Since the fungus grows on wood in nature, mixtures of lignocellulosic materials such as sawdust and cereal bran have been utilized as substrates in the commercial production of the mushroom. However, knowledge of the bioconversion of these lignocellulosics by G. frondosa during cultivation is still very limited. More detailed information would be helpful to improve the cultivation conditions for e⁄cient production of the mushroom. During the course of cellulose degradation, many wood-
* Corresponding author. Tel. : +81 (3) 5841 5255; Fax : +81 (3) 5841 5273. E-mail address : [email protected] (M. Samejima).
rotting fungi produce the extracellular £avocytochrome cellobiose dehydrogenase (CDH), as well as other hydrolytic enzymes [2^4]. CDH oxidizes the reducing-end groups of cellobiose and cellodextrins to the corresponding lactones in the presence of various electron acceptors [2,5,6]. Although the physiological role of this enzyme is still uncertain, the localization of CDH on cellulose in vivo [7] and the synergistic interaction with cellobiohydrolase [8] indicate an involvement in cellulose degradation. Recently, Vallim and co-workers [9] detected CDH transcripts during wood degradation. Moreover, Dumonceaux and co-workers [10] have reported that a CDH-de¢cient strain of the wood-rotting fungus Trametes versicolor grows poorly on wood. These results clearly indicate the importance of the enzyme in wood degradation, as well as cellulose degradation. In the present work, therefore, we cloned cDNA encoding CDH from G. frondosa (GfrCDH) and investigated CDH gene transcription in cellulolytic cultures by reverse transcription-polymerase chain reaction (RT-PCR) in or-
0378-1097 / 02 / $22.00 : 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 1 0 6 8 - 6
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der to achieve a better understanding of lignocellulose degradation by G. frondosa.
amino acid sequences of actins from several microorganisms. The PCR products were separated on a 2% agarose gel and stained with ethidium bromide. The PCR products were subcloned and sequenced as described above.
2. Materials and methods 2.1. Cultivation
3. Results and discussion
G. frondosa M-51 was cultivated on a potato dextrose agar plate at 26.5‡C for 14 days. The mycelium on the plate was punched out and inoculated into 400 ml of Kremer and Wood medium [6] containing 2% cellulose powder (Whatman CF11) in a 1-l Erlenmeyer £ask. The culture was incubated at 26.5‡C with rotary shaking (150 rpm, diameter 20 mm).
Many cellulolytic fungi produce CDH, which is the only extracellular £avocytochrome reported, as far as we are aware [2^4]. The two prosthetic groups, FAD and b-type heme, are located in di¡erent domains of this enzyme [11,12]. The cDNA encoding CDH has already been cloned from ¢ve cellulolytic fungi [13^18]. Analysis of the amino acid sequences encoded by these clones showed that all CDHs retain the glucose-methanol-choline (GMC) oxidoreductase family motif in the £avin domain [19]. Therefore, to clone CDH cDNA from G. frondosa, we designed a degenerate primer for the consensus sequence of the GMC oxidoreductase family motif (primer R1 in Fig. 1). A fragment of 125 bp was obtained by the ¢rst PCR using degenerate primers (primers F1 and R1 in Fig. 1). The amino acid sequence deduced from the fragment showed high degrees of identity (82.9^92.7%) with those of CDH from three basidiomycetes (Phanerochaete chrysosporium, T. versicolor and Pycnoporus cinnabarinus), indicating that the fragment is a part of the CDH cDNA. The full-length sequence involving this fragment was then cloned by ¢ve steps of PCR. As shown in Fig. 2, the cDNA consisted of 2469 bp nucleotides, including an open reading frame encoding 768 amino acid residues. The ¢rst 18 amino acid residues in the N-terminal region are expected to be a signal peptide [20]. Thus, the mature protein consists of 750 amino acids with a molecular mass
2.2. Cloning of cDNA encoding CDH After 30 days cultivation, the mycelium was harvested by centrifugation and then ground to ¢ne powder using a pestle in liquid nitrogen. Total RNA was extracted from 100 mg of the powder using E.Z.N.A. Fungal RNA Kit (Omega Bio-tek, USA). First-strand cDNA was synthesized by a Ready-To-Go1 T-primed First-strand cDNA Synthesis Kit (Amersham Biosciences, USA) for three steps of PCR and 3P rapid ampli¢cation of cDNA ends (RACE), and by a SMART RACE cDNA Ampli¢cation Kit (Clontech, USA) for 5PRACE. A schematic diagram of the nucleotide sequences of primers used for these reactions and their locations on the cloned cDNA is shown in Fig. 1. The nucleotide sequence including the coding region was con¢rmed by PCR ampli¢cation using speci¢c primers (F4 and R4 in Fig. 1). All PCR products were ligated into pGEM-T Easy Vectors (Promega, USA) followed by sequencing with a LongRead Tower sequencer (Visible Genetics, USA) using a Thermo Sequenase Cy 5.5 dye terminator cycle sequencing kit (Visible Genetics). Database search using the deduced amino acid sequences was performed using the NCBI online program BLAST for protein sequences stored in GenBank (http://www.ncbi.nlm.nih.gov/BLAST/). 2.3. Detection of CDH transcription by RT-PCR Total RNA and ¢rst-strand cDNA was prepared from the mycelium grown in Kremer and Wood medium [6] supplemented with glucose, cellobiose or cellulose as a sole source of carbon. The ¢rst-strand cDNA from each sample was used as a template and ampli¢cation was performed with a pair of primers (RT-F : 5P-ATGCAGGCTCGACTTCCTAG and RT-R: 5P-TCGATACTCGGATGAGTGAA). To ensure equal loading of samples, an actin cDNA fragment was ampli¢ed with two degenerate primers, L-actin-F: 5P-GACATGGARAAGATCTGGCA-3P, and L-actin-R : 5P-TTCTCCTTGATRTCACGGACRATTTC-3P, which were designed based on the
Fig. 1. Schematic diagram of CDH cDNA showing locations and nucleotide sequences of primers used for PCR. Arrowheads represent primers and underlines between them indicate cDNA fragments ampli¢ed with these primers. F and R indicate the forward and reverse directions, respectively.
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Fig. 2. The nucleotide and deduced amino acid sequences of cDNA encoding GfrCDH precursor. The potential signal peptide sequence is double-underlined. The potential N-glycosylation sites are boxed. The nucleotide sequence have been deposited in DDBJ/EMBL/GenBank databases and assigned the accession number AB083245.
of 79.6 kDa and a pI value of 4.32. The amino acid sequence reveals the presence of eight possible sites of N-glycosylation, Asn-X-Thr/Ser, in which X is a residue other than proline. In addition, there are numerous possible sites for O-glycosylation. The amino acid sequence of GfrCDH was compared with those of previously known CDHs. As shown in Fig. 3, GfrCDH has high degrees of identity (72.8^ 75.7%) with CDHs from basidiomycetes, but only limited identity (37.3^38.8%) with CDHs from ascomycetes (Humicola insolens and Sporotrichum thermophile). By com-
parison with other CDHs, the amino acid sequence of GfrCDH appears to contain an N-terminal heme domain (residues 1^185) and a C-terminal £avin domain (residues 211^750) with a hydrophilic linker region (residues 186^ 210). Two amino acid residues, Met61 and His158, for the heme ligands [21] are preserved in GfrCDH, as well as other CDHs. In a database search for each domain, no signi¢cantly homologous sequences to the heme domain were found, except among other CDHs. On the other hand, the £avin domain includes not only GMC oxidoreductase motifs 1 and 2 (in aa 294^317 and aa 462^476,
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Fig. 3. Multiple alignment of the amino acid sequences of GfrCDH and other known CDHs. Perfect matches are enclosed in boxes with a dark background. Identical amino acids in basidiomycetes are open-boxed. Arrowheads indicate Met and His that may be associated with the heme ligands. GMC oxidoreductase signatures are underlined. The £avin-binding motif is double-underlined. Abbreviations: Gfr., G. frondosa ; Pch., P. chrysosporium ; Tve., T. versicolor ; Pci., P. cinnabarinus; Hin., H. insolens ; Sth., S. thermophile.
respectively), but also a £avin-binding motif (GxGxxG [22] in aa 218^223), as found in other CDHs. All these observations on GfrCDH cDNA suggest that the GfrCDH has similar structural features to other fungal CDHs. The e¡ect of carbon source on GfrCDH transcription was investigated by RT-PCR analysis of total RNA extracted from the mycelium grown on glucose, cellobiose or cellulose. As shown in Fig. 4, transcripts of the CDH gene
were clearly detected in the mycelium grown on both cellobiose and cellulose, whereas no signi¢cant band was observed in the case of the mycelium grown on glucose. This suggests that GfrCDH transcription is promoted under conditions favoring cellulose degradation and is regulated by carbon catabolite repression. In previous reports, CDH transcripts were not detected in cellobiose culture of P. chrysosporium and P. cinnabarinus [14,16].
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1
2
3 [2]
1353 1078 872
[3]
603
[4]
310
[5]
[6]
603 [7]
310 (bp)
[8]
Fig. 4. RT-PCR analysis of GfrCDH (upper) and actin (lower) gene transcription in mycelium grown in di¡erent carbon sources. Firststrand cDNA was synthesized from total RNA of mycelia cultured with glucose (lane 1), cellobiose (lane 2) or cellulose (lane 3) as a sole source of carbon. The PCR products were separated on 2% agarose gel.
[9]
[10]
However, Szabo and co-workers [13] have demonstrated production of cellulases and CDH by P. chrysosporium using cellobiose as a sole source of carbon, and noted that CDH production was related to cellobiose concentration. Consequently, the di¡erence in the responses to cellobiose between G. frondosa and other basidiomycetes seems to be due to di¡erences of cellobiose concentration in the culture, rather than di¡erences of transcriptional response among fungal species. In conclusion, we have cloned a cDNA encoding CDH from the wood-rotting fungus G. frondosa and demonstrated by RT-PCR analysis that its transcription occurs speci¢cally in cellulolytic cultures. This technique could be used to monitor the fungal activity of cellulose utilization during the commercial cultivation of G. frondosa, and to ¢nd optimum growth conditions for e⁄cient production of maitake mushroom.
[11]
[12]
[13]
[14]
[15]
[16]
Acknowledgements This research was partly supported by the MAFF Project of Bioenergy Conversion to M.S. and by a Research Fellowship (No. 08446) from the Japan Society for the Promotion of Science to M.Y.
[17]
[18]
References [1] Shen, Q. and Royse, D.J. (2001) E¡ects of nutrient supplements on
[19]
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biological e⁄ciency, quality and crop cycle time of maitake (Grifola frondosa). Appl. Microbiol. Biotechnol. 57, 74^78. Ayers, A.R., Ayers, S.B. and Eriksson, K.-E. (1978) Cellobiose oxidase, puri¢cation and partial characterization of a hemoprotein from Sporotrichum pulverulentum. Eur. J. Biochem. 90, 171^181. Eriksson, K.-E.L., Habu, N. and Samejima, M. (1993) Recent advances in fungal cellobiose oxidoreductases. Enzyme Microb. Technol. 15, 1002^1008. Henriksson, G., Johansson, G. and Pettersson, G. (2000) A critical review of cellobiose dehydrogenases. J. Biotechnol. 78, 93^113. Samejima, M. and Eriksson, K.-E.L. (1992) A comparison of the catalytic properties of cellobiose :quinone oxidoreductase and cellobiose oxidase from Phanerochaete chrysosporium. Eur. J. Biochem. 207, 103^107. Kremer, S.M. and Wood, P.M. (1992) Evidence that cellobiose oxidase from Phanerochaete chrysosporium is primarily an Fe(III) reductase. Kinetic comparison with neutrophil NADPH oxidase and yeast £avocytochrome b2 . Eur. J. Biochem. 205, 133^138. Igarashi, K., Samejima, M., Saburi, Y., Habu, N. and Eriksson, K.-E.L. (1997) Localization of cellobiose dehydrogenase in cellulose-grown cultures of Phanerochaete chrysosporium. Fungal Genet. Biol. 21, 214^222. Igarashi, K., Samejima, M. and Eriksson, K.-E.L. (1998) Cellobiose dehydrogenase enhances Phanerochaete chrysosporium cellobiohydrolase I activity by relieving product inhibition. Eur. J. Biochem. 253, 101^106. Vallim, M.A., Janse, B.J.H., Gaskell, J., Kleiner, A.A.P. and Cullen, D. (1998) Phanerochaete chrysosporium cellobiohydrolase and cellobiose dehydrogenase transcripts in wood. Appl. Environ. Microbiol. 64, 1924^1928. Dumonceaux, T., Bartholomew, K., Valeanu, L., Charles, T. and Archibald, F. (2001) Cellobiose dehydrogenase is essential for wood invasion and nonessential for kraft pulp deligni¢cation by Trametes versicolor. Enzyme Microb. Technol. 29, 478^489. Morpeth, F.F. (1985) Some properties of cellobiose oxidase from the white-rot fungus Sporotrichum pulverulentum. Biochem. J. 228, 557^ 564. Henriksson, G., Pettersson, G., Johansson, G., Ruiz, A. and Uzcategui, E. (1991) Cellobiose oxidase from Phanerochaete chrysosporium can be cleaved by papain into two domains. Eur. J. Biochem. 196, 101^106. Raices, M., Paifer, E., Cremata, J., Montesino, R., Stahlberg, J., Divne, C., Szabo, I.J., Henriksson, G., Johansson, G. and Pettersson, G. (1995) Cloning and characterization of a cDNA encoding a cellobiose dehydrogenase from the white rot fungus Phanerochaete chrysosporium. FEBS Lett. 369, 233^238. Li, B., Nagalla, S.R. and Renganathan, V. (1996) Cloning of a cDNA encoding cellobiose dehydrogenase, a hemo£avoenzyme from Phanerochaete chrysosporium. Appl. Environ. Microbiol. 62, 1329^1335. Dumonceaux, T.J., Bartholomew, K.A., Charles, T.C., Moukha, S.M. and Archibald, F.S. (1998) Cloning and sequencing of a gene encoding cellobiose dehydrogenase from Trametes versicolor. Gene 210, 211^219. Moukha, S.M., Dumonceaux, T.J., Record, E. and Archibald, F.S. (1999) Cloning and analysis of Pycnoporus cinnabarinus cellobiose dehydrogenase. Gene 234, 23^33. Subramaniam, S.S., Nagalla, S.R. and Renganathan, V. (1999) Cloning and characterization of a thermostable cellobiose dehydrogenase from Sporotrichum thermophile. Arch. Biochem. Biophys. 365, 223^ 230. Xu, F., Golightly, E.J., Duke, K.R., Lassen, S.F., Knusen, B., Christensen, S., Brown, K.M., Brown, S.H. and Schulein, M. (2001) Humicola insolens cellobiose dehydrogenase: cloning, redox chemistry, and ‘logic gate’-like dual functionality. Enzyme Microb. Technol. 28, 744^753. Cavener, D.R. (1992) GMC oxidoreductases. A newly de¢ned family
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of homologous proteins with diverse catalytic activities. J. Mol. Biol. 223, 811^814. [20] von Heijne, G. (1986) A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 14, 4683^4690. [21] Cox, M.C., Rogers, M.S., Cheesman, M., Jones, G.D., Thomson, A.J., Wilson, M.T. and Moore, G.R. (1992) Spectroscopic identi¢ca-
tion of the haem ligands of cellobiose oxidase. FEBS Lett. 307, 233^ 236. [22] Wierenga, R.K., Terpstra, P. and Hol, W.G. (1986) Prediction of the occurrence of the ADP-binding beta alpha beta-fold in proteins, using an amino acid sequence ¢ngerprint. J. Mol. Biol. 187, 101^ 107.
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Molecular cloning and characterization of a cDNA encoding cellobiose dehydrogenase from the wood-rotting fungus Grifola frondosa Makoto Yoshida, Tsuyoshi Ohira, Kiyohiko Igarashi, Hiromichi Nagasawa, Masahiro Samejima Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan Received 20 September 2002; accepted 17 October 2002 First published online 6 November 2002
Abstract Cloning of a cDNA encoding cellobiose dehydrogenase (CDH) from the wood-rotting fungus Grifola frondosa, which produces the edible maitake mushroom, was performed using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends. The CDH cDNA consisted of 2469 bp, including an open reading frame encoding the 18-amino acid signal peptide at the N-terminal region and the 750-amino acid mature protein with a predicted molecular mass of 79.6 kDa and a pI value of 4.32. Analysis of the amino acid sequence revealed that it contains a flavin-binding motif, two glucose-methanol-choline oxidoreductase motifs, and two possible residues for heme ligand binding (Met61 and His58). The amino acid sequence of G. frondosa CDH (GfrCDH) has a high degree of identity with three known CDHs from basidiomycetes, but not with two CDHs from ascomycetes. In addition, transcription of the CDH gene in G. frondosa grown on several carbon sources was analyzed by RT-PCR. mRNA of GfrCDH was detected from mycelia grown on cellobiose and cellulose, but not on glucose. Consequently, transcription of the GfrCDH gene seems to be promoted under conditions favoring cellulose degradation, and to be regulated by carbon catabolite repression. : 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Cellobiose dehydrogenase; Grifola frondosa; Cellulose degradation ; Edible maitake mushroom
1. Introduction The fruiting body of the wood-rotting fungus Grifola frondosa is the edible maitake mushroom, for which there is a substantial consumer demand in Japan, China and the USA [1]. Since the fungus grows on wood in nature, mixtures of lignocellulosic materials such as sawdust and cereal bran have been utilized as substrates in the commercial production of the mushroom. However, knowledge of the bioconversion of these lignocellulosics by G. frondosa during cultivation is still very limited. More detailed information would be helpful to improve the cultivation conditions for e⁄cient production of the mushroom. During the course of cellulose degradation, many wood-
* Corresponding author. Tel. : +81 (3) 5841 5255; Fax : +81 (3) 5841 5273. E-mail address : [email protected] (M. Samejima).
rotting fungi produce the extracellular £avocytochrome cellobiose dehydrogenase (CDH), as well as other hydrolytic enzymes [2^4]. CDH oxidizes the reducing-end groups of cellobiose and cellodextrins to the corresponding lactones in the presence of various electron acceptors [2,5,6]. Although the physiological role of this enzyme is still uncertain, the localization of CDH on cellulose in vivo [7] and the synergistic interaction with cellobiohydrolase [8] indicate an involvement in cellulose degradation. Recently, Vallim and co-workers [9] detected CDH transcripts during wood degradation. Moreover, Dumonceaux and co-workers [10] have reported that a CDH-de¢cient strain of the wood-rotting fungus Trametes versicolor grows poorly on wood. These results clearly indicate the importance of the enzyme in wood degradation, as well as cellulose degradation. In the present work, therefore, we cloned cDNA encoding CDH from G. frondosa (GfrCDH) and investigated CDH gene transcription in cellulolytic cultures by reverse transcription-polymerase chain reaction (RT-PCR) in or-
0378-1097 / 02 / $22.00 : 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 1 0 6 8 - 6
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der to achieve a better understanding of lignocellulose degradation by G. frondosa.
amino acid sequences of actins from several microorganisms. The PCR products were separated on a 2% agarose gel and stained with ethidium bromide. The PCR products were subcloned and sequenced as described above.
2. Materials and methods 2.1. Cultivation
3. Results and discussion
G. frondosa M-51 was cultivated on a potato dextrose agar plate at 26.5‡C for 14 days. The mycelium on the plate was punched out and inoculated into 400 ml of Kremer and Wood medium [6] containing 2% cellulose powder (Whatman CF11) in a 1-l Erlenmeyer £ask. The culture was incubated at 26.5‡C with rotary shaking (150 rpm, diameter 20 mm).
Many cellulolytic fungi produce CDH, which is the only extracellular £avocytochrome reported, as far as we are aware [2^4]. The two prosthetic groups, FAD and b-type heme, are located in di¡erent domains of this enzyme [11,12]. The cDNA encoding CDH has already been cloned from ¢ve cellulolytic fungi [13^18]. Analysis of the amino acid sequences encoded by these clones showed that all CDHs retain the glucose-methanol-choline (GMC) oxidoreductase family motif in the £avin domain [19]. Therefore, to clone CDH cDNA from G. frondosa, we designed a degenerate primer for the consensus sequence of the GMC oxidoreductase family motif (primer R1 in Fig. 1). A fragment of 125 bp was obtained by the ¢rst PCR using degenerate primers (primers F1 and R1 in Fig. 1). The amino acid sequence deduced from the fragment showed high degrees of identity (82.9^92.7%) with those of CDH from three basidiomycetes (Phanerochaete chrysosporium, T. versicolor and Pycnoporus cinnabarinus), indicating that the fragment is a part of the CDH cDNA. The full-length sequence involving this fragment was then cloned by ¢ve steps of PCR. As shown in Fig. 2, the cDNA consisted of 2469 bp nucleotides, including an open reading frame encoding 768 amino acid residues. The ¢rst 18 amino acid residues in the N-terminal region are expected to be a signal peptide [20]. Thus, the mature protein consists of 750 amino acids with a molecular mass
2.2. Cloning of cDNA encoding CDH After 30 days cultivation, the mycelium was harvested by centrifugation and then ground to ¢ne powder using a pestle in liquid nitrogen. Total RNA was extracted from 100 mg of the powder using E.Z.N.A. Fungal RNA Kit (Omega Bio-tek, USA). First-strand cDNA was synthesized by a Ready-To-Go1 T-primed First-strand cDNA Synthesis Kit (Amersham Biosciences, USA) for three steps of PCR and 3P rapid ampli¢cation of cDNA ends (RACE), and by a SMART RACE cDNA Ampli¢cation Kit (Clontech, USA) for 5PRACE. A schematic diagram of the nucleotide sequences of primers used for these reactions and their locations on the cloned cDNA is shown in Fig. 1. The nucleotide sequence including the coding region was con¢rmed by PCR ampli¢cation using speci¢c primers (F4 and R4 in Fig. 1). All PCR products were ligated into pGEM-T Easy Vectors (Promega, USA) followed by sequencing with a LongRead Tower sequencer (Visible Genetics, USA) using a Thermo Sequenase Cy 5.5 dye terminator cycle sequencing kit (Visible Genetics). Database search using the deduced amino acid sequences was performed using the NCBI online program BLAST for protein sequences stored in GenBank (http://www.ncbi.nlm.nih.gov/BLAST/). 2.3. Detection of CDH transcription by RT-PCR Total RNA and ¢rst-strand cDNA was prepared from the mycelium grown in Kremer and Wood medium [6] supplemented with glucose, cellobiose or cellulose as a sole source of carbon. The ¢rst-strand cDNA from each sample was used as a template and ampli¢cation was performed with a pair of primers (RT-F : 5P-ATGCAGGCTCGACTTCCTAG and RT-R: 5P-TCGATACTCGGATGAGTGAA). To ensure equal loading of samples, an actin cDNA fragment was ampli¢ed with two degenerate primers, L-actin-F: 5P-GACATGGARAAGATCTGGCA-3P, and L-actin-R : 5P-TTCTCCTTGATRTCACGGACRATTTC-3P, which were designed based on the
Fig. 1. Schematic diagram of CDH cDNA showing locations and nucleotide sequences of primers used for PCR. Arrowheads represent primers and underlines between them indicate cDNA fragments ampli¢ed with these primers. F and R indicate the forward and reverse directions, respectively.
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227
Fig. 2. The nucleotide and deduced amino acid sequences of cDNA encoding GfrCDH precursor. The potential signal peptide sequence is double-underlined. The potential N-glycosylation sites are boxed. The nucleotide sequence have been deposited in DDBJ/EMBL/GenBank databases and assigned the accession number AB083245.
of 79.6 kDa and a pI value of 4.32. The amino acid sequence reveals the presence of eight possible sites of N-glycosylation, Asn-X-Thr/Ser, in which X is a residue other than proline. In addition, there are numerous possible sites for O-glycosylation. The amino acid sequence of GfrCDH was compared with those of previously known CDHs. As shown in Fig. 3, GfrCDH has high degrees of identity (72.8^ 75.7%) with CDHs from basidiomycetes, but only limited identity (37.3^38.8%) with CDHs from ascomycetes (Humicola insolens and Sporotrichum thermophile). By com-
parison with other CDHs, the amino acid sequence of GfrCDH appears to contain an N-terminal heme domain (residues 1^185) and a C-terminal £avin domain (residues 211^750) with a hydrophilic linker region (residues 186^ 210). Two amino acid residues, Met61 and His158, for the heme ligands [21] are preserved in GfrCDH, as well as other CDHs. In a database search for each domain, no signi¢cantly homologous sequences to the heme domain were found, except among other CDHs. On the other hand, the £avin domain includes not only GMC oxidoreductase motifs 1 and 2 (in aa 294^317 and aa 462^476,
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Fig. 3. Multiple alignment of the amino acid sequences of GfrCDH and other known CDHs. Perfect matches are enclosed in boxes with a dark background. Identical amino acids in basidiomycetes are open-boxed. Arrowheads indicate Met and His that may be associated with the heme ligands. GMC oxidoreductase signatures are underlined. The £avin-binding motif is double-underlined. Abbreviations: Gfr., G. frondosa ; Pch., P. chrysosporium ; Tve., T. versicolor ; Pci., P. cinnabarinus; Hin., H. insolens ; Sth., S. thermophile.
respectively), but also a £avin-binding motif (GxGxxG [22] in aa 218^223), as found in other CDHs. All these observations on GfrCDH cDNA suggest that the GfrCDH has similar structural features to other fungal CDHs. The e¡ect of carbon source on GfrCDH transcription was investigated by RT-PCR analysis of total RNA extracted from the mycelium grown on glucose, cellobiose or cellulose. As shown in Fig. 4, transcripts of the CDH gene
were clearly detected in the mycelium grown on both cellobiose and cellulose, whereas no signi¢cant band was observed in the case of the mycelium grown on glucose. This suggests that GfrCDH transcription is promoted under conditions favoring cellulose degradation and is regulated by carbon catabolite repression. In previous reports, CDH transcripts were not detected in cellobiose culture of P. chrysosporium and P. cinnabarinus [14,16].
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1
2
3 [2]
1353 1078 872
[3]
603
[4]
310
[5]
[6]
603 [7]
310 (bp)
[8]
Fig. 4. RT-PCR analysis of GfrCDH (upper) and actin (lower) gene transcription in mycelium grown in di¡erent carbon sources. Firststrand cDNA was synthesized from total RNA of mycelia cultured with glucose (lane 1), cellobiose (lane 2) or cellulose (lane 3) as a sole source of carbon. The PCR products were separated on 2% agarose gel.
[9]
[10]
However, Szabo and co-workers [13] have demonstrated production of cellulases and CDH by P. chrysosporium using cellobiose as a sole source of carbon, and noted that CDH production was related to cellobiose concentration. Consequently, the di¡erence in the responses to cellobiose between G. frondosa and other basidiomycetes seems to be due to di¡erences of cellobiose concentration in the culture, rather than di¡erences of transcriptional response among fungal species. In conclusion, we have cloned a cDNA encoding CDH from the wood-rotting fungus G. frondosa and demonstrated by RT-PCR analysis that its transcription occurs speci¢cally in cellulolytic cultures. This technique could be used to monitor the fungal activity of cellulose utilization during the commercial cultivation of G. frondosa, and to ¢nd optimum growth conditions for e⁄cient production of maitake mushroom.
[11]
[12]
[13]
[14]
[15]
[16]
Acknowledgements This research was partly supported by the MAFF Project of Bioenergy Conversion to M.S. and by a Research Fellowship (No. 08446) from the Japan Society for the Promotion of Science to M.Y.
[17]
[18]
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