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Components of the ligninolytic system of Fusarium oxysporum and Trichoderma atroviride
FUEL PROCESSING TECHNOLOGY ELSEVIER
Fuel Processing
Technology
52 (1997) 73-77
Components of the ligninolytic system of Fusarium oxysporum and Trichoderma atroviride Heike Mijnkemann *, Udo Hiilker, Milan Hijfer Botanisches Institut der lJnic;ersitiitBonn, Kirschallee I, 53115 Bonn, Germany
Abstract The ligninolytic system in the two deuteromycetous fungi Fusarium oxysporum and Trichoderma atrouiride, which are able to solubilize low-rank coal [l], has been proved to have several components. Analysis of the chromosomal DNA of these fungi revealed distinct bands with probes coding for three ligninase isoenzymes (H7, H8, HlO), glyoxal oxidase and arylalcohol dehydrogenase of the basidiomycete Phanerochaete chrysosporium. These data constitute a strong indication for the existence in F. oxysporum and T. atrouiride of a ligninolytic system comparable to that in P. chrysosporium that may be involved in the process of coal solubilization. 0 1997 Elsevier Science B.V. Keywords: Coal solubilization; Lignin peroxidases; oxysporum; Trichodenna atrouiride
Glyoxal
oxidase;
Arylalcohol
dehydrogenase;
Fusarium
1. Introduction
The
two
deuteromycetes
Fusarium
oxysporum
shown to solubilize low-rank coal. Moreover, both on the carbon source used for cultivation [1,2]. low-rank coal constitutively, in T. atrouiride the induced [2]. Ligninolytic enzymes have as yet been in T. atroviride. Phanerochaete chrysosporium, a wood-attacking
’ Corresponding
and Trichoderma atroviride were fungi solubilize coal in dependence Whereas F. oxysporum solubilizes ability to solubilize coal has to be found neither in F. oxysporum nor and lignin-degrading
author.
037%3820/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SO378-3820(97)000 17-9
white-rot fun-
74
H. Miinkemann et al. /Fuel
Processing Technology 52 (1997) 73-77
gus, has been extensively studied. The ligninolytic system of this basidiomycete consists of multiple lignin peroxidases [3], manganese peroxidase [4], glyoxal oxidase [5], and lactase [6]. It was also found that the transcript level of arylalcohol dehydrogenase correlates with those of lignin peroxidase isoenzymes in ligninolytic cultures of P. chrysosporium [7]. With the exception of lactase, DNA sequences of ligninolytic enzymes have already been determined in P. chrysosporium [3-71. A possible role or even an interaction of ligninolytic enzymes in lignite solubilization still remains to be proven. In this report we demonstrate the existence of several components of the P. chrysosporium ligninolytic system in F. oxysporum and T. atroviride.
2. Materials and methods 2. I. Fungal material F. oxysporum was kindly supplied by R.M. Fakoussa of the Institute for Microbiology and Biotechnology, University of Bonn. It was collected in an open-cast mining area near Cologne. T. atroviride was isolated in the authors’ laboratory from stored low-rank coal. F, oxysporum and T. atroviride were grown at 30°C and 18°C respectively, as described previously by Htilker [2]. 2.2. Genomic DNA
Chromosomal DNA from 50 ml F. oxysporum and T. atroviride cultures was isolated using a method developed by Hoffmann [8]. 2.3. Southern blot analysis 30 pg chromosomal DNA was hydrolyzed overnight with 50 U of restriction endonuclease EcoRI in a total volume of 100 ~1 at 37°C. The hydrolyzed DNA was separated according to its molecular weight on 0.8% agarose gel. After denaturation (1.5 M NaCl and 0.5 M NaOH, 2 X 15 min) and neutralization (1.0 M Tris and 1.5 M NaCl (pH 7.4), 3 X 20 min), the DNA was transferred to a GeneScreenPlus membrane (DuPont, Bad Homburg, Germany) by overnight capillary transfer with 10 X SSC. The DNA was then covalently bound to the membrane by incubation for 5 min in 0.05 M NaOH and neutralized for 5 min in 2 X SSC.
2.4. Probes Genomic fragments from P. chrysosporium coding for ligninase isoenzymes H7, H8 and HlO were kindly supplied by D. Cullen of the Department of Bacteriology, University of Wisconsin, USA [9]. cDNA probes encoding glyoxal oxidase and arylalcoho1 dehydrogenase, both from P. chrysosporium, were kindly supplied by P.J. Kersten of the Department of Agriculture, University of Wisconsin, USA [5] and J. Reiser of the
H. Miinkemann et al. / Fuel Processing Technology 52 (1997) 73-77
15
National Institutes of Health, NII, Bethesda, MD, USA [7], respectively. The above mentioned fragments were labelled with a random fluorescein labelling kit (DuPont) and used for hybridization experiments according to the manufacturer’s instructions. The colour reaction was carried out accordingly. 2.5. Chemicals All chemicals
were of analytical
grade from Boehringer,
Merck and Serva.
3. Results Detection amplification
of a gene coding for ligninase isoenzyme H8 in F. oxysporum by PCR [lo] led to further Southern blot analysis. Genomic DNA was isolated
1
2
1
2
1
2
Fig. 1. Ligninase isoenzymes of F. oxyspomm and T. atrouiride. Southern blot analysis of genomic DNA from F. oxysporum (lane 1) and T. atrouiride (lane 2) hybridized with ligninase isoenzyme H7 (a), ligninase isoenzyme H8 (b) and ligninase isoenzyme HlO (c) genomic DNA fragments from P. chrysosporium, respectively.
Fig. 2. Glyoxal oxidase of F. oxysporum and T. atrouiride. Southern blot analysis of genomic DNA from F. onysporum (lane 1) and T. atrouiride (lane 2) hybridized with glx-lc cDNA from P. chrysosporium.
16
H. Miinkemann et al. /Fuel
Processing Technology 52 (1997) 73-77
Fig. 3. Arylalcohol dehydrogenase of F. oxysporum and T. atrouiride. Southern blot analysis of genomic DNA from F. oxysporum (lane 1) and T. atrouiride (lane 2) hybridized with arylalcohol dehydrogenase (AAD) cDNA from P. chrysospon’um.
from both F. oxysporum and T. atroviride, digested with restriction endonuclease EcoRI, separated on agarose gels, and blotted to GeneScreenPlus membranes. For hybridization, we focused on lignin peroxidase (lip) genes which are highly expressed under carbon and nitrogen deficiency in ligninolytic P. chrysosporium cultures (ligninase isoenzymes H7, H8 and HlO), as well as on glyoxal oxidase, and arylalcohol dehydrogenase. Hybridization and washing steps (2.0 X SSC and 0.1% SDS; 0.2 X SSC and 0.1% SDS) were performed at 65°C. This high temperature and the ion concentration of the washing buffers resulted in high stringency, which allowed only the annealing of homologous DNA sequences. Distinct signals of the three lip genes were detected in the genomic DNA of both fungi, F. oxysporum and T. atroviride (Fig. la-c). With the complete glyoxal oxidase-coding region from the basidiomycete P. chrysosporium, bands were obtained with the genomic DNA from both fungi (Fig. 2). Probing the blotted chromosomal DNA from the two fungi with a cDNA of P. chrysosporium encoding arylalcohol dehydrogenase resulted in bands of different intensities in both DNA preparations (Fig. 3). For hybridization experiments of the last two enzymes, the same high stringency conditions were applied.
4. Discussion The chemical structure of low-rank coal shows similarity to lignin. Several basidiomycetes are known to possess a ligninolytic system. The best characterized system is that of P. chrysosporium, a white-rot fungus which is able to decompose wood. Since these enzymes are involved in lignin degradation by the fungus also, the coal-active molds F. oxysporum and T. atroviride could use these enzymes for coal solubilization. Enzymatic assays, however, have as yet revealed no corresponding enzyme activity [ 111. In this communication, we demonstrate the existence of genetic information in the two coal-active molds for some of the potentially coal-solubilizing enzymes, viz. lignin
H. Mb;nkemann et al./ Fuel Processing Technology 52 (1997) 73-77
II
peroxidase isoenzymes (H7, H8, HlO), glyoxal oxidase and arylalcohol dehydrogenase. These ligninolytic enzymes do not cross-hybridize with other published fungal DNA sequences, hence, these results imply the existence of a homologous ligninolytic system in F. oxysporum and T. atrouiride. The results obtained with the ligninase isoenzyme H8 confirm previous findings acquired by PCR amplification of F. oxysporum genomic DNA and by Northern blot analysis [lo]. Because the two mold genera acquired the ability to solubilize coal depending on the growth substrate [1,2], analysis of the mRNA expression of the individual enzymes under appropriate growth conditions will provide unambiguous answers to the question of enzyme participation in the process of coal solubilization.
Acknowledgements This work was supported by the BMBF, grant no. 0320620B
to M.H.
References [l] U. HBlker, R.M. Fakoussa, M. Hofer, Growth substrates control the ability of Fusarium oxysporum to solubilize low-rank coal, Appl. Microbial. Biotechnol. 44 (1995) 351-355. [2] U. Hiilker, H. Monkemann, M. HGfer, A test system to analyze the complex physiological states of coal-solubilizing fungi, Fuel Processing Technol., 1996 (in press). [3] J. Gaskell, P. Stewart, P. Kersten, S. Covert, J. Reiser, D. Cullen, Establishment of genetic linkage by allele-specific polymerase chain reaction: Application to the lignin peroxidase gene family of Phanerochaete chrysosporium, Bio/Technology 12 (1994) 1372-1375. [4] B.J. Godfrey, M.B. Mayfield, J.A. Brown, M.H. Gold, Characterization of a gene encoding a manganese peroxidase from Phanerochaete chrysosporium, Gene 93 (1990) 119-124. [5] P.J. Kersten, D. Cullen, Cloning and characterization of a cDNA encoding glyoxal oxidase, a H,O,-producing enzyme from the lignin-degrading basidiomycete Phanerochaete chrysosporium, Proc. Natl. Acad. Sci. USA 90 (1993) 7411-7413. [6] C. Srinivasan, T.M. D’Souza, K. Boominathan, CA. Reddy, Demonstration of a lactase in the white-rot basidiomycete Phanerochaete chrysosporium BKM-F1767, Appl. Environ, Microbial. 61 (1995) 42744277. [7] J. Reiser, A. Muheim, M. Hardegger, G. Frank, A. Fiechter, Arylalcohol dehydrogenase from the white-rot fungus Phanerochaete chtysosporium: Gene cloning, sequence analysis, expression and purification of the recombinant enzyme, J. Biol. Chem. 269 (1994) 28152-28159. [S] C.S. Hoffmann, F. Winston, A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli, Gene 57 (1987) 267-272. [9] R.T. Lamar, B. Schoenike, A. van den Wymelenberg, P. Stewart, D.M. Dietrich, D. Cullen, Quantification of fungal mRNA’s in complex substrates by reverse transcription PCR and its application to Phanerochaete chrysosporium-colonized soil, Appl. Environ. Microbial. 61 (1995) 2122-2126. [lo] H. Monkemann, U. Hiilker, 0. Golubnichaya-Labudova, H. Lichtenberg-Fratt, M. Hiifer, Molecular evidence of a lignin peroxidase H8 homologue in Fusurium oxysporum, Folia Microbial., 1996 (in press). 1111 E. Hammer, Ph.D. thesis, 1995, Ernst-Moritz-Arndt-Universitat, Greifswald.
Fuel Processing
Technology
52 (1997) 73-77
Components of the ligninolytic system of Fusarium oxysporum and Trichoderma atroviride Heike Mijnkemann *, Udo Hiilker, Milan Hijfer Botanisches Institut der lJnic;ersitiitBonn, Kirschallee I, 53115 Bonn, Germany
Abstract The ligninolytic system in the two deuteromycetous fungi Fusarium oxysporum and Trichoderma atrouiride, which are able to solubilize low-rank coal [l], has been proved to have several components. Analysis of the chromosomal DNA of these fungi revealed distinct bands with probes coding for three ligninase isoenzymes (H7, H8, HlO), glyoxal oxidase and arylalcohol dehydrogenase of the basidiomycete Phanerochaete chrysosporium. These data constitute a strong indication for the existence in F. oxysporum and T. atrouiride of a ligninolytic system comparable to that in P. chrysosporium that may be involved in the process of coal solubilization. 0 1997 Elsevier Science B.V. Keywords: Coal solubilization; Lignin peroxidases; oxysporum; Trichodenna atrouiride
Glyoxal
oxidase;
Arylalcohol
dehydrogenase;
Fusarium
1. Introduction
The
two
deuteromycetes
Fusarium
oxysporum
shown to solubilize low-rank coal. Moreover, both on the carbon source used for cultivation [1,2]. low-rank coal constitutively, in T. atrouiride the induced [2]. Ligninolytic enzymes have as yet been in T. atroviride. Phanerochaete chrysosporium, a wood-attacking
’ Corresponding
and Trichoderma atroviride were fungi solubilize coal in dependence Whereas F. oxysporum solubilizes ability to solubilize coal has to be found neither in F. oxysporum nor and lignin-degrading
author.
037%3820/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SO378-3820(97)000 17-9
white-rot fun-
74
H. Miinkemann et al. /Fuel
Processing Technology 52 (1997) 73-77
gus, has been extensively studied. The ligninolytic system of this basidiomycete consists of multiple lignin peroxidases [3], manganese peroxidase [4], glyoxal oxidase [5], and lactase [6]. It was also found that the transcript level of arylalcohol dehydrogenase correlates with those of lignin peroxidase isoenzymes in ligninolytic cultures of P. chrysosporium [7]. With the exception of lactase, DNA sequences of ligninolytic enzymes have already been determined in P. chrysosporium [3-71. A possible role or even an interaction of ligninolytic enzymes in lignite solubilization still remains to be proven. In this report we demonstrate the existence of several components of the P. chrysosporium ligninolytic system in F. oxysporum and T. atroviride.
2. Materials and methods 2. I. Fungal material F. oxysporum was kindly supplied by R.M. Fakoussa of the Institute for Microbiology and Biotechnology, University of Bonn. It was collected in an open-cast mining area near Cologne. T. atroviride was isolated in the authors’ laboratory from stored low-rank coal. F, oxysporum and T. atroviride were grown at 30°C and 18°C respectively, as described previously by Htilker [2]. 2.2. Genomic DNA
Chromosomal DNA from 50 ml F. oxysporum and T. atroviride cultures was isolated using a method developed by Hoffmann [8]. 2.3. Southern blot analysis 30 pg chromosomal DNA was hydrolyzed overnight with 50 U of restriction endonuclease EcoRI in a total volume of 100 ~1 at 37°C. The hydrolyzed DNA was separated according to its molecular weight on 0.8% agarose gel. After denaturation (1.5 M NaCl and 0.5 M NaOH, 2 X 15 min) and neutralization (1.0 M Tris and 1.5 M NaCl (pH 7.4), 3 X 20 min), the DNA was transferred to a GeneScreenPlus membrane (DuPont, Bad Homburg, Germany) by overnight capillary transfer with 10 X SSC. The DNA was then covalently bound to the membrane by incubation for 5 min in 0.05 M NaOH and neutralized for 5 min in 2 X SSC.
2.4. Probes Genomic fragments from P. chrysosporium coding for ligninase isoenzymes H7, H8 and HlO were kindly supplied by D. Cullen of the Department of Bacteriology, University of Wisconsin, USA [9]. cDNA probes encoding glyoxal oxidase and arylalcoho1 dehydrogenase, both from P. chrysosporium, were kindly supplied by P.J. Kersten of the Department of Agriculture, University of Wisconsin, USA [5] and J. Reiser of the
H. Miinkemann et al. / Fuel Processing Technology 52 (1997) 73-77
15
National Institutes of Health, NII, Bethesda, MD, USA [7], respectively. The above mentioned fragments were labelled with a random fluorescein labelling kit (DuPont) and used for hybridization experiments according to the manufacturer’s instructions. The colour reaction was carried out accordingly. 2.5. Chemicals All chemicals
were of analytical
grade from Boehringer,
Merck and Serva.
3. Results Detection amplification
of a gene coding for ligninase isoenzyme H8 in F. oxysporum by PCR [lo] led to further Southern blot analysis. Genomic DNA was isolated
1
2
1
2
1
2
Fig. 1. Ligninase isoenzymes of F. oxyspomm and T. atrouiride. Southern blot analysis of genomic DNA from F. oxysporum (lane 1) and T. atrouiride (lane 2) hybridized with ligninase isoenzyme H7 (a), ligninase isoenzyme H8 (b) and ligninase isoenzyme HlO (c) genomic DNA fragments from P. chrysosporium, respectively.
Fig. 2. Glyoxal oxidase of F. oxysporum and T. atrouiride. Southern blot analysis of genomic DNA from F. onysporum (lane 1) and T. atrouiride (lane 2) hybridized with glx-lc cDNA from P. chrysosporium.
16
H. Miinkemann et al. /Fuel
Processing Technology 52 (1997) 73-77
Fig. 3. Arylalcohol dehydrogenase of F. oxysporum and T. atrouiride. Southern blot analysis of genomic DNA from F. oxysporum (lane 1) and T. atrouiride (lane 2) hybridized with arylalcohol dehydrogenase (AAD) cDNA from P. chrysospon’um.
from both F. oxysporum and T. atroviride, digested with restriction endonuclease EcoRI, separated on agarose gels, and blotted to GeneScreenPlus membranes. For hybridization, we focused on lignin peroxidase (lip) genes which are highly expressed under carbon and nitrogen deficiency in ligninolytic P. chrysosporium cultures (ligninase isoenzymes H7, H8 and HlO), as well as on glyoxal oxidase, and arylalcohol dehydrogenase. Hybridization and washing steps (2.0 X SSC and 0.1% SDS; 0.2 X SSC and 0.1% SDS) were performed at 65°C. This high temperature and the ion concentration of the washing buffers resulted in high stringency, which allowed only the annealing of homologous DNA sequences. Distinct signals of the three lip genes were detected in the genomic DNA of both fungi, F. oxysporum and T. atroviride (Fig. la-c). With the complete glyoxal oxidase-coding region from the basidiomycete P. chrysosporium, bands were obtained with the genomic DNA from both fungi (Fig. 2). Probing the blotted chromosomal DNA from the two fungi with a cDNA of P. chrysosporium encoding arylalcohol dehydrogenase resulted in bands of different intensities in both DNA preparations (Fig. 3). For hybridization experiments of the last two enzymes, the same high stringency conditions were applied.
4. Discussion The chemical structure of low-rank coal shows similarity to lignin. Several basidiomycetes are known to possess a ligninolytic system. The best characterized system is that of P. chrysosporium, a white-rot fungus which is able to decompose wood. Since these enzymes are involved in lignin degradation by the fungus also, the coal-active molds F. oxysporum and T. atroviride could use these enzymes for coal solubilization. Enzymatic assays, however, have as yet revealed no corresponding enzyme activity [ 111. In this communication, we demonstrate the existence of genetic information in the two coal-active molds for some of the potentially coal-solubilizing enzymes, viz. lignin
H. Mb;nkemann et al./ Fuel Processing Technology 52 (1997) 73-77
II
peroxidase isoenzymes (H7, H8, HlO), glyoxal oxidase and arylalcohol dehydrogenase. These ligninolytic enzymes do not cross-hybridize with other published fungal DNA sequences, hence, these results imply the existence of a homologous ligninolytic system in F. oxysporum and T. atrouiride. The results obtained with the ligninase isoenzyme H8 confirm previous findings acquired by PCR amplification of F. oxysporum genomic DNA and by Northern blot analysis [lo]. Because the two mold genera acquired the ability to solubilize coal depending on the growth substrate [1,2], analysis of the mRNA expression of the individual enzymes under appropriate growth conditions will provide unambiguous answers to the question of enzyme participation in the process of coal solubilization.
Acknowledgements This work was supported by the BMBF, grant no. 0320620B
to M.H.
References [l] U. HBlker, R.M. Fakoussa, M. Hofer, Growth substrates control the ability of Fusarium oxysporum to solubilize low-rank coal, Appl. Microbial. Biotechnol. 44 (1995) 351-355. [2] U. Hiilker, H. Monkemann, M. HGfer, A test system to analyze the complex physiological states of coal-solubilizing fungi, Fuel Processing Technol., 1996 (in press). [3] J. Gaskell, P. Stewart, P. Kersten, S. Covert, J. Reiser, D. Cullen, Establishment of genetic linkage by allele-specific polymerase chain reaction: Application to the lignin peroxidase gene family of Phanerochaete chrysosporium, Bio/Technology 12 (1994) 1372-1375. [4] B.J. Godfrey, M.B. Mayfield, J.A. Brown, M.H. Gold, Characterization of a gene encoding a manganese peroxidase from Phanerochaete chrysosporium, Gene 93 (1990) 119-124. [5] P.J. Kersten, D. Cullen, Cloning and characterization of a cDNA encoding glyoxal oxidase, a H,O,-producing enzyme from the lignin-degrading basidiomycete Phanerochaete chrysosporium, Proc. Natl. Acad. Sci. USA 90 (1993) 7411-7413. [6] C. Srinivasan, T.M. D’Souza, K. Boominathan, CA. Reddy, Demonstration of a lactase in the white-rot basidiomycete Phanerochaete chrysosporium BKM-F1767, Appl. Environ, Microbial. 61 (1995) 42744277. [7] J. Reiser, A. Muheim, M. Hardegger, G. Frank, A. Fiechter, Arylalcohol dehydrogenase from the white-rot fungus Phanerochaete chtysosporium: Gene cloning, sequence analysis, expression and purification of the recombinant enzyme, J. Biol. Chem. 269 (1994) 28152-28159. [S] C.S. Hoffmann, F. Winston, A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli, Gene 57 (1987) 267-272. [9] R.T. Lamar, B. Schoenike, A. van den Wymelenberg, P. Stewart, D.M. Dietrich, D. Cullen, Quantification of fungal mRNA’s in complex substrates by reverse transcription PCR and its application to Phanerochaete chrysosporium-colonized soil, Appl. Environ. Microbial. 61 (1995) 2122-2126. [lo] H. Monkemann, U. Hiilker, 0. Golubnichaya-Labudova, H. Lichtenberg-Fratt, M. Hiifer, Molecular evidence of a lignin peroxidase H8 homologue in Fusurium oxysporum, Folia Microbial., 1996 (in press). 1111 E. Hammer, Ph.D. thesis, 1995, Ernst-Moritz-Arndt-Universitat, Greifswald.