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Characterization of the xylanase regulator protein gene, xlnR, in Talaromyces cellulolyticus (formerly known as Acremonium cellulolyticus) a
Tatsuya Fujii , Hiroyuki Inoue & Kazuhiko Ishikawa a
Biomass Refinery Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Higashi-Hiroshima, Japan Published online: 16 Jun 2014.
To cite this article: Tatsuya Fujii, Hiroyuki Inoue & Kazuhiko Ishikawa (2014) Characterization of the xylanase regulator protein gene, xlnR, in Talaromyces cellulolyticus (formerly known as Acremonium cellulolyticus), Bioscience, Biotechnology, and Biochemistry, 78:9, 1564-1567, DOI: 10.1080/09168451.2014.923298 To link to this article: http://dx.doi.org/10.1080/09168451.2014.923298
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Bioscience, Biotechnology, and Biochemistry, 2014 Vol. 78, No. 9, 1564–1567
Note
Characterization of the xylanase regulator protein gene, xlnR, in Talaromyces cellulolyticus (formerly known as Acremonium cellulolyticus) Tatsuya Fujii*, Hiroyuki Inoue and Kazuhiko Ishikawa Biomass Refinery Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Higashi-Hiroshima, Japan Received January 24, 2014; accepted April 1, 2014
Downloaded by [New York University] at 10:20 17 October 2014
http://dx.doi.org/10.1080/09168451.2014.923298
We cloned a putative Talaromyces cellulolyticus (formerly known as Acremonium cellulolyticus) xlnR gene and isolated a xlnR disruptant strain. XlnR protein was localized in the nucleus. Xylanase production by the xlnR disruptant was lower than in the control strain at both the enzyme and transcriptional level. These data suggest that the XlnR protein regulates xylanase production in T. cellulolyticus. Key words:
xylanase; cellulase; Acremonium cellulolyticus; Talaromyces cellulolyticus
Cellulase and hemicellulase are the major enzymes that hydrolyze cellulose and hemicellulose to monomeric sugars. The filamentous fungus Talaromyces cellulolyticus (formerly known as Acremonium cellulolyticus) was isolated in 1982 from soil in Japan and was shown to be a cellulose-degrading organism.1) The taxon of this organism was recently revised from the genus Acremonium to the genus Talaromyces.2) T. cellulolyticus is one of several fungi that hold promise as an alternative to Hypocrea jecorina for use in the industrial production of cellulase. The enzymes from T. cellulolyticus reportedly produce glucose more rapidly from various lignocellulosic materials than the enzymes from H. jecorina.3) Over 40 reports or patents related to T. cellulolyticus have been published, making it one of the best characterized cellulase-producing organisms. Furthermore, a genomic database (unpublished data) and transformation system for T. cellulolyticus4) have been constructed by our group. We successfully overexpressed the cellulase and hemicellulase genes in this organism and constructed a starchinducible homologous expression system.5,6) The expression of cellulase and hemicellulase genes is regulated by various transcriptional factors in filamentous fungi.7–10) The transcriptional factor XlnR/ Xyr1 was isolated as an inducer of xylanase genes in Aspergillus niger.10) In H. jecorina, a XlnR/Xyr1 gene disruptant strain is incapable of both xylanase and cellulase production, suggesting that XlnR/Xyr1 functions as a main regulator of cellulase and hemicellulase gene *Corresponding author. Email: [email protected] © 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry
expression.11) Although CreA, a carbon catabolite repressor protein, represses cellulase and xylanase production in T. cellulolyticus,12) no other transcriptional factors involved in cellulase and hemicellulase production have been identified to date. In the present study, we cloned a putative xlnR gene from T. cellulolyticus, and then isolated a recombinant strain in which the xlnR gene was disrupted. XlnR protein was found to localize in the nucleus. Although the level of cellulase production by the xlnR disruptant was similar to that of the control strain, the production of xylanase by the xlnR disruptant was drastically lower than that of the control strain at both the enzyme and transcriptional level. These data suggest that the cloned putative xlnR gene encodes a xylanase regulator protein. T. cellulolyticus YP-4,5) which is a uracil auxotrophic strain derived from T. cellulolyticus Y-941) (CBS 136886, FERM BP-5826), was maintained on potato dextrose agar (Difco, Detroit, MI) plates containing 1 g/L uracil and 1 g/L uridine. The transformants were maintained on MM plates (1% glucose, 10 mM NH4Cl, 10 mM potassium phosphate (pH 6.5), 7 mM KCl, and 2 mM MgSO4). First, we searched the T. cellulolyticus Y-94 genome database for a putative xlnR gene (unpublished data). A 2831-bp nucleotide sequence (including putative introns) was found that encodes an 898-amino acid protein. A database search revealed that the amino acid sequence of this protein was similar to that of the XlnR protein of T. marneffei (XP_002145389), Aspergillus acleatus (BAL72831), and H. jecorina (EGR48040), at 90, 62, and 47% identity, respectively. The predicted protein contains a zinc-finger domain that is conserved among the xlnR proteins. These results suggest that the nucleotide sequence encodes a T. cellulolyticus Y-94 ortholog of the xlnR gene. The nucleotide and amino acid sequence of xlnR from Y-94 will be deposited in the GenBank/EMBL/DDBJ nucleotide database under accession No. AB915784. Next, we isolated an xlnR gene disruptant strain to investigate XlnR function. Gene disruption was carried out by homologous recombination, and we confirmed that xlnR was replaced with the pyrF marker, as shown in Fig. 1. Plasmids used for disrupting the xlnR gene
XlnR of Talaromyces cellulolyticus
were constructed by inserting DNA fragments carrying the 5′ and 3′ regions of xlnR into the upstream and downstream regions of the pyrF gene in pbs-pyrF.4) DNA fragments carrying the 5′ regions fused with appropriate restriction sites were amplified using the primers 5′-ccgcggccgcagcggttgaaggacagtcag-3′ and 5′cctctagatggccgcataatttggtgac-3′, digested with NotI and XbaI, and ligated with pBS-pyrF already spliced with the same restriction enzymes. The 3′ region of each gene was amplified using the primers 5′-ccgaattcgttcttgcactttatcgctgga-3′ and 5′-ccgtcgaccgttaggtgagtcccaagct-3′, digested with EcoRI and SalI, and inserted into the same restriction sites of the resulting plasmids to generate pDXlnR. The plasmid was digested with NotI before using fungal transformation. Fungal transformation was carried out as described previously.4) Total
Downloaded by [New York University] at 10:20 17 October 2014
(A)
7.2 kb
YP-4
probe
P
P
xlnR
pyrF
YDX P
P
probe
P
pyrF 4.3 kb YDX
7.2 kb
4.3 kb
(B)
YP-4
GFP
DAPI
Xylan
Cellulose
Fig. 1. Disruption of the xlnR Gene and Cellular Localization of the XlnR Protein. Notes: (A) The strategy for homologous recombination into the xlnR locus to construct xlnR gene disruptants is shown. Total DNA was isolated and digested with PstI before Southern blotting. (B) YXGFP was observed under a fluorescence microscope. Fluorescence emitted by GFP is indicated by an arrowhead.
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fungal DNA was Southern blotted and analyzed using a DIG DNA labeling and detection kit (Roche, Basel, Switzerland) according to the manufacturer’s instructions. Southern blotting of total DNA from the YP-4 and YDX (xlnR disruptant) strains revealed a 7.2 kb PstI DNA fragment specific for YP-4 xlnR but not for YDX xlnR. YDX generated a 4.3-kb band, indicating deletion of the xlnR gene (Fig. 1(A)). The cellular localization of XlnR protein was investigated by observation of XlnR-green fluorescent protein (GFP) fusion protein fluorescence. The pXlnGFP plasmid used for production of the XlnR-GFP fusion was constructed as follows: the DNA fragment encoding GFP was amplified from pGFPuv (Takara Bio, Otsu, Japan) using the primers 5′-aattgtcgacatgagtaaaggagaagaacttttcac-3′ and 5′-aattgggcccctatttgtatagttcatccatgcc-3′, digested with SalI and ApaI, and ligated with pBS-pyrF already spliced with the same restriction enzymes. The xlnR DNA fragment containing the promoter region was amplified using the primers 5′-aattgaattcgttggttatacagggcttgc-3′ and 5′-aattgtcgacgatgaatgtatgtggcaacg-3′, digested with EcoRI and SalI, and inserted into the same restriction sites of the resulting plasmids to generate pXlnGFP. Strain YP-4 carrying pXlnGFP (YXGFP) was cultured in basic medium12) containing 5% cellulose (Solka Floc; Fiber Sales & Development, Urbana, OH) or 5% xylan (birch-wood xylan; SIGMA, St. Louis, MO) at 30 °C for 24 h, after which the cells were collected and incubated with 1 mM 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, Lonza, Walkersville, MD) and then analyzed under a fluorescence microscope (ZEISS, Oberkochen, Germany) to determine the fluorescence excitation of GFP and DAPI. Fig. 1(B) shows that fluorescence was emitted by GFP in DAPI-stained nuclei, indicating that XlnR protein was localized in the nucleus. YPyrF, which harbors a single copy of pbs-pyrF in the pyrF locus of the YP-4 genome,12) and YDX was cultured in medium containing cellulose or xylan, and enzyme production by the strains was then examined in time-course experiments. The strains were cultivated in 10 mL of basic medium12) supplemented with 2 g/L urea and 40 g/L glycerol in 100 mL Erlenmeyer flasks at 30 °C for 72 h on a rotary shaker operated at 230 rpm. The cells were washed three times with saline, and then aliquots of the washed cells were inoculated into 10 mL of basic medium supplemented with 4 g/L urea and 50 g/L carbon source in 100 mL Erlenmeyer flasks which were then incubated at 30 °C on a rotary shaker at 230 rpm. Cellulose and xylan were used as the carbon source. Filter-paper degrading enzyme (FPase) and xylanase activities were measured as previously described.3) When cellulose was used as the sole carbon source, YDX produced 51% of the xylanase activity of YPyrF after 120 h of cultivation (Fig. 2(A)), although the FPase activities of both strains were similar (YDX produced 101% of the FPU activity of YPyrF). When xylan was used as the sole carbon source, the xylanase activities of both strains were clearly lower than when the strains were cultured with cellulose (Fig. 2(A)). Furthermore, the xylanase activity of YDX cultured with xylan was very low. These data suggest that the xlnR gene contributes to xylanase production in T. cellulolyticus.
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(A) Relative activity (%) R
120 100 80 60 40 20 0
(B)
0
Relative expression ratio
8
Relative expression ratio
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100
0 50 Culturing time (h)
YPyrF
7
100
150
YDX
6 5 4 3 2 1 0
(C)
50
Bioscience, Biotechnology, and Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbbb20
Characterization of the xylanase regulator protein gene, xlnR, in Talaromyces cellulolyticus (formerly known as Acremonium cellulolyticus) a
Tatsuya Fujii , Hiroyuki Inoue & Kazuhiko Ishikawa a
Biomass Refinery Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Higashi-Hiroshima, Japan Published online: 16 Jun 2014.
To cite this article: Tatsuya Fujii, Hiroyuki Inoue & Kazuhiko Ishikawa (2014) Characterization of the xylanase regulator protein gene, xlnR, in Talaromyces cellulolyticus (formerly known as Acremonium cellulolyticus), Bioscience, Biotechnology, and Biochemistry, 78:9, 1564-1567, DOI: 10.1080/09168451.2014.923298 To link to this article: http://dx.doi.org/10.1080/09168451.2014.923298
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Bioscience, Biotechnology, and Biochemistry, 2014 Vol. 78, No. 9, 1564–1567
Note
Characterization of the xylanase regulator protein gene, xlnR, in Talaromyces cellulolyticus (formerly known as Acremonium cellulolyticus) Tatsuya Fujii*, Hiroyuki Inoue and Kazuhiko Ishikawa Biomass Refinery Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Higashi-Hiroshima, Japan Received January 24, 2014; accepted April 1, 2014
Downloaded by [New York University] at 10:20 17 October 2014
http://dx.doi.org/10.1080/09168451.2014.923298
We cloned a putative Talaromyces cellulolyticus (formerly known as Acremonium cellulolyticus) xlnR gene and isolated a xlnR disruptant strain. XlnR protein was localized in the nucleus. Xylanase production by the xlnR disruptant was lower than in the control strain at both the enzyme and transcriptional level. These data suggest that the XlnR protein regulates xylanase production in T. cellulolyticus. Key words:
xylanase; cellulase; Acremonium cellulolyticus; Talaromyces cellulolyticus
Cellulase and hemicellulase are the major enzymes that hydrolyze cellulose and hemicellulose to monomeric sugars. The filamentous fungus Talaromyces cellulolyticus (formerly known as Acremonium cellulolyticus) was isolated in 1982 from soil in Japan and was shown to be a cellulose-degrading organism.1) The taxon of this organism was recently revised from the genus Acremonium to the genus Talaromyces.2) T. cellulolyticus is one of several fungi that hold promise as an alternative to Hypocrea jecorina for use in the industrial production of cellulase. The enzymes from T. cellulolyticus reportedly produce glucose more rapidly from various lignocellulosic materials than the enzymes from H. jecorina.3) Over 40 reports or patents related to T. cellulolyticus have been published, making it one of the best characterized cellulase-producing organisms. Furthermore, a genomic database (unpublished data) and transformation system for T. cellulolyticus4) have been constructed by our group. We successfully overexpressed the cellulase and hemicellulase genes in this organism and constructed a starchinducible homologous expression system.5,6) The expression of cellulase and hemicellulase genes is regulated by various transcriptional factors in filamentous fungi.7–10) The transcriptional factor XlnR/ Xyr1 was isolated as an inducer of xylanase genes in Aspergillus niger.10) In H. jecorina, a XlnR/Xyr1 gene disruptant strain is incapable of both xylanase and cellulase production, suggesting that XlnR/Xyr1 functions as a main regulator of cellulase and hemicellulase gene *Corresponding author. Email: [email protected] © 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry
expression.11) Although CreA, a carbon catabolite repressor protein, represses cellulase and xylanase production in T. cellulolyticus,12) no other transcriptional factors involved in cellulase and hemicellulase production have been identified to date. In the present study, we cloned a putative xlnR gene from T. cellulolyticus, and then isolated a recombinant strain in which the xlnR gene was disrupted. XlnR protein was found to localize in the nucleus. Although the level of cellulase production by the xlnR disruptant was similar to that of the control strain, the production of xylanase by the xlnR disruptant was drastically lower than that of the control strain at both the enzyme and transcriptional level. These data suggest that the cloned putative xlnR gene encodes a xylanase regulator protein. T. cellulolyticus YP-4,5) which is a uracil auxotrophic strain derived from T. cellulolyticus Y-941) (CBS 136886, FERM BP-5826), was maintained on potato dextrose agar (Difco, Detroit, MI) plates containing 1 g/L uracil and 1 g/L uridine. The transformants were maintained on MM plates (1% glucose, 10 mM NH4Cl, 10 mM potassium phosphate (pH 6.5), 7 mM KCl, and 2 mM MgSO4). First, we searched the T. cellulolyticus Y-94 genome database for a putative xlnR gene (unpublished data). A 2831-bp nucleotide sequence (including putative introns) was found that encodes an 898-amino acid protein. A database search revealed that the amino acid sequence of this protein was similar to that of the XlnR protein of T. marneffei (XP_002145389), Aspergillus acleatus (BAL72831), and H. jecorina (EGR48040), at 90, 62, and 47% identity, respectively. The predicted protein contains a zinc-finger domain that is conserved among the xlnR proteins. These results suggest that the nucleotide sequence encodes a T. cellulolyticus Y-94 ortholog of the xlnR gene. The nucleotide and amino acid sequence of xlnR from Y-94 will be deposited in the GenBank/EMBL/DDBJ nucleotide database under accession No. AB915784. Next, we isolated an xlnR gene disruptant strain to investigate XlnR function. Gene disruption was carried out by homologous recombination, and we confirmed that xlnR was replaced with the pyrF marker, as shown in Fig. 1. Plasmids used for disrupting the xlnR gene
XlnR of Talaromyces cellulolyticus
were constructed by inserting DNA fragments carrying the 5′ and 3′ regions of xlnR into the upstream and downstream regions of the pyrF gene in pbs-pyrF.4) DNA fragments carrying the 5′ regions fused with appropriate restriction sites were amplified using the primers 5′-ccgcggccgcagcggttgaaggacagtcag-3′ and 5′cctctagatggccgcataatttggtgac-3′, digested with NotI and XbaI, and ligated with pBS-pyrF already spliced with the same restriction enzymes. The 3′ region of each gene was amplified using the primers 5′-ccgaattcgttcttgcactttatcgctgga-3′ and 5′-ccgtcgaccgttaggtgagtcccaagct-3′, digested with EcoRI and SalI, and inserted into the same restriction sites of the resulting plasmids to generate pDXlnR. The plasmid was digested with NotI before using fungal transformation. Fungal transformation was carried out as described previously.4) Total
Downloaded by [New York University] at 10:20 17 October 2014
(A)
7.2 kb
YP-4
probe
P
P
xlnR
pyrF
YDX P
P
probe
P
pyrF 4.3 kb YDX
7.2 kb
4.3 kb
(B)
YP-4
GFP
DAPI
Xylan
Cellulose
Fig. 1. Disruption of the xlnR Gene and Cellular Localization of the XlnR Protein. Notes: (A) The strategy for homologous recombination into the xlnR locus to construct xlnR gene disruptants is shown. Total DNA was isolated and digested with PstI before Southern blotting. (B) YXGFP was observed under a fluorescence microscope. Fluorescence emitted by GFP is indicated by an arrowhead.
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fungal DNA was Southern blotted and analyzed using a DIG DNA labeling and detection kit (Roche, Basel, Switzerland) according to the manufacturer’s instructions. Southern blotting of total DNA from the YP-4 and YDX (xlnR disruptant) strains revealed a 7.2 kb PstI DNA fragment specific for YP-4 xlnR but not for YDX xlnR. YDX generated a 4.3-kb band, indicating deletion of the xlnR gene (Fig. 1(A)). The cellular localization of XlnR protein was investigated by observation of XlnR-green fluorescent protein (GFP) fusion protein fluorescence. The pXlnGFP plasmid used for production of the XlnR-GFP fusion was constructed as follows: the DNA fragment encoding GFP was amplified from pGFPuv (Takara Bio, Otsu, Japan) using the primers 5′-aattgtcgacatgagtaaaggagaagaacttttcac-3′ and 5′-aattgggcccctatttgtatagttcatccatgcc-3′, digested with SalI and ApaI, and ligated with pBS-pyrF already spliced with the same restriction enzymes. The xlnR DNA fragment containing the promoter region was amplified using the primers 5′-aattgaattcgttggttatacagggcttgc-3′ and 5′-aattgtcgacgatgaatgtatgtggcaacg-3′, digested with EcoRI and SalI, and inserted into the same restriction sites of the resulting plasmids to generate pXlnGFP. Strain YP-4 carrying pXlnGFP (YXGFP) was cultured in basic medium12) containing 5% cellulose (Solka Floc; Fiber Sales & Development, Urbana, OH) or 5% xylan (birch-wood xylan; SIGMA, St. Louis, MO) at 30 °C for 24 h, after which the cells were collected and incubated with 1 mM 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, Lonza, Walkersville, MD) and then analyzed under a fluorescence microscope (ZEISS, Oberkochen, Germany) to determine the fluorescence excitation of GFP and DAPI. Fig. 1(B) shows that fluorescence was emitted by GFP in DAPI-stained nuclei, indicating that XlnR protein was localized in the nucleus. YPyrF, which harbors a single copy of pbs-pyrF in the pyrF locus of the YP-4 genome,12) and YDX was cultured in medium containing cellulose or xylan, and enzyme production by the strains was then examined in time-course experiments. The strains were cultivated in 10 mL of basic medium12) supplemented with 2 g/L urea and 40 g/L glycerol in 100 mL Erlenmeyer flasks at 30 °C for 72 h on a rotary shaker operated at 230 rpm. The cells were washed three times with saline, and then aliquots of the washed cells were inoculated into 10 mL of basic medium supplemented with 4 g/L urea and 50 g/L carbon source in 100 mL Erlenmeyer flasks which were then incubated at 30 °C on a rotary shaker at 230 rpm. Cellulose and xylan were used as the carbon source. Filter-paper degrading enzyme (FPase) and xylanase activities were measured as previously described.3) When cellulose was used as the sole carbon source, YDX produced 51% of the xylanase activity of YPyrF after 120 h of cultivation (Fig. 2(A)), although the FPase activities of both strains were similar (YDX produced 101% of the FPU activity of YPyrF). When xylan was used as the sole carbon source, the xylanase activities of both strains were clearly lower than when the strains were cultured with cellulose (Fig. 2(A)). Furthermore, the xylanase activity of YDX cultured with xylan was very low. These data suggest that the xlnR gene contributes to xylanase production in T. cellulolyticus.
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(A) Relative activity (%) R
120 100 80 60 40 20 0
(B)
0
Relative expression ratio
8
Relative expression ratio
Downloaded by [New York University] at 10:20 17 October 2014
100
0 50 Culturing time (h)
YPyrF
7
100
150
YDX
6 5 4 3 2 1 0
(C)
50
