Enhancing cellulase and hemicellulase production by genetic modification of the carbon catabolite repressor gene, creA, in Acremonium cellulolyticus
© Fujii et al.; licensee Springer. 2013
Received: 5 December 2013
Accepted: 12 December 2013
Published: 20 December 2013
Acremonium cellulolyticus is one of several fungi that offer promise as an alternative to Trichoderma reesei for use in industrial cellulase production. However, the mechanism of cellulase production has not been studied at the molecular level because adequate genetic engineering tools for use in A. cellulolyticus are lacking. In the present study, we developed a gene disruption method for A. cellulolyticus, which needs a longer homologous region length. We cloned a putative A. cellulolyticus creA gene that is highly similar to the creA genes derived from other filamentous fungi, and isolated a creA disruptant strain by using the disruption method. Growth of the creA disruptant on agar plates was slower than that of the control strain. In the wild-type strain, the CreA protein was localized in the nucleus, suggesting that the cloned gene encodes the CreA transcription factor. Cellulase and xylanase production by the creA disruptant were higher than that of the control strain at the enzyme and transcription levels. Furthermore, the creA disruptant produced cellulase and xylanase in the presence of glucose. These data suggest both that the CreA protein functions as a catabolite repressor protein, and that disruption of creA is effective for enhancing enzyme production by A. cellulolyticus.
Lignocellulosic biomass is a promising material for use in biorefining because it contains a large amount of sugar in the form of cellulose and hemicellulose (Lynd 1996). Cellulase and hemicellulase are the two major families of enzymes that hydrolyze cellulose and hemicellulose (a lignocellulose) to monomeric sugars. Some filamentous fungi, such as Trichoderma reesei, secrete large amounts of cellulase and hemicellulase (Goyal et al. 1991; Krogh et al. 2004; Sehnem et al. 2006; Wen et al. 2005). The cellulases produced by fungi include three major groups of enzymes: endoglucanases, which randomly hydrolyze internal glycosidic linkages; cellobiohydrolases, which produce cellobiose from cellulose chain ends; and β-glucosidases, which convert cellobiose into glucose (Goyal et al. 1991).
The filamentous fungus, Acremonium cellulolyticus, which was isolated in 1982 from soil in Japan, is a cellulose-degrading organism (Yamanobe et al. 1987) and is one of several fungi that offer promise as an alternative to T. reesei for the industrial production of cellulase. A cellulase mixture produced by A. cellulolyticus is commercially sold as ‘Acremonium cellulase’ by Meiji Seika Pharma Co.. Strains TN, C-1, and CF-2612, which are cellulase hyper-producing mutants, were isolated from the wild type strain Y-94 by random mutagenesis (Fang et al. 2009; Yamanobe et al. 2003). The enzymes from A. cellulolyticus reportedly produce glucose more rapidly from various lignocellulosic materials than the enzymes from T. reesei (Fujii et al. 2009). Over 40 reports or patents related to A. cellulolyticus have been published, making it one of the best characterized cellulase-producing organisms. Furthermore, a genomic database (unpublished data) and transformation system for A. cellulolyticus (Fujii et al. 2012) have been constructed by our group. We successfully overexpressed cellulase and hemicellulase genes in this organism and constructed a starch-inducible homologous expression system (Inoue et al. 2013; Kanna et al. 2011), thus making available genetic engineering tools suitable for A. cellulolyticus. However, the development of these tools is not sufficient because gene targeting, such as gene disruption by homologous recombination, is difficult in A. cellulolyticus.
Several transcription factors have been reported as regulators of cellulase and hemicellulase gene expression in other filamentous fungi, e.g., XlnR/Xyr1 for genes encoding cellulase, hemicellulase, and accessory enzymes involved in xylan degradation in Aspergillus niger and T. reesei (Stricker et al. 2006; van Peij et al. 1998); Ace2 for cellulase genes in T. reesei (Aro et al. 2001); BglR for the β-glucosidase gene in T. reesei (Nitta et al. 2012); and AraR for the L-arabinose reductase gene in A. niger and Aspergillus nidulans (Battaglia et al. 2011). These transcription factors specifically regulate the expression of cellulase and hemicellulase genes. On the other hand, some transcription factors regulate a wide range of genes including cellulase and hemicellulase genes. These factors include CreA (Dowzer and Kelly 1989; Ilmen et al. 1996;: Nakari-Setälä et al. 2009; Wang et al. 2013), which is involved in catabolite repression; AreA (Lockington et al. 2002), which is involved in nitrogen source assimilation; and the Hap complex (Tsukagoshi et al. 2001), which regulates various genes. Although a number of transcription factors involved in regulating cellulase and hemicellulase gene expression in other filamentous fungi have been analyzed, the regulation of these gene expressions in A. cellulolyticus has not been investigated. Because A. cellulolyticus is an industrially important fungal species, how cellulase and hemicellulase gene expression is regulated in this organism is crucial for the development of more efficient enzyme production methods.
In the present study, we cloned a putative creA gene from A. cellulolyticus that is highly similar to the creA genes of other filamentous fungi and then isolated a recombinant strain in which the creA gene was disrupted. The length of the homologous region was important for gene disruption. Growth of the creA disruptant on agar plates was slower than that of the control strain, and CreA protein was found to localize in the nucleus. The production of cellulase and xylanase by the creA disruptant was higher than that of the control strain at both the enzyme and transcription level. Furthermore, the creA disruptant produced cellulase and xylanase in the presence of glucose. These data suggest that the cloned putative creA gene encodes a catabolite repressor protein, and that disruption of creA leads to enhanced enzyme productivity.
Materials and methods
Strains, cultures, and media
Characteristics of the A. cellulolyticus strains and plasmids used in this study
Strain or Plasmid
A. cellulolyticus Y-94
Wild type (FERM BP-5826)
Yamanobe et al. (1987)
Uracil auxotroph mutant derived from Y-94
Inoue et al. (2013)
YP-4 prototrophic transformant horboring pDCre2500, creA disruptant.
YP-4 prototrophic transformant horboring a single copy pbs-pyrF in pyrF loci.
YP-4 prototrophic transformant harboring pCreGFP
Ampr PyrFr; pBluescript KS(+) derivative containing 2.7-kb fragment harboring pyrF from Y-94
Fujii et al. (2012)
Ampr PyrFr; pbs-pyrF derivative containing 2.5 kb upstream and 2.5 kb downstream regions of creA
Ampr PyrFr; pbs-pyrF derivative containing 1.0 kb upstream and 1.0 kb downstream regions of creA
Ampr PyrFr; pbs-pyrF derivative containing GFP and creA fused gene
Plasmid construction and fungal transformation
Nucleotide primers used in this study
For plasmids construction
For quantitative PCR
Total RNA was extracted from disrupted fungal cells. Single-stranded cDNA was synthesized and then real-time quantitative PCR proceeded as described previously (Fujii et al. 2010). Table 1 lists the gene-specific primers used. The primers were designed according to the sequence of each gene in the database: endoglucanase (cel5A), HV540858; endoglucanase (cel5B), HV540855; cellobiohydrolase II (cel6A), AB022429; cellobiohydrolase I (cel7A), E39854; endoglucanase (cel7B), HV540856; and xylanase (xyl11B), E39857. The expression of each gene was normalized against that of the glyceraldehyde dehydrogenase gene (gpdA). Results are shown as relative expression.
YPyrF and YCreGFP were cultured in MM medium 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.
Filter-paper degrading enzyme (FPase) and xylanase activities were measured as previously described (Fujii et al. 2009). The concentration of soluble protein was determined using the method of Lowry et al. (Lowry et al. 1951). The glucose concentration was determined using an HPLC system equipped with an RI-2031 Plus refractive index detector (Jasco, Tokyo, Japan) and an Aminex HPX-87P column (Bio-Rad, Hercules, CA) fitted with a Carbo-P micro-guard cartridge (Bio-Rad). The mobile phase was double-deionized water, the flow rate was 1.0 mL/min, and the column temperature was 80°C.
The nucleotide and amino acid sequences of creA and gpdA from Y-94 are to appear in the GenBank/EMBL/DDBJ nucleotide database under accession nos. AB847424 and AB847425, respectively.
Characterization of the putative creA gene
Isolation of a creA disruptant strain
Efficiency of A. cellulolyticus creA gene disruption
Number of transformants
Number of creA disruptants
Gene disruption efficiency (%)
The role of creA in cellulase and hemicellulase production
Next, we measured the activity of the enzymes FPase and xylanase in the culture supernatant after 48 h of cultivation under various conditions. The activity in the YPyrF supernatant when the cells were grown only on cellulose (5:0) was taken as 100% (Figure 4B). The relative FPase activity in YpyrF supernatants was 142% (cellulose:glucose = 4:1), 45% (2.5:2.5), and 5% (1:4), whereas the relative xylanase activity was 155% (4:1), 101% (2.5:2.5), and 3% (1:4) (Figure 4B). When the glucose concentration was high (1:4), the FPase and xylanase activities of YPyrF were very low, indicating that cellulase and xylanase production were repressed by glucose. The relative FPase activity in YDCre supernatants was 301% (5:0), 324% (4:1), 173% (2.5:2.5), and 168% (1:4), whereas the relative xylanase activity was 201% (5:0), 176% (4:1), 175% (2.5:2.5), and 85% (1:4) (Figure 4B). The activity of both enzymes was higher than that of the YPyrF enzymes under all conditions tested. Furthermore, although the activity of both enzymes in the YPyrF culture was very low at the highest glucose:cellulose ratio examined, their activity was much higher in the YDCre culture grown under the same conditions. When xylan was used as the sole carbon source, YDCre produced higher FPase and xylanase activities than YPyrF (Figure 4B). These data suggest that creA is involved in repression of cellulase and xylanase production.
The role of creA in cellulase and hemicellulase gene expression
In this study, we characterized the function of the A. cellulolyticus gene, creA. The deduced amino acid sequence of CreA showed that it is highly similar to CreAs produced by other filamentous fungi. In addition, CreA was found to localize in the nucleus of A. cellulolyticus. Strain YDCre showed higher cellulase and xylanase activity than strain YPyrF, and repression of enzyme activity by glucose was abolished in YDCre. These activities were regulated at the transcription level; taken together, these data suggest that CreA is a transcription factor involved in carbon catabolite repression. The present study is the first report demonstrating improved enzyme production following modification of transcriptional regulation in A. cellulolyticus.
In our previous study, we noticed that exogenous DNA can be commonly integrated into the A. cellulolyticus genome through non-homologous recombination (Fujii et al. 2012). In fact, introduction of pDCre1000 into YP-4 precluded the generation of a creA disruptant, indicating that pDCre1000 was integrated through non-homologous recombination. However, introduction of pDCre2500 into YP-4 resulted in the isolation of creA disruptants with satisfactory frequency (27%). Furthermore, we obtained a transformant that carried pbs-pyrF in the pyrF locus by introducing digested pbs-pyrF (pyrF internal digestion) (Fujii et al. 2012). These data suggest that rigorous conditions for introducing DNA are necessary for gene targeting in A. cellulolyticus; however, a future study will make rapid progress by using a the gene disruption strategy based on the present study.
The cellulase and xylanase activities (Figures 4 and 5) and the levels of the corresponding mRNAs produced (Figure 6) were higher in YDCre than in YPyrF. Enhanced enzyme activity and gene expression were observed in both glucose-containing medium and medium containing only cellulose. Furthermore, the higher cellulase and xylanase activities of YDCre were observed when the strains were cultured with xylan. These data suggest that the repression involving creA is responsive to various carbon sources. Enzyme production in a T. reesei, creA-knockout strain was shown to be higher than in the parental strain under certain conditions (Nakari-Setälä et al. 2009), which is consistent with our results described above.
In other filamentous fungi, CreA protein regulates gene expression by binding to the promoter region (Ilmen et al. 1996). Three binding sequences of CreA protein (5′-SYGGRG-3′) in other fungi were identified in a 1300 bp upstream region of cel7A of A. cellulolyticus (data not shown). Furthermore, the binding sequences were found in the upstream region of other genes analyzed in Figure 6 (data not shown). These data imply that the CreA protein of A. cellulolyticus represses gene expression by binding to the promoter regions. In T. reesei, transcription of Xyr1, which is currently considered a main inducer of cellulase and xylanase production, and of Ace1, which is a specific repressor for cellulase and hemicellulase production, were repressed by carbon catabolite repression involving CreA protein (Mach-Aigner et al. 2008; Portnoy et al. 2011). These data suggest that CreA protein of T. reesei regulates not only cellulase and hemicellulase genes but also their transcription factors. CreA protein of A. cellulolyticus may regulate other transcription factors involving cellulase and hemicellulase production as T. reesei; however, no transcription factors other than CreA of A. cellulolyticus have been investigated. Hence, further experiments, such as the identification of other transcription factors, are needed to address the mechanism of regulation of cellulase and hemicellulase production by A. cellulolyticus.
The results obtained in this study strongly indicate that disruption of creA leads to elevated cellulase and hemicellulase production in A. cellulolyticus. We are currently analyzing other transcription factors that are expected to regulate the production of these enzymes, and intend to further improve cellulase and hemicellulase production by A. cellulolyticus by modifying these factors.
We thank Dr. Shigeki Sawayama of Kyoto University for helpful discussions. This study was supported by the New Energy and Industrial Technology Development Organization (NEDO) of Japan.
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