SHORT COMMUNICATION - Hyperexpression of xylanase from 2-deoxyglucose (2-DG) resistant mutant of Chaetomium thermophilum.
Summary: Xylanases have got central importance in the utilization of lignocellulosic material in industry. Filamentous fungi have got tremendous importance for the production of xylanases. The expression of genes encoding the enzymes is regulated by different factors. The presence of readily metabolizable carbon sources like glucose, cellobiose, xylobiose, or xylose represses the synthesis of xylanase enzymes for the utilization of certain carbon sources such as xylan or cellulose and the process is known as catabolite repression. In filamentous ascomycetes, glucose repression is known to be mediated by Cre1. In present study, mutagenesis was applied to a thermophilic fungus Chaetomium thermophilum (ATCC 28076) by treating spores with N-ethyl-N-nitrosourea (ENU) and UV radiations. The mutagenized conidia were grown in liquid minimal medium having 2% xylose and 0.5% (w/v) 2-deoxyglucose (2-DG).
Colonies were selected with clear zones of dissolved xylan in toxic 2-DG presence as catabolite repressor on agar screening plates containing Rose Bengal. After mutation, 36 mutants were selected and checked for xylanase activity. One mutant strain named as CTM/xyn-MBL gave 2.5 fold higher activity than the wild one in the presence of repressor glucose. The C. thermophilum strain with higher xylanase activity was successfully selected with 2-DG after random mutation with ENU. This procedure for xylanase hyperexpression can be used for other fungal strains as well.
Keywords: 2-DG; C. thermophilum; ENU; Hyperexpression; Xylanase.
Xylan is one of the major carbohydrate components of hemicellulose , comprised of [beta]-1,4 backbone of xylopyranosyl repeating units having substitutions of acetyl, arabinocyl and glucronyl residues . The backbone of xylan is hydrolyzed by xylanases and other de-branching enzymes . Xylanases are very important chemicals and secreted by variety of microorganisms, including bacteria, yeasts and filamentous fungi [4,5]. Thermostable celluloses are produced by filamentous fungus Chaetomium thermophilum which is widely found in the soil [6,7]. C. thermophylum is a target for the production of thermostable xyalanases. Thermostable enzymes showing optimal activities between 60-70AdegC have been reproduced from C. thermophilum  and Phialophora sp. .
The production of xylanases and cellulases is regulated by complex mechanisms of induction and repression, which depends upon the nature of the carbon source [10,11]. The synthesis of xylanases and cellulases is repressed if easily metabolizable carbon source like glucose, cellobiose, xylose, or xylobiose is present . Glucose and related sugars repress the transcription of genes required to utilize certain carbon sources such as xylan or cellulose by carbon catabolite repression (CCR) . The repression effects can be decreased by isolation of mutants for hyper production, construction of efficient enzymes, good optimization of culture conditions and use of genetic engineering. The selection of mutants has provided good results. The use of different strategies and methods has allowed the screening and selection of good number of mutants with required changes. These methods have assured the improvement in the production of cellulases and xylanases .
One of the selection strategies in mutagenesis is the use of simple sugar like glucose and non-metabolizable analogs like 2-deoxyglucose (2-DG), it increases the enzyme activity and provides feedback resistance . A toxic analogue of glucose i.e. 2-deoxy-D-glucose (2-DG) was used for the isolation of improved strains of Humicola lanuginosa which were resistant to this compound . Some mutant strains of Aspergillus niger overproduced pectinases when compared to wild type fungal strains in different media. The strains were also found to be less sensitive to CCR by glucose when compared to the wild strain C28B25. The finding was in accordance with reports of Gosh et al.  and Kirimura et al.  who isolated 2-DG-resistant strains that were also less sensitive to CCR. It is important to fully understand the mechanism controlling the expression of xylanase genes to genetically improve the production of xylanase from different organisms.
Therefore, generation of random or site-specific mutations in the promoter regions or in cre1 gene involved in CCR can be useful to get mutant fungi with enhanced xylanase expression.
The objective of the present study was to isolate mutant of C. thermophilum with hyper production of xylanase and minimum CCR. The selected mutant was characterized for the production of xylanases and phenomenon of CCR by glucose and xylose was also studied.
Microorganism and culture conditions for xylanase production
Chaetomium thermophilum strain ATCC 28076 was employed for mutation. Eggins and Pugh  medium [g/l (KH2PO4 1; (NH4)2SO4 0.5; CaCl2 0.1; KCl 0.5; MgSO4 0.2; [alpha]-aspargine 0.5; yeast extract 0.5; carbon source)] was used to grow the fungus. The pH of the medium was adjusted to 5 and the culture was set to grow for 4 days at 45AdegC under shaking (150 rpm) and harvested by centrifugation (10,000 rpm, 20 min and 4 AdegC) .
One milliliter of appropriately diluted enzyme was incubated with 1% birch wood xylan in citrate phosphate buffer at 50 AdegC for 30 min. The releasing sugars were determined spectrophoto-metrically at regular intervals after treating with 3,5-Dinitrosalysilic acid reagent . The amount of xylanase that released 1 umol of reducing sugars equivalent xylose per min was taken as one unit of enzyme activity.
Mutagenesis and enrichment
Mutagenesis was performed following the protocol of Labudova and Fakas . Spores (108/ml) were suspended in a Petri dish with 10 mL of 0.05 M Tris-HCl buffer having 0.2% (w/v) Tween 80 and 0.05% (w/v) N-ethyl-N-nitrosourea (ENU). Spores were irradiated for 5 min under a UV-lamp (40 W) at a distance of 20 cm. After irradiation conidial suspension was incubated at 45 AdegC for 6 h. Mutagenized conidia were grown in the Eggin's and Pugh medium with 2% xylose and 0.5% (w/v) 2DG. The conidial suspension was incubated at 45 AdegC until the first germinated conidia (micro colonies) were appeared visible to the naked eye. The micro colonies were separated from ungerminated conidia by filtration through a G-1 sintered glass filter of 120-160 um pore size. Separated colonies were washed with sterile water. Germinated conidia separated after filtration were resuspended and spread on sporulation agar (minimal medium plus 2% glucose and grown at 45 AdegC).
Conidia were isolated from the sporulation medium and grown in liquid minimal medium with 0.5% (w/v) xylan plus 0.5 % (w/v) 2-DG.
Screening of mutant
Conidia grown on agar plates were isolated and spread on minimal medium agar screening plates with 1% xylan, Rose Bengal (50 mg/ml) and 5% (w/v) fructose. After grown at 45 AdegC the colonies were isolated with clear zones of dissolved xylan in the presence of catabolite repressor.
Results and Discussion
Induction of xylanase
In present study, production of xylanase by wild type C. thermophilum was determined in growth medium containing xylan, xylose or glucose as carbon source (Table-1). Good xylanase activity was observed with 1% and 2% xylan as sole carbon source. But the use of xylose (xylan breakdown product) as the only carbon source resulted in good fungal growth with low enzyme activity. Similarly, good growth of fungus was obtained with less enzyme activity in the presence of glucose as the sole carbon source. These results revealed that xylan and xylose are good inducers of xylanase while simple sugar glucose is not a good inducer.
Xylanase repression by glucose
Wild C. themophilum was grown in two different conditions. First in the culture medium with xylan as the sole carbon source, second in the medium containing xylan as carbon source with glucose as repressor. Xylanase activity was calculated after an interval of 24 h for both the samples and maximum activity was observed after 120 h (Fig. 1). There was a constant increase in xylanase activity with xylan as the only carbon source while no any detectable xylanase activity was observed when the repressor glucose was present in medium with xylan. The results from this study showed that the use of glucose along with xylan in the culture medium exhibited strong repression of xylanase gene expression.
Table-1: Activity of xylanase and protein content of culture filtrate from wild type C. thermophilum.
Sr. No.###Carbon source###Enzyme Activity (IU/mL) +- SD###p-value###Protein content (g/ml) +- SD###p-value
In this study mutagenesis approach was used to reduce the catabolite repression effects of simple sugar glucose. The mutagenic treatment of C. thermophilum ATCC 28076 produced 36 colonies which presented higher hydrolysis zones in Xylan-2DG plates than in the parent strain. These mutants were checked for growth and xylanase activity. The xylanase activity was found to be increased in only one strain which was named as mutant CTM/xyn-MBL (Fig. 2) as compared to wild type parent strain.
After isolation of mutant colonies, wild type parent strain and mutant colonies were grown in liquid minimal medium in the presence of xylan as sole carbon source. Xylanase activity was measured and compared for both the fungal strains (Fig. 3). The mutant produced good amount of xylanase as compared to wild type strain. It shows that mutation has increased the xylanase gene expression level. To further check that the mutant has the ability to overexpress the xylanase gene in the presence of glucose or not, both wild type and mutant strains were grown in the presence of xylan as carbon source plus glucose as repressor in the culture medium. Xylanase activity was calculated for both the samples shown in Fig. 4. A constant increase in xylanase activity was found in the presence of repressor (glucose) with xylan as carbon source for the mutant strain. Xylanase activity was three-fold higher in the mutant strain than the enzyme activity by the wild type strain.
These results confirmed that mutation has not only increased the xylanase expression level with normal carbon source xylan but it has also increased the xylanase expression in the presence of easily metabolizable sugar glucose.
The results from present study showed that xylanase expression is induced by xylan and xylose and end product of xylan degradation. These results are in agreement with previous studies where cellulolytic and xylanolytic activities were found significantly high with xylan, cellulose or mixtures of plant polymers [23,24]. Previously, Orejas et al.  revealed that the expression of xlnA gene was also controlled at transcriptional level like other fungal xylanase genes and they also observed induction of xylnase gene with xylan and its metabolic product xylose. For this study, xylanase activity was checked after every 24 h and maximum activity was found with birch wood xylan after 120 h (Fig.1). Katapodis et al.  used xylan as carbon source for xylanase production from C. thermophilum. They got maximum activity at 4% (w/v) substrate concentration. Loera and Cordova  used xylan as carbon source for xylanase production from A. niger and got maximum activity after 88 h.
Different researchers have reported the repression of xylanase expression in the presence of simple sugars in the medium. In this study, expression of xylanase gene was repressed when glucose was added in the medium along with xylan. Previously, Perez-Gonzales et al.  found that in A. nidulans xlnD gene was induced by xylan and D-xylose but repressed by D-glucose. Likewise, Zeilinger et al.  demonstrated the xln2 transcription at a low basal level when the fungus was grown on glucose as sole carbon source. The production of xylanases and cellulases by microorganisms is limited when the amount of simple sugars is increased in the culture medium and it is called as carbon catabolite repression (CCR) . Similar results were also obtained by Rizzatti et al.  who observed the xylanase gene was induced by xylan, xylose or [beta]-methylxyloside in Aspergillus phoenicis while xylanase production was repressed by the presence of 1% glucose in the medium containing xylan or xylose.
In present study, mutant of C. thermophilum was produced for high production of xylanase even in the presence of glucose thus minimizing the CCR. The 2-deoxyglucose was used to isolate large number of putative mutants but one mutant CTM/xyn-MBL showed highest xylanase activity. Fernandez-Espinar et al.  demonstrated that xylanase synthesis in A. nidulans was under CCR and by using different cre mutants they suggested the role of intact creA gene repressed by glucose and induced by xylan and arabinoxylan. Later on, Ilmen et al.  reported the repression of cellulose expression was mediated by Cre1 (glucose repressor protein) in T. reesei. Another report about the involvement of Cre1 was from Mach et al.  who stated that the presence of glucose in the medium interferes with biosynthesis of xylanase1 by T. reesei and accumulation of xyn1 mRNA was regulated by the carbon catabolite repressor Cre1.
Presence of glucose repressed xylose utilization in the wild type strain growing on the sugar mixture while no repression observed in creA deleted (carbon catabolite de-repressed) strain of A. nidulans .
Xylanase was produced from A. niger in a medium with xylan as a carbon source in the presence or absence of glucose as a repressing sugar . They demonstrated that glucose presence in culture medium displayed strong repression of xylanases and no important enzymatic activity was noticed. Many researchers have used different strategies to reduce CCR effects. Ilyes et al.  overcome glucose triggered CCR by growing A. nidulans at defined low growth rates thus indicated that growth rate was determinant of CCR in A. nidulans. Robledo-Monterrubio et al.  also used UV irradiation to isolate series of 2-deoxy-D-glucose resistant mutants from wild type Beauveria bassiana 88 (Bb 88). UV radiation was used by De Nicolas-Santiago et al.  to produce mutant strains of A. niger to enhance their hemicellulolytic and cellulolytic activities, production and improving their potential industrial applications.
Loera and Cordova  used parasexual recombination to isolate diploid strain (D4) from two xylanase overproducing mutants of A. niger. The xylanase production was nearly 100% higher as compared to wild strain. The diploid D4 showed less sensitivity towards glucose CCR as no decrease in maximum xylanase expression level was detected in glucose presence. Rajoka and Khan  isolated 2-deoxy-D-glucose and cycloheximide resistant mutant of Kluyveromyces marxianus PPY125 to study the production of [beta]-xylosidase in growth medium containing different carbon sources. The mutant produced maximum 1.5 to 2-fold more [beta]-xylosidase than that produced by the wild cells.
Our approach involving two steps method using ENU and 2-DG for the isolation of mutant C. thermophilum strain with enhanced xylanase activity in the presence of repressor glucose is successful. It is suggested that the methodology used in this study can be equally good for other fungal strains and would be helpful to improve the production of xylanases by technology development.
The work was supported by a grant from Higher Education Commission, Government of Pakistan.
1. G. Mustafa, S. Kousar, M. I. Rajoka and A. Jamil, Molecular Cloning and Comparative Sequence Analysis of Fungal [beta]-Xylosidases, AMB Express., 6, 1 (2016).
2. M. Dashtban, H. Schraft and W. Qin, Fungal Bioconversion of Lignocellulosic Residues; Opportunities and Perspectives, Int. J. Biol. Sci., 5, 578 (2009).
3. T. Collins, C. Gerday and G. Feller, Xylanases, Xylanase Families and Extremophilic Xylanases, FEMS Microbiol. Rev., 29, 3 (2005).
4. S. Ahmed, S. Riaz and A. Jamil, Molecular Cloning of Xylanases: an Overview, Appl. Microbiol. Biotechnol., 84, 19 (2009).
5. S. Kousar, G. Mustafa and A. Jamil, Microbial Xylosidases: Production and Biochemical Characterization, Pak. J. Life Soc. Sci., 11, 85 (2013).
6. A. Ghaffar, S. A. Khan, Z. Mukhtar, M. I. Rajoka and F. Latif, Heterologous Expression of a Gene for Thermostable Xylanase from Chaetomium thermophilum in Pichia pastoris GS115, Mol. Biol. Rep., 38, 3227 (2011).
7. D. C. Li, M. Lu, Y. L. Li and J. Lu, Purification and Characterization of an Endocellulase from the Thermophilic Fungus Chaetomium thermophilum CT2, Enzyme Microb. Technol., 33, 932 (2003).
8. S. Ahmed, S. S. Imdad and A. Jamil, Comparative Study for the Kinetics of Extracellular Xylanases from Trichoderma harzianum and Chaetomium thermophilum, Electron. J. Biotechn., 15, 3 (2012).
9. F. Zhang, P. Shi, Y. Bai, H. Luo, T. Yuan, H. Huang, P. Yang, L. Miao and B. Yao, An Acid and Highly Thermostable Xylanase from Phialophora sp. G5, Appl. Microbiol. Biot., 89, 1851 (2011).
10. M. Suto and F. Tomita, Induction and Catabolite Repression Mechanisms of Cellulase in Fungi, J. Biosci. Bioeng., 92, 305 (2001).
11. Y. H. P. Zhang and L. R. Lynd, Regulation of Cellulase Synthesis in Batch and Continuous Cultures of Clostridium thermocellum, J. Bacteriol., 187, 99 (2005).
12. A. R. Mach-Aigner, M. E. Pucher and R. L. Mach, D-Xylose as a Repressor or Inducer of Xylanase Expression in Hypocrea jecorina (Trichoderma reesei), Appl. Environ. Microbol., 76, 1770 (2010).
13. T. M. Mello-De-Sousa, R. Gorsche, A. Rassinger, M. J. Pocas-Fonseca, R. L. Mach and A. R. Mach-Aigner, A Truncated form of the Carbon Catabolite Repressor 1 Increases Cellulase Production in Trichoderma reesei, Biotechnol. Biofuels., 7, 129 (2014).
14. G. M. Zhang, J. Huang, G. R. Huang, L. X. Ma and X. E. Zhang, Molecular Cloning and Heterologous Expression of a New Xylanase Gene from Plectosphaerella cucumerina, Appl. Microbiol. Biotechnol., 74, 339 (2006).
15. A. M. El-Bondkly, Molecular identification using ITS Sequences and Genome Shuffling to Improve 2-deoxyglucose Tolerance and Xylanase Activity of Marine-Derived Fungus, Aspergillus sp. NRCF5, Appl. Biochem. Biotech., 167, 2160 (2012).
16. S. A. I. Bokhari, F. Latif, M. W. Akhtar and M. I. Rajoka, Characterization of a [beta]-Xylosidase Produced by a Mutant Derivative of Humicola lanuginosa in Solid State Fermentation, Ann. Microbiol., 60, 21 (2010).
17. A. Gosh, B. Chatterjee and A. Das, Production of Glucoamylase by 2-deoxy-D-glucose Resistant Mutant of Aspergillus terreus, Biotech. Lett., 13, 515 (1991).
18. K. Kirimura, S. Saragbin, S. Rugsaseel and S. Usami, Citric Acid Production by 2-deoxyglucose Resistant Mutant Strain of Aspergillus niger, Appl. Microbiol. Biotechnol., 36, 573 (1992).
19. H. O. W. Eggins and G. J. F. Pugh, Isolation of Cellulose Decomposing Fungi from Soil, Nature., 193, 94 (1962).
20. S. Ahmed, A. Jabeen and A. Jamil, Xylanase from Trichoderma harzianum: Enzyme Characterization and Gene Isolation, J. Chem. Soc. Pak., 29, 176 (2007).
21. A. A. N. Saqib and P. J. Whitney, Differential Behaviour of the Dinitrosalicylic Acid (DNS) Reagent Towards Mono-and Di-Saccharide Sugars, Biomass Bioenergy., 35, 4748 (2011).
22. L. Labudova and V. Farks, Enrichment Technique for the Selection of Catabolite Repression-Resistant Mutants of Trichoderma as Producer of Cellulose, FEMS Microbiol Lett., 20, 211 (1983).
23. M. F. Eida, T. Nagaoka, J. Wasaki and K. Kouno, Evaluation of Cellulolytic and Hemicellulolytic Abilities of Fungi Isolated from Coffee Residue and Sawdust Composts, Microbes Environ., 26, 220 (2011).
24. A. J. D. Silva, D. P. Gomez-Mendoza, M. Junqueira, G. B. Domont, X. Ferreira Filho, M. V. de Sousa and C. A. O. Ricart, Blue native-PAGE analysis of Trichoderma harzianum Secretome Reveals Cellulases and Hemicellulases Working as Multienzymatic Complexes, Proteomics., 12, 2729 (2012).
25. M. Orejas, A. P. MacCabe, J. A. Perez Gonzalez, S. Kumar and D. Ramon, Carbon Catabolite Repression of the Aspergillus nidulans xlnA gene, Mol. Microbiol., 31, 177 (1999).
26. P. Katapodis, V. Christakopoulou, D. Kekos and P. Christakopoulos, Optimization of Xylanase Production by Chaetomium thermophilum in Wheat Straw Using Response Surface Methodology, Biochem Eng J., 35, 136 (2007).
27. O. Loera and J. Cordova, Improvement of Xylanase Production by a Parasexual Cross Between Aspergillus niger strains, Braz. Arch. Biol. Technol., 46, 177 (2003).
28. J. A. Perez-Gonzalez, N. N. van Peij, A. Bezoen, A. P. Maccabe, D. Ramon and L. H. de Graaff, Molecular Cloning and Transcriptional Regulation of the Aspergillus nidulans xlnD Gene Encoding a [beta]-xylosidase, Appl. Environ. Microbiol., 64, 1412 (1998).
29. S. Zeilinger, L. M. Robert, M. Schindler, P. Herzog and C. P. Kubicek, Different Inducibility of Expression of the Two Xylanase Genes xyn1 and xyn2 in Trichoderma reesei, Biol. Chem., 271, 25624 (1996).
30. A. C. S. Rizzatti, F. Z. Freitas, M. C. Bertolini, S. C. Peixoto-Nogueira, H. F. Terenzi, J. A. Jorge, M. de Lourdes and T. de M. Polizeli, Regulation of Xylanase in Aspergillus phoenicis: a Physiological and Molecular Approach, J. Ind. Microbiol. Biotechnol., 35, 237 (2008).
31. M. Fernandez-Espinar, F. Pinaga, L. De Graaff, J. Visser, D. Ramon and S. Valles, Purification, Characterization and Regulation of the Synthesis of an Aspergillus nidulans Acidic xylanase, Appl. Microbiol. Biotechnol., 42, 555 (1994).
32. R. L. Mach, J. Strauss, S. Zeilinger, M. Schindler and C. P. Kubicek, Carbon Catabolite Repression of xylanase I (xyn1) Gene Expression in Trichoderma reesei, Mol. Microbiol., 21, 1273 (1996).
33. W. Prathumpai, M. Mcintyre and J. Nielsen, The effect of CreA in glucose and xylose catabolism in Aspergillus nidulans, Appl. Microbiol. Biotechnol., 63, 748 (2004).
34. H. Ilyes, E. Fekete, L. Karaffa, E. Fekete, E. Sandor, A. Szentirmai and C. P. Kubicek, CreA mediated carbon catabolite repression of [beta]- galactosidase formation in Aspergillus nidulans is growth rate dependent, FEMS Microbiol. Letts., 235, 147 (2004).
35. M. Robledo-Monterrubio, R. Alatorre-Rosas, G. Viniegra-Gonzalez and O. Loera, Selection of improved Beauveria bassiana (Bals.) Vuill. strains based on 2-deoxy-d-glucose resistance and physiological analysis, J. Invertebr. Pathol., 101, 222 (2009).
36. S. De Nicolas-Santiago, C. Regalado-Gonzalez, B. Garcia-Almendarez, F. J. Fernandez, A. Tellez-Jurado and S. Huerta-Ochoa, Physiological, morphological, and mannanase production studies on Aspergillus niger uam-gs1 mutants, Electron. J. Biotechnol., 9, 50 (2006).
37. M. I. Rajoka and S. Khan, Hyper-production of a thermotolerant [beta]-xylosidase by a deoxy-D-glucose and cycloheximide resistant mutant derivative of Kluyveromyces marxianus PPY 125, Electron. J. Biotechnol., 8, 58 (2005).
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|Author:||Shaheen, Sumera; Mustafa, Ghulam; Jamil, Amer|
|Publication:||Journal of the Chemical Society of Pakistan|
|Date:||Aug 31, 2017|
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