REGULATION OF XYLANASE GENE EXPRESSION

The mechanism of control of xylanolytic enzymes synthesis varies considerably among different organisms. Individual xylanases from an organism might be under different control (de Vries et al., 2001). In general, xylanase expression in fungi is subjected to substrate induction and glucose or catabolite repression (Gómez-Gómez et al., 2002; Tonukari et al., 2002). The constitutive xylanase expression has been reported in Cellulomonas fimi (Khanna and Gauri 1993), Bacillus stearothermophilus (Khasin et al., 1993), Bacillus subtilus (Lindner et al., 1994), and Streptomyces cynneus (Zhao et al., 1997). Therefore, induction, catabolic repression, growth rate and other environmental factors can influence the activity of xylanolytic enzymes.

Induction

Xylanase induction is a complex phenomenon. The induction model proposed by Thompson (1993), Subramaniyan and Prema (2002) and Mach and Zeilinger (2003) suggest that high molecular weight xylan cannot enter the cells and therefore cannot directly induce the synthesis of xylanolytic enzymes. The low molecular mass fragments of xylan (xylose, xylobiose and xylo-oligosaccharides) are known to play an important role in xylanase biosynthesis. These small soluble (signal) fragments are released by the action of low amount of constitutively produced xylanases, which degrade the xylan to xylo-oligosaccharides and xylobiose that are further taken up by the cell and induce other xylanase genes. The inducible xylanases degrade xylan to xylo-oligosaccharides and xylobiose. The b- xylosidases, which may be produced constitutively and/or inducible, convert xylobiose to xylose and may subsequently transglycosylate it to Xylß1-2Xyl and Glcß1-2Xyl. These compounds are believed to be taken up by the cell and act as additional inducers of genes encoding xylanases allowing the utilization of xylan (Tsujibo et al., 2004). Studies on Aspergillus and Trichoderma spp. at the cellular and molecular level indicate that xylanase gene expressions are regulated at the transcriptional level (de Graaff et al., 1994; Margolles-Clark et al., 1997) and that a transcriptional activator XlnR (positive acting element)  regulates induction of xylanase expression. Characterization of XlnR showed that it was responsible for the expression of genes encoding endoxylanase and β-xylosidase. Analysis of the promoter region of these genes identified a putative XlnR binding site, GGCTAAA, of which the second G was determined to be essential for XlnR binding by band mobility shift assays and in vivo analysis (Gielkens, etal., 1999). In addition to its role as a xylanolytic activator, XlnR also regulates the expression of some, but not all, genes encoding cellulolytic enzymes (van Peij et al., 1998b; Gielkens et al., 1999b).. Analysis of the promoter regions of the genes that are regulated by XlnR demonstrated that the third A in the consensus for the binding site is variable, and the consensus sequence was therefore shortened to GGCTAA. However, the presence of a putative XlnR binding site does not automatically imply regulation by XlnR.

Carbon catabolic repression

Carbon catabolic repression in microorganisms is a means to control the synthesis of a range of enzymes required for the utilization of less favored carbon source when more readily utilizable carbon sources are available in the medium. Microorganisms are reported to turn off a large number of genes in the presence of glucose as an energy saving response, as it primarily affects enzymes used to metabolize other carbon sources which are dispensable in the presence of glucose (Ronne et al., 1995). Gene encoding the glucose repressors has been isolated from numerous filamentous fungi including Trichoderma and Aspergillus sp. (Strauss et al., 1995; Iimen et al., 1996; Takashima et al., 1996), Humicola grisea (Takashima et al., 1998), Sclerotinia sclerotiorium (Vautard et al., 1999) and Botrytis cinerea (gene bank accession no 094130).  Catabolite repression by glucose is a common phenomenon observed in xylanase biosynthesis as reported in Cellulomonase flavigens (Ponce and Torre, 2001) and Aspergillus nidulans (Prathumpai et al., 2004). It has been shown that the carbon catabolite repressor protein CreA is involved in transcriptional repression of xylanase-encoding (de Graaff et al 1994) and arabinanase encoding genes in Aspergillus species (Ruijter et al., 1997). It has been demonstrated that in Trichoderma reesei the CreA counterpart Cre1 causes repression of transcription of xylanase-encoding genes (Margolles-Clark et al., 1997) and cellulase encoding genes (Iimen et al., 1996).

It has been shown that in A. niger CreA modulates the XlnR-induced transcription of genes encoding xylanolytic enzymes when the fungus is grown on D xylose. The transcription of the xlnB, xlnD, aguA and faeA genes on D-xylose was studied in a wild-type strain and in a creAd mutant. A decrease was observed in transcription levels of all four genes with increasing D-xylose concentrations, whereas the transcription levels were unaffected in the creAd mutant strain. The results indicated that the transcription levels of these xylanolytic genes were partially repressed at D-xylose concentrations higher than 1 mM (de vries et al., 1999). Both a specific regulator and the CreA repressor protein regulate transcription of these genes. Presence and concentration of the carbon source determine the balance between induction and repression controlled by these regulatory proteins. This is also illustrated by the influence of D-glucose concentrations on the regulation of cellulase biosynthesis, as the end product of cellulose hydrolysis, D-glucose, inhibits further synthesis of cellulases. In T. reesei, it has been shown that D-glucose interferes with cellulase biosynthesis by blocking the uptake of diglucosides that can act as an inducer (Kubicek et al., 1993) and by repression of de novo biosynthesis of cellulases via the transcriptional repressor protein Cre1. So far only one negatively acting factor CreA (carbon catabolic repression, discussed above) and one positively acting factor (a transcription activator, XlnR) have been studied in detail. The actual level of expression of xylanase genes appears to be influenced by the balance between the induction by XlnR and repression by CreA.