Clarification of cellulase and xylanase induction mechanism to direct toward enzymatic saccharification of cellulosics

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Xylanase III in Trichoderma reesei has been found to be co-induced with the cellulases and not to be induced by xylan and its derivatives which are general inducers in xylanase expression. Moreover, the enzyme gene (xyn3) was expressed in T. reesei PC-3-7 but not in T. reesei QM9414, the parent strain of the former, although both strains have the same chromosomal gene and are able to express the cellulase genes similarly.Firstly, the nucleotide sequences of the promoter region of the genes of both strains were determined to clear the regulatory mechanism of cellulase and xylanase induction in T. reesei. The both sequences were completely the same, and the putative binding region with the known regulatory factors, cellulase gene-relating ACE I, ACE II and catabolite repressor Cre I, were presented in the promoter, suggesting that one or some unknown regulatory factors were deleted in T. reesei QM9414 or PC-3-7.Next, the promoter region (ca. 3. 4 kbp) of endoglucanase III gene (eg13) was cloned and sequenced, which is expressed extremely lower than are the other main cellulase genes such as cellobiohydrolase I and II genes in T. reesei. Furthermore, the homologous transformation system in T. reesei was confirmed using amdS gene as a maker.From these results, the deleted promoter regions of xyn3 and eg13 followed by the reporter gene (α-glucronidase gene) were constructed, and the in vivo reporter assay are now being tried in T. reesei transformed with the deleted promoter regions using the homologous recombination system. On the other hand, in vivo interactions between the promoters and nuclear proteins containing reguratory factors are also being investigated using band-shift assays.