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

研究发现，里氏木霉的木聚糖酶III与纤维素酶共诱导，而不受木聚糖及其衍生物的诱导。木聚糖酶是木聚糖酶表达的一般诱导剂。此外，酶基因（xyn3）在T. reesei PC-3-7中表达，而在T. reesei QM9414（前者的亲本菌株）中不表达，尽管这两株菌株具有相同的染色体基因，并且能够相似地表达纤维素酶基因。首先，测定了两菌株基因启动子区的核苷酸序列，明确了木聚糖酶和纤维素酶诱导的调控机制。这两个序列完全相同，并且在启动子中出现了已知调节因子（纤维素酶基因相关的ACE I、ACE II和分解代谢抑制因子Cre I）的推定结合区，这表明T. reesei QM9414或PC-3-7中缺失了一个或几个未知的调节因子。接下来，启动子区域(约3。克隆并测序了4 kbp)的内切葡聚糖酶III基因（eg13），该基因的表达量远低于其他主要的纤维素酶基因，如纤维生物水解酶I和II基因。此外，还以amdS基因为标记物，确定了柽柽树的同源转化体系。根据这些结果，构建了xyn3和eg13缺失的启动子区域，然后是报告基因（α-葡萄糖苷酶基因），目前正在利用同源重组系统对缺失启动子区域进行转化的T. reesei进行体内报告基因检测。另一方面，体内启动子和含有调节因子的核蛋白之间的相互作用也正在使用带移法进行研究。

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.