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

澄清纤维素酶和木聚糖酶诱导机制以指导纤维素酶糖化

基本信息

批准号：
13650851
负责人：
MORIKAWA Yasushi
金额：
$ 2.24万
依托单位：
Nagaoka University of Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13650851/
关键词：
Cellulase Xylanase Induction Promoter Trichoderma reesei Homologous recombination Gene Trichoderma reesei 相同組み換え誘導 xyn3 eg13 転写活性化因子

项目摘要

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.

里氏木霉的木聚糖酶III与纤维素酶共诱导，而不受木聚糖酶及其衍生物的诱导，后者是木聚糖酶表达的一般诱导剂。此外，酶基因(Xyn3)在里氏酵母PC-3-7中有表达，而在里氏酵母的亲本菌株QM9414中没有表达，尽管两株菌株具有相同的染色体基因，并且能够表达类似的纤维素酶基因。这两个序列完全相同，并且在启动子中存在与纤维素酶基因相关的已知调控因子ACE I、ACE II和分解代谢抑制因子Cre I的可能结合区，这表明里氏酵母QM9414或PC-3-7中缺失了一个或一些未知的调控因子。接下来，克隆了里氏酵母内切葡聚糖酶III基因(Eg13)的启动子区域(约3.4kbp)，其表达水平远远低于其他主要纤维素酶基因，如纤维素酶I和II基因。在此基础上，以amds基因为标记，构建了缺失的Xyn3、eg13启动子区域和报告基因(α-葡萄糖苷酶基因)，并利用同源重组系统对缺失的启动子区域转化里氏木霉进行了体内报告实验。另一方面，启动子和含有调节因子的核蛋白之间的体内相互作用也正在用带移分析进行研究。