Regulatory mechanisms of xpression of glycosidase genes in filamentous fungi and their applications

以米曲霉为代表的丝状真菌在日本发酵工业中发挥着重要作用。本研究以空心拟酵母菌为中间寄主，检测了从稻瘟病菌中分离的taka -淀粉酶A基因的表达特性。在诱导条件下，CCAAT序列和CCAAT结合蛋白AnCP在提高taka -淀粉酶A基因表达中发挥了重要作用。本项目通过对纤维素酶和木聚糖酶基因进行鉴定，阐明其调控机制，以及taka -淀粉酶基因，为有效利用各种生殖自然资源，特别是淀粉、纤维素和木聚糖等碳水化合物开辟新的途径。在过去的两年中，我们取得了以下成果：HAPC是AnCP的组成部分，通过在大肠杆菌中表达malE-hapC融合基因制备了HAPC抗体。利用重组HAPC及其抗体，我们成功地证明了AnCP由包括HAPC 2在内的三个亚基组成。证明CCAAT序列不参与该基因的诱导。淀粉反应元件（SRE）及其结合蛋白SREB被认为在Taka-amylaseA基因的诱导中起重要作用。从芽孢杆菌中分离到CMC诱导型纤维素酶基因eglA，并对其进行了测序。我们还对启动子区域进行了初步分析，以寻找诱导或抑制egla的元件。木聚糖酶基因cgxA是一种分离自薄壁藤本植物的木聚糖酶基因，其抑制元件和抑制样因子可识别13bp的抑制元件。此外，还确定了a.m oryzae木聚糖酶基因xynFI的诱导元件。一个35bp的元件似乎参与了该基因的诱导表达。

Filamentous fungi represented by Aspergillus oryzae have been playd important roles in Japanese fermentation industries. We have examined the expression properties of Taka-amylase A gene isolated from A.oryzae using A.nidulans as an intermediate host. CCAAT sequence and AnCP,CCAAT binding protein, have been shown to play important roles in enhancement of Taka-amylase A gene expression under the inducible conditions.In this project, cellulase and xylanase genes have been characterized to elucidate their regulatory mechanisms in addition to Taka-amylase gene in order to develop a novel way to utilize efficiently various reproductive natural resources, especially carbohydrates such as starch, cellulose and xylan. We have obtained the following results during last two years.1. HAPC,a compoment of AnCP,was prepared by expressing malE-hapC fusion gene in E.coli and used to raise an antibody to HAPC.With recombinant HAPC and its antibody, we succedeed to prove that AnCP comprised three subunits including HAPC.2. CCAAT sequence was proved not to be involved in the induction of the gene. SRE (starch responsive element) and its binding protein, SREB,were suggested to play a vital role in induction of Taka-amylaseA gene.3. CMC inducible cellulase gene, eglA,was isolated from A.nidulans and sequenced. Preliminary analysis as to the promoter region was also conducted to find any elements for induction or reperssion of eglA.4. Xylanase gene, cgxA,isolated from C.gracile was characterized for itsrepressive element and repressor-likefactor which recognized the 13bp repressive element. Furthermore, the A.oryzae xylanase gene, xynFI,was also determined for its inducible element. A 35bp element appeared to be involved in inductive expression of the gene.