Molecular Characterization and Expression Mechanism of Maltose-Utilization Gene Clusters in a Koji-Mold, Aspergillus oryzae

在米曲霉EST数据库中发现了一个与酵母麦芽糖酶基因(MAL62)同源的候选EST克隆。当以该克隆为探针筛选米曲霉基因组文库时，分离到两个可能与麦芽糖利用有关的不同基因簇。一个是麦芽糖酶同系物，命名为MALT，以及一个与酵母麦芽糖渗透酶基因(MAL61)高度同源的基因，命名为MALP。此外，一个可能的转录调控基因MalR位于麦芽下游，其N端具有典型的锌指基序。转录分析表明，该MAL簇中的MALP和MALT基因是由麦芽糖诱导的，而不是由葡萄糖或甘油诱导的，而MALR基因是结构性表达的。它们的表达方式与淀粉分解基因相似。将MalP基因导入麦芽糖渗透酶缺陷型酵母中，证实其编码一种能够掺入麦芽糖的蛋白质。对米曲霉MalP和MalR基因的中断分析表明，MalP编码该真菌中的一个主要麦芽糖转运体，而MalR是有效表达MalP和MALT基因所必需的。另一个基因簇与寄生曲霉中可能的糖利用基因簇具有很高的同源性。这个Glc簇具有编码α-葡萄糖苷酶(GLCA)、糖转运蛋白(HxtA)和转录激活因子(SuGR)的基因，并位于黄曲霉毒素生物合成基因簇的一端。然而，与紧邻GLC簇之前的NADA和stcQ同源的推定基因有缺失或移码的突变，因此可能是不活跃的。此外，到目前为止还没有观察到包含在GLC簇中的基因被转录。

One candidate EST clone homologous to the yeast maltase gene (MAL62) was found in the Aspergillus oryzae EST database. When an A. oryzae genomic library was screened with the clone as a probe, two different gene clusters probably involved in maltose utilization have been isolated. One is consisted of maltase homologue itself, designated malT, and a gene highly homologous to the yeast maltose permease gene (MAL61), designated malP. In addition, a putative transcriptional regulator gene designated malR which has a typical zinc finger motif at N-terminus is located at downstream of the malT. The transcriptional analysis showed that the malP and malT genes within this MAL cluster were induced by maltose but not by glucose or glycerol whereas the malR gene was expressed constitutively. The manner of their expression is similar to the amylolytic genes. The malP gene was introduced to maltose permease deficient yeast and was proved to encode a protein with a capability of incorporating maltose. Disruption analyses of the malP and malR genes in A. oryzae showed that the malP encodes a major maltose transporter in this fungus and that the malR is required for the efficient expression of the malP and malT genes. Another gene cluster is extensively highly homologous to the putative sugar utilization gene cluster in Aspergillus parasiticus. This GLC cluster has genes encoding α-glucosidase (glcA), a sugar transporter (hxtA), and a transcriptional activator (sugR) and is located at one end of the aflatdxin biosynthetic gene cluster, as in A. parasiticus. However, putative genes homologous to the nadA and stcQ located immediately prior to the GLC cluster have mutations of deletion or frame-shift, and thus may be inactive. In addition, it was not observed so far that the genes contained in the GLC cluster are transcribed.