Dalbøge H, Heldt-Hansen H P
Novo Nordisk A/S, Bioindustrial Group, GeneExpress, Novo Alle, Denmark.
Mol Gen Genet. 1994 May 10;243(3):253-60. doi: 10.1007/BF00301060.
We have developed a method for fast and efficient isolation of enzyme genes from filamentous fungi by combining the ability of Saccharomyces cerevisiae to express heterologous genes with the utilisation of sensitive and reliable enzyme assays. A cDNA library from the fungus Humicola insolens was constructed in a S. cerevisiae/Escherichia coli shuttle vector in E. coli. Sub-pools of the library were subsequently screened for enzyme activity in S. cerevisiae. More than 130 clones were identified as positive in either an endo-beta-glucanase or an endo-xylanase assay. Based on a partial characterization of the DNA sequence of the individual clones, they could be grouped into five distinct types of endo-beta-glucanases and three types of endo-xylanases. A representative cDNA from each type was sub-cloned in an Aspergillus vector and expressed in A. oryzae. The new cloning method may be an important alternative to traditional cloning methods based on amino acid sequence information.
我们开发了一种从丝状真菌中快速高效分离酶基因的方法，该方法结合了酿酒酵母表达异源基因的能力与灵敏可靠的酶活性测定方法。在大肠杆菌中，利用酿酒酵母/大肠杆菌穿梭载体构建了来自腐质霉的cDNA文库。随后在酿酒酵母中筛选文库的亚库以检测酶活性。在β-1,4-内切葡聚糖酶或内切木聚糖酶测定中，超过130个克隆被鉴定为阳性。根据单个克隆DNA序列的部分特征，它们可分为五种不同类型的β-1,4-内切葡聚糖酶和三种类型的内切木聚糖酶。每种类型的一个代表性cDNA被亚克隆到曲霉载体中并在米曲霉中表达。这种新的克隆方法可能是基于氨基酸序列信息的传统克隆方法的重要替代方法。
