A novel method for efficient expression cloning of fungal enzyme genes.
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.
Dalbøge
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Novo Nordisk A/S, Bioindustrial Group, GeneExpress, Novo Alle, Denmark.
Heldt-Hansen
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D000595:Amino Acid Sequence; D001230:Aspergillus / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary; D043364:Endo-1,4-beta Xylanases; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics*; D005658:Fungi / Q000201:enzymology*; D005800:Genes, Fungal; D005822:Genetic Vectors; D004693:Glucan Endo-1,3-beta-D-Glucosidase / Q000235:genetics; D006026:Glycoside Hydrolases / Q000235:genetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000235:genetics
D018076:DNA, Complementary; D005656:Fungal Proteins; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; D004693:Glucan Endo-1,3-beta-D-Glucosidase; D043364:Endo-1,4-beta Xylanases