Cloning and characterization of the EXG1 gene from the yeast Yarrowia lipolytica

Abstract

The YlEXG1 gene of Yarrowia lipolytica, encoding an exo-1,3-β-glucanase, was isolated by screening a genomic library with a DNA probe obtained by PCR amplification, using oligonucleotides designed according to conserved regions in the EXG1, EXG2 and SSG1 genes from Saccharomyces cerevisiae. YlEXG1 consists of a 1263 bp open reading frame encoding a protein of 421 amino acids with a calculated molecular weight of 48,209 Da. Northern blot analysis revealed a unique YlEXG1-specific transcript, 1.4 kb long. A putative pre(signal)-peptide of 15 amino acids is proposed at the N-terminal domain of the primary translation product. The deduced amino acid sequence shares a high degree of homology with exo-1,3-β-glucanases from other yeast species, including S. cerevisiae, Kluyveromyces lactis, Pichia angusta and Debaryomyces occidentalis. YlExg1p contains the invariant amino acid positions which have been shown to be important in the catalytic function of family 5 glycosyl hydrolases. Chromoblot analysis indicated that YlEXG1 is located on chromosome VI. Disruption of YlEXG1 did not result in a phenotype under laboratory conditions and did not prevent the yeast-hypha transition. The sequence data reported in this paper have been assigned EMBL Accession No. Z46872.