Cloning and characterization of 1,3-beta-glucanase-encoding genes from non-conventional yeasts

The molecular cloning of 1,3-beta-glucanase-encoding genes from different y east species was achieved by screening genomic libraries with DNA probes ob tained by PCR-amplification using oligonucleotides designed according to co nserved regions in the EXG1, EXG2 and SSG1 genes from Saccharomyces cerevis iae. The nucleotide sequence of the KlEXG1 (Kluyveromyces lactis), HpEXG1 ( Hansenula polymorpha) and SoEXG1 (Schwanniomyces occidentalis) genes was de termined. KlEXG1 consists of a 1287 bp open reading frame encoding a protei n of 429 amino acids (49,815 Da). HpEXG1 specifies a 435-amino acid polypep tide (49,268 Da) which contains two potential N-glycosylation sites. SoEXG1 encodes a protein of 425 residues (49,132 Da) which contains one potential site for N-linked glycosylation. Expression in S. cerevisiae of KlEXG1, So EXG1 or HpEXG1 under control of their native promoters resulted in the secr etion of active 1,3-beta-glucanases. Disruption of KlEXG1 did not result in a phenotype under laboratory conditions. Comparison of the primary transla tion products encoded by KlEXG1, HpEXG1 and SoEXG1 with the previously char acterized exo-1,3-beta-glucanases from S. cerevisiae and C. albicans reveal s that enzymes with this type of specificity constitute a family of highly conserved proteins in yeasts. K1Exg1p, HpExg1p and SoExg1p contain the inva riant amino acid positions which have been shown to be important in the cat alytic function of family 5 glycosyl hydrolases. The sequence data reported in this paper have been deposited in the EMBL data bank under Accession Nu mbers Z46868, Z46869 and Z46871. Copyright (C) 1999 John Wiley & Sons, Ltd.