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 yeast species was achieved by screening genomic libraries with DNA probes obtained by PCR-amplification using oligonucleotides designed according to conserved regions in the EXG1, EXG2 and SSG1 genes from Saccharomyces cerevisiae. The nucleotide sequence of the KlEXG1 (Kluyveromyces lactis), HpEXG1 (Hansenula polymorpha) and SoEXG1 (Schwanniomyces occidentalis) genes was determined. K1EXG1 consists of a 1287 bp open reading frame encoding a protein of 429 amino acids (49,815 Da). HpEXG1 specifies a 435-amino acid polypeptide (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, SoEXG1 or HpEXG1 under control of their native promoters resulted in the secretion of active 1,3-beta-glucanases. Disruption of KlEXG1 did not result in a phenotype under laboratory conditions. Comparison of the primary translation products encoded by KlEXG1, HpEXG1 and SoEXG1 with the previously characterized exo-1,3-beta-glucanases from S. cerevisiae and C. albicans reveals that enzymes with this type of specificity constitute a family of highly conserved proteins in yeasts. KlExg1p, HpExg1p and SoExg1p contain the invariant amino acid positions which have been shown to be important in the catalytic function of family 5 glycosyl hydrolases.
Departamento de Microbiología, Universidad de Salamancal/CSIC, Spain.
P F
PF
Esteban
C R
CR
Vazquez de Aldana
F
F
del Rey
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D017353:Gene Deletion; D005800:Genes, Fungal; D043326:Glucan 1,3-beta-Glucosidase; D007716:Kluyveromyces / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D020161:Physical Chromosome Mapping; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D015183:Restriction Mapping; D004718:Saccharomycetales / Q000201:enzymology / Q000235:genetics; D017422:Sequence Analysis, DNA; D015003:Yeasts / Q000201:enzymology* / Q000235:genetics*; D001617:beta-Glucosidase / Q000235:genetics* / Q000378:metabolism*
D001617:beta-Glucosidase; D043326:Glucan 1,3-beta-Glucosidase