Cloning and sequence analysis of the gene encoding invertase from the yeast Schwanniomyces occidentalis

Abstract

The gene encoding invertase (INV) has been cloned from Schwanniomyces occidentalis. The enzyme consists of 533 amino acids, 8 potential glycosylation sites and has a 45% identity with the invertase from Saccharomyces cerevisiae. The proenzyme has a 22 amino acid signal sequence that has a high alpha-helical transmembrane potential which differs significantly from that predicted for the Saccharomyces cerevisiae enzyme.

Development of a reporter system for the yeast Schwanniomyces occidentalis: influence of DNA composition and codon usage

“…Standard bacterial and yeast cultivation media and temperatures were used (Sambrook et al, 1989;Ausubel et al, 1987). The plasmids pINV2-A and pINV2-B (Klein et al, 1989) were the sources for isolation of promoter and terminator sequences of the invertase-encoding gene INV from S. occidentalis (Figure 1). The plasmid pADE (Klein and Favreau, 1988) consisting of a 4.5 kb Sau3A fragment of the chromosomal DNA of S. occidentalis complementing the ade2 mutation, cloned into the BamHI site of the Saccharomyces cerevisiae expression vector, pYcDE8 (McKnight et al, 1985) served as a basis for construction of expression vectors for S. occidentalis.…”

“…In expression vectors constructed in this study, we used different-length fragments of the 5 untranslated region (UTR) of the homologous invertase-encoding gene (INV ). The INV gene of S. occidentalis is regulated by glucose catabolite repression and was cloned and sequenced by Klein et al (1989), although its transcription-regulating regions have not been precisely defined. Only the nucleotide sequences of 620 bp of the INV 5 UTR and 200 bp of the 3 UTR were known when we started this work; however, longer fragments were available on the plasmids pINV2-A and pINV2-B carrying the parts of the INV gene (Klein et al, 1989).…”

“…The INV gene of S. occidentalis is regulated by glucose catabolite repression and was cloned and sequenced by Klein et al (1989), although its transcription-regulating regions have not been precisely defined. Only the nucleotide sequences of 620 bp of the INV 5 UTR and 200 bp of the 3 UTR were known when we started this work; however, longer fragments were available on the plasmids pINV2-A and pINV2-B carrying the parts of the INV gene (Klein et al, 1989). Therefore, we isolated the sequenced 620 bp fragment of the INV promoter region by PCR using the primers Inv1 and Inv2 at first.…”

“…To be able to direct secretion of proteins from S. occidentalis cells, three fragments (0.7 kb, 1.5 kb and 3.7 kb) containing parts of the 5 UTR and the invertase signal peptide sequence (first 22 amino acids of the protein sequence; Klein et al, 1989) were amplified and cloned using the primer Inv3, matching the invertase nucleotide sequence downstream from the predicted signal peptidase cleavage site, and the universal reverse sequencing primer ( Figure 1A).…”

“…At least 21 S. occidentalis genes encoding 14 different proteins have been cloned so far (Wang et al, 1999). The isolated genes code for industrially interesting enzymes such as α-amylase, glucoamylase or invertase (Dohmen et al, , 1990Klein et al, 1989), biosynthetic genes, e.g. ADE2 (Klein and Favreau, 1988;Gourdon et al, 1995), LEU2 (Iserentant and Verachtert, 1995) and metabolic enzymes such as GDH (De Zoysa et al, 1991), HX K (Rose, 1995) or ATPases encoding genes ENA1 and ENA2 (Banuelos and RodriguezNavarro, 1998).…”