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Cloning, sequencing and amplification of the alkaline extracellular protease (XPR2) gene of the yeast Yarrowia lipolytica

1989; Elsevier BV; Volume: 12; Issue: 3-4 Linguagem: Inglês

10.1016/0168-1656(89)90048-5

1873-4863

Jean‐Marc Nicaud, Emmanuelle Fabre, J. M. Beckerich, Philippe Fournier, C. Gaillardin,

RNA and protein synthesis mechanisms

The alkaline extracellular protease (AEP) gene (XPR2) of the yeast Yarrowia lipolytica was cloned using synthetic oligonucleotide probes based on the N-terminal amino acid sequence of mature AEP. The XPR2 gene codes for a predicted precursor polypeptide of 46903 Da and a mature AEP of 30524 Da. We have increased XPR2 gene copy number by successive integration of XPR2-LYS5 and XPR2-URA3 vectors obtaining strains with one, two and three copies of the XPR2 gene at the XPR2 locus. A disrupting vector xpr2::LYS5 was used to construct a control strain with no XPR2 gene. We have also used a XPR2-LEU2-ars18 replicative vector to transform the disrupted strain. By measuring AEP produced by these strains, we observed a linear increase of AEP secretion with gene copy number.