title: Production of glucoamylase in pyruvate decarboxylase deletion mutants of the yeast Kluyveromyces lactis.
abstract: Yeasts are widely used as hosts for the production of diverse heterologous proteins ranging from laboratory scale to industrial scale. The aim of this work is to provide new tools for the production of heterologous proteins in the yeast Kluyveromyces lactis. The promoter of the single gene (KlPDC1) encoding pyruvate decarboxylase is strong, inducible, and responsive to the presence of fermentable sugars and anoxic conditions in this yeast. Expression of KlPDC1 is repressed by ethanol and by autoregulation, a mechanism that involves protein KlPdc1. We constructed a heterologous gene expression cassette for a secreted protein (glucoamylase, GAM) under the control of the KlPDC1 promoter on a stable multicopy plasmid. GAM production by wild-type transformed strains was compared with that of klpdc1-deleted transformants. We obtained higher GAM production in the latter strains, which was due to continued expression of the GAM gene during the stationary phase rather than due to GAM transcription levels higher than the wild-type strains during growth phase. This finding opens new perspectives on the physiology of the stationary phase in K. lactis and suggests the possibility of using high-cell-density approaches for the efficient production of heterologous proteins with this yeast.
authors.affiliation: Centre of Excellence in Molecular Biology and Medicine, University of Rome La Sapienza, Rome, 00185, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Salani
authors.forename: Michele M
authors.initials: MM
authors.lastname: Bianchi
mesh_terms: D019149:Bioreactors; D005285:Fermentation; D017353:Gene Deletion; D018628:Gene Dosage; D015966:Gene Expression Regulation, Fungal; D005822:Genetic Vectors; D005087:Glucan 1,4-alpha-Glucosidase / Q000096:biosynthesis*; D007716:Kluyveromyces / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D011767:Pyruvate Decarboxylase / Q000235:genetics*; D012331:RNA, Fungal / Q000032:analysis / Q000302:isolation & purification; D012333:RNA, Messenger / Q000032:analysis / Q000302:isolation & purification; D014170:Transformation, Genetic
chemical_list: D012331:RNA, Fungal; D012333:RNA, Messenger; D005087:Glucan 1,4-alpha-Glucosidase; D011767:Pyruvate Decarboxylase