title: Protein expression and secretion in the yeast Yarrowia lipolytica.
abstract: Strains and vectors for protein expression and secretion have been developed in the yeast Yarrowia lipolytica. Host strains were constructed with non-reverting auxotrophic markers, deletions of protease-encoding genes, and carrying a docking platform. To drive transcription, either the synthetic hp4d or the inducible POX2 promoter were used. Protein secretion is either directed by the targeting sequence of the alkaline extracellular protease or the extracellular lipase (LIP2p) signal sequence. We describe a set of vectors based on these promoters, targeting sequences and two URA3 alleles as selection markers. The wild-type URA3 allele, ura3d1, was used for single-copy integration and a mutant URA3 allele, ura3d4, was used to select for multi-copy integration into the genome. These vectors were used to express the Y. lipolytica extracellular lipase LIP2p and the Aspergillus oryzae leucine amino peptidase II. Lipase production under the control of the hp4d promoter by a strain containing a single copy reached 1000 U ml(-1) in shake flasks, while a strain containing multiple integrations reached 2000 U ml(-1) in shake flasks, 11500 U ml(-1) in batch and 90500 U ml(-1) in fed batch. Leucine amino peptidase production under the control of the hp4d promoter reached 320 mU ml(-1) in batch with a mono-copy lapA integrant and 28000 mU ml(-1) in fed batch with a multi-copy transformant.
authors.affiliation: Laboratoire de Génétique des Microorganismes, Centre National de la Recherche Scientifique, 78850 Thiverval-Grignon, France. jean-marc.nicaud@grignon.inra.fr
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Nicaud
authors.forename: Catherine
authors.initials: C
authors.lastname: Madzak
authors.forename: Peter
authors.initials: P
authors.lastname: van den Broek
authors.forename: Christof
authors.initials: C
authors.lastname: Gysler
authors.forename: Philippe
authors.initials: P
authors.lastname: Duboc
authors.forename: Peter
authors.initials: P
authors.lastname: Niederberger
authors.forename: Claude
authors.initials: C
authors.lastname: Gaillardin
mesh_terms: D001426:Bacterial Proteins; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics; D005784:Gene Amplification; D015870:Gene Expression; D005800:Genes, Fungal; D005822:Genetic Vectors; D008049:Lipase / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011995:Recombination, Genetic; D025062:Yarrowia / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; C408971:LIP2 protein, Yarrowia lipolytica; D008049:Lipase