title: A set of aspartyl protease-deficient strains for improved expression of heterologous proteins in Kluyveromyces lactis.
abstract: Secretion of recombinant proteins is a common strategy for heterologous protein expression using the yeast Kluyveromyces lactis. However, a common problem is degradation of a target recombinant protein by secretory pathway aspartyl proteases. In this study, we identified five putative pfam00026 aspartyl proteases encoded by the K. lactis genome. A set of selectable marker-free protease deletion mutants was constructed in the prototrophic K. lactis GG799 industrial expression strain background using a PCR-based dominant marker recycling method based on the Aspergillus nidulans acetamidase gene (amdS). Each mutant was assessed for its secretion of protease activity, its health and growth characteristics, and its ability to efficiently produce heterologous proteins. In particular, despite having a longer lag phase and slower growth compared with the other mutants, a Δyps1 mutant demonstrated marked improvement in both the yield and the quality of Gaussia princeps luciferase and the human chimeric interferon Hy3, two proteins that experienced significant proteolysis when secreted from the wild-type parent strain.
authors.affiliation: Division of Gene Expression, New England Biolabs, Ipswich, MA 01938-2723, USA.
authors.forename: Mehul B
authors.initials: MB
authors.lastname: Ganatra
authors.forename: Saulius
authors.initials: S
authors.lastname: Vainauskas
authors.forename: Julia M
authors.initials: JM
authors.lastname: Hong
authors.forename: Troy E
authors.initials: TE
authors.lastname: Taylor
authors.forename: John-Paul M
authors.initials: JP
authors.lastname: Denson
authors.forename: Dominic
authors.initials: D
authors.lastname: Esposito
authors.forename: Jeremiah D
authors.initials: JD
authors.lastname: Read
authors.forename: Hana
authors.initials: H
authors.lastname: Schmeisser
authors.forename: Kathryn C
authors.initials: KC
authors.lastname: Zoon
authors.forename: James L
authors.initials: JL
authors.lastname: Hartley
authors.forename: Christopher H
authors.initials: CH
authors.lastname: Taron
mesh_terms: D028023:Arecaceae / Q000201:enzymology; D057055:Aspartic Acid Proteases / Q000172:deficiency*; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics; D017353:Gene Deletion; D015870:Gene Expression; D007716:Kluyveromyces / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011994:Recombinant Proteins / Q000378:metabolism*; D017422:Sequence Analysis, DNA
chemical_list: D004271:DNA, Fungal; D005656:Fungal Proteins; D011994:Recombinant Proteins; D008156:Luciferases; D057055:Aspartic Acid Proteases