title: High-throughput transformation method for Yarrowia lipolytica mutant library screening.
abstract: As a microorganism of major biotechnological importance, the oleaginous yeast Yarrowia lipolytica is subjected to intensive genetic engineering and functional genomic analysis. Future advancements in this area, however, require a system that will generate a large collection of mutants for high-throughput screening. Here, we report a rapid and efficient method for high-throughput transformation of Y. lipolytica in 96-well plates. We developed plasmids and strains for the large-scale screening of overexpression mutant strains, using Gateway® vectors that were adapted for specific locus integration in Y. lipolytica. As an example, a collection of mutants that overexpressed the alkaline extracellular protease (AEP) was obtained in a single transformation experiment. The platform strain that we developed to receive the overexpression cassette was designed to constitutively express a fluorescent protein as a convenient growth reporter for screening in non-translucid media. An example of growth comparison in skim milk-based medium between AEP overexpression and deletion mutants is provided.
authors.affiliation: INRA, UMR1319 Micalis, F-78352 Jouy-en-Josas, France AgroParisTech, UMR Micalis, F-78352 Jouy-en-Josas, France.
authors.forename: Christophe
authors.initials: C
authors.lastname: Leplat
authors.affiliation: INRA, UMR1319 Micalis, F-78352 Jouy-en-Josas, France AgroParisTech, UMR Micalis, F-78352 Jouy-en-Josas, France.
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Nicaud
authors.affiliation: INRA, UMR1319 Micalis, F-78352 Jouy-en-Josas, France AgroParisTech, UMR Micalis, F-78352 Jouy-en-Josas, France tristan.rossignol@grignon.inra.fr.
authors.forename: Tristan
authors.initials: T
authors.lastname: Rossignol
mesh_terms: D015723:Gene Library; D018014:Gene Transfer Techniques; D005820:Genetic Testing / Q000379:methods*; D005827:Genetics, Microbial / Q000379:methods*; D057166:High-Throughput Screening Assays; D008967:Molecular Biology / Q000379:methods*; D010957:Plasmids; D014170:Transformation, Genetic; D025062:Yarrowia / Q000235:genetics*
keywords: Gateway®; XPR2; Yarrowia lipolytica; overexpression; transformation method, mutant screening