title: Golden Gate Assembly system dedicated to complex pathway manipulation in Yarrowia lipolytica.
abstract: In this study, we have adopted Golden Gate modular cloning strategy to develop a robust and versatile DNA assembly platform for the nonconventional yeast Yarrowia lipolytica. To this end, a broad set of destination vectors and interchangeable building blocks have been constructed. The DNA modules were assembled on a scaffold of predesigned 4 nt overhangs covering three transcription units (each bearing promoter, gene and terminator), selection marker gene and genomic integration targeting sequences, constituting altogether thirteen elements. Previously validated DNA modules (regulatory elements and selection markers) were adopted as the Golden Gate bricks. The system's operability was demonstrated based on synthetic pathway of carotenoid production. This technology greatly enriches a molecular biology toolbox dedicated to this industrially relevant microorganism enabling fast combinatorial cloning of complex synthetic pathways.
authors.affiliation: Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences, ul. Wojska Polskiego 48, 60-627, Poznan, Poland.
authors.forename: Ewelina
authors.initials: E
authors.lastname: Celińska
authors.affiliation: Micalis Institute, INRA, AgroParisTech, Université Paris-Saclay, 78350, Jouy-en-Josas, France.
authors.forename: Rodrigo
authors.initials: R
authors.lastname: Ledesma-Amaro
authors.affiliation: Micalis Institute, INRA, AgroParisTech, Université Paris-Saclay, 78350, Jouy-en-Josas, France.
authors.forename: Macarena
authors.initials: M
authors.lastname: Larroude
authors.affiliation: Micalis Institute, INRA, AgroParisTech, Université Paris-Saclay, 78350, Jouy-en-Josas, France.
authors.forename: Tristan
authors.initials: T
authors.lastname: Rossignol
authors.affiliation: Institute of System and Synthetic Biology, Universite d'Evry vald'Essonnes, Bt. Geneavenir 6 Genopole Campus 1, 5 rue Henry Desbrueres, 91000, Evry, France.
authors.forename: Cyrille
authors.initials: C
authors.lastname: Pauthenier
authors.affiliation: Micalis Institute, INRA, AgroParisTech, Université Paris-Saclay, 78350, Jouy-en-Josas, France.
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Nicaud
mesh_terms: D002338:Carotenoids / Q000378:metabolism; D005827:Genetics, Microbial / Q000379:methods*; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways; D025062:Yarrowia / Q000235:genetics* / Q000378:metabolism*
chemical_list: D002338:Carotenoids