Golden Gate Assembly system dedicated to complex pathway manipulation in Yarrowia lipolytica.

Ewelina Celińska (1) , Rodrigo Ledesma Amaro (2, 3) , Macarena Larroudé (2, 3) , Tristan Rossignol (2, 3) , Cyrille Pauthenier (4) , Jean-Marc Nicaud (2, 3)

1 Department of Biotechnology and Food Microbiology
2 MICALIS - MICrobiologie de l'ALImentation au Service de la Santé
3 Université Paris-Saclay
4 ISSB - Institut de biologie systémique et synthétique

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.

Domaines: Sciences du Vivant [q-bio]

Ewelina Celińska, Rodrigo Ledesma Amaro, Macarena Larroudé, Tristan Rossignol, Cyrille Pauthenier, et al.. Golden Gate Assembly system dedicated to complex pathway manipulation in Yarrowia lipolytica.. Microbial Biotechnology, 2017, 10 (2), pp.450-455. ⟨10.1111/1751-7915.12605⟩. ⟨hal-01602609⟩