Open AccessGolden Gate Assembly system dedicated to complex pathway manipulation in Yarrowia lipolytica
Author(s) -
Celińska Ewelina,
LedesmaAmaro Rodrigo,
Larroude Macarena,
Rossignol Tristan,
Pauthenier Cyrille,
Nicaud JeanMarc
Publication year - 2017
Publication title -
microbial biotechnology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.287
H-Index - 74
ISSN - 1751-7915
DOI - 10.1111/1751-7915.12605
Subject(s) - yarrowia , synthetic biology , golden gate , computational biology , biology , cloning (programming) , terminator (solar) , modular design , gene , genetics , computer science , engineering , ionosphere , civil engineering , physics , astronomy , span (engineering) , programming language , operating system
Summary 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.