Syntetic biology toolkit for engineering the non-conventional yeast Yarrowia lipolytica
Résumé
The non-conventional yeast Yarrowia lipolytica is a well established biotechnological chassis for the production of numerous valuable bioproducts, especially based on its ability to produce high amounts of lipids and lipid derivatives. Despites the availability of numerous tools for genetic engineering, there is still a need for faster and versatile new tools to speed up and multiplex engineering of this yeast. Due to the recent development of DNA assembly techniques for metabolic pathway engineering, a great world-wide effort is now being pursued towards establishing such cloning platforms for an individual organism of interest. Golden Gate modular cloning system, relying on type IIs restriction enzymes, appears as one of the most robust techniques within this field We have used the Golden Gate modular cloning strategy to develop a robust and versatile DNA assembly platform for Y. lipolytica. To this end, a broad set of destination vectors and interchangeable building blocks have been constructed allowing the assembly of up to 3 transcriptional units in one vector. The DNA modules were assembled on a scaffold of pre-designed 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. A broad range of strength promoter have been implemented in this system for fine tuning regulation and combinatorial expression of each transcription unit. The efficiency of the developed Golden Gate strategy was demonstrated by assembling the synthetic pathway for carotenoids’ production. Through this modular cloning strategy a replicative vector containing the CRISPR-Cas9 system was constructed. In addition to knock-out genes by the microdeletion caused by CRISPR-Cas9 system, heterologous pathways could be inserted at specific locus. This system has been used to genetically modify wild-type strains of Y. lipolytica. The combination of these two technologies greatly enriches the molecular biology toolbox dedicated for this industrially relevant microorganism, leading to a new efficient way to engineer wild-type strains. Fast combinatorial cloning of complex synthetic pathways, combined with efficient marker-less locus insertion and traditional metabolic engineering strategies can be of great importance for the development of Y. lipolytica as cell factory.