New Yarrowia lipolytica Recipient Strains Equipped with a Zeta Docking Platform Allow Efficient Integration of a Series of New and Existent Zeta-Based Auto-Cloning Vectors for Multiple Biotechnological Applications

Yarrowia lipolytica has a long history of industrial use. With its high secretion capacity, GRAS status and wide range of engineering tools, this yeast has become a powerful host for heterologous protein production as well as a favourite candidate for cellular factory design. Among integration vectors mostly used worldwide for Y. lipolytica engineering, zeta-based auto-cloning vectors took advantage of their bordering sequences (LTRs of Ylt1 retrotransposon) to integrate either by homology in Ylt1-bearing strains or non-homologously into any strain devoid of this retrotransposon. As such non-homologous integrations were less efficient and reliable, there was a need for designing zeta-bearing derivatives of widely used Y. lipolytica strains. Namely, the W29 wild-type strain and its genetically engineered derivative Po1f strain were equipped with a zeta docking platform in order to allow a more efficient and reliable integration of any zeta-based vector. The presence of this new docking platform allows to compare directly the performances of transformants obtained with different constructs, enabling to perform high-throughput screening or protein engineering. The efficiency of these new Po1z and Po1zP strains was demonstrated through the surexpression of two native proteins, a chitinase and a protease. At last, new zeta-based auto-cloning vectors with more powerful promoters were added to this new expression system.