Travail d'études : Heterologous protein production in Yarrowia lipolytica: laccase as a case study.

Abstract

Recombinant proteins (rProt) are essential in industrial, pharmaceutical and diagnostic applications. Their efficient production often requires not only high expression of their encoding gene, but also effective rProt secretion, which simplifies the downstream processing. Protein secretion relies on a so called signal peptide (SP), located at the N-terminal sequence of the protein. It is mainly composed of a pre-leader and a pro-region that have distinctive functions in the secretion pathway. The yeast Yarrowia lipolytica is commonly used for rProt production due to its ability to express genes at a high level and its effective secretion machinery. However, the secretion efficiency for a given rProt strongly depends on the specificity of the SP sequence. This thesis explores the influence of different SPs on the extracellular production of a model protein, the Lac Vader laccase. Using the Golden Gate Assembly technology, several variants of SP that combined diverse pre- and pro-regions were constructed, including some derived from Lip2 and Xpr2 proteins, as well as synthetic parts. The efficiency of those SP on the secretion of the model protein was evaluated by monitoring the laccase activity in the culture supernatant for the different SP-rProt constructs. The results revealed that extended pro-regions (like Lip2 pro-region) significantly enhanced secretion. However, secretion efficiency varied between the different constructs, highlighting that not all signal sequences contributed equally to secretion. The study showed that protein export in Y. lipolytica is highly dependent on the combination of pre- and pro-region used. Importantly, by comparing secretion performance in Y. lipolytica and Pichia pastoris, the results demonstrated that Y. lipolytica generally achieved higher secretion levels. These findings confirm that secretion tags are critical for protein expression in Y. lipolytica and demonstrate how their choice directly influences secretion efficiency. In comparison, P. pastoris showed lower secretion levels, underscoring the superior performance of Y. lipolytica.