A Modular Toolkit for Generating Pichia pastoris Secretion Libraries.

Yeasts are powerful eukaryotic hosts for the production of recombinant proteins due to their rapid growth to high cell densities and ease of genetic modification. For large-scale industrial production, secretion of a protein offers the advantage of simple and efficient downstream purification that avoids costly cell rupture, denaturation and refolding. The methylotrophic yeast Pichia pastoris (Komagataella phaffi) is a well-established expression host that has the ability to perform post-translational modifications and is generally regarded as safe (GRAS). Nevertheless, optimization of protein secretion in this host remains a challenge due to the multiple steps involved during secretion and a lack of genetic tools to tune this process. Here, we developed a toolkit of standardized regulatory elements specific for Pichia pastoris allowing the tuning of gene expression and choice of protein secretion tag. As protein secretion is a complex process, these parts are compatible with a hierarchical assembly method to enable the generation of large and diverse secretion libraries in order to explore a wide range of secretion constructs, achieve successful secretion, and better understand the regulatory factors of importance to specific proteins of interest. To assess the performance of these parts, we built and characterized the expression and secretion efficiency of 124 constructs that combined different regulatory elements with two fluorescent reporter proteins (RFP, yEGFP). Intracellular expression from our promoters was comparatively independent of whether RFP or yEGFP, and whether plasmid-based expression or genomically integrated expression, was used. In contrast, secretion efficiency significantly varied for different genes expressed using identical regulatory elements, with differences in secretion efficiency of >10-fold observed. These results highlight the importance of generating diverse secretion libraries when searching for optimal expression conditions, and demonstrate that our toolkit is a valuable asset for the creation of efficient microbial cell factories.