Evaluation of strategies for construction of protease libraries in yeast.

Abstract: Proteases are a group of enzymes that are involved in breaking down larger proteins into smaller polypeptides or single amino acids, and they are used in various applications. In this project, the potential of budding yeast Saccharomyces cerevisiae as host for the construction of protease libraries is evaluated. Three model proteases of bacterial origin are used to evaluate transformation efficiency, protease gene expression level, and yeast cell fitness. A plasmid-based system was compared with a CRISPR/Cas9-based chromosomal integration system in terms of transformation efficiency. It was found that the plasmid system had 5-fold higher transformation efficiency. To study expression of the protease genes they were fused to green fluorescent protein (GFP), enabling easy detection of the proteins by measuring fluorescence. A new experimental protocol to screen the GFP expression level of the yeast colonies in microtiter plates based on fluorometry and spectrometry, to measure fluorescence and optical density, respectively, was developed. Gene expression and viability for yeast strains were also studied using the flow cytometry. Growth and metabolic profiles of yeast strains were studied in different conditions to understand possible effects of proteases on yeast fitness. It was found that two out of three proteases were expressed at high level and yeast still displayed high fitness, demonstrating its potential as host for proteases with a certain substrate specificity. On the other hand, the results indicate that recombinant proteases can also have a detrimental effect on yeast fitness thereby restricting the possibility to build a broad specificity protease gene expression library.