Improved β-glucosidase expressing in Saccharomyces cerevisiae An-α by YPS1/YPS2 gene inactivation

Abstract:

[Background] Low level expression and poor secretion efficiency are the common problems in the production of secretory recombinant proteins by industrial yeast. [Objective] To investigate the effect of inactivating yapsin proteases Yps1p and Yps2p on β-glucosidase expression in Saccharomyces cerevisiae An-α, a yeast strain isolated from Angel industrial yeasts. [Methods] By utilizing CRISPR/Cas9-based gene editing technology, we firstly constructed a UPR-indicator strain An-α(leu2::UPRE-lacZ), designated as An-αL. Next, the YPS1 and YPS2 genes were inactivated, followed by transformation of the β-glucosidase-expressing plasmid BG that was constructed on the YEplac195 vector. The growth and enzymatic activities of the resultant strains were evaluated. [Results] Inactivation of the YPS1 or YPS2 gene of the strain An-αL did not affect the cell growth in YPD medium. The presence of the plasmid BG increased maximum OD₆₀₀ values in the YPC medium by 21.9% and 7.4%, respectively. The maximum enzymatic activities were 0.087 5 and 0.068 6 U/(mL·OD₆₀₀), which were 2.268 and 1.778 times of the control value, respectively. The ratio of the secreted protein was also increased by 19.4% and 22.2%, respectively. There was a good correlation between the β-glucosidase activity level and the UPR signal response value as represented by the β-galactosidase activity level. [Conclusion] Inactivation of YPS1 and YPS2 could improve the secretory β-glucosidase yield of the strain An-α.

Key words:YPS1/YPS2 gene inactivation;Saccharomyces cerevisiae An-α;β-glucosidase;secretory expression