Systematic genetic modifications of cell wall biosynthesis enhanced the secretion and surface-display of polysaccharide degrading enzymes in Saccharomyces cerevisiae

Abstract

Saccharomyces cerevisiae is a robust cell factory to secrete or surface-display cellulase and amylase for the conversion of agricultural residues into valuable chemicals. Engineering the secretory pathway is a well-known strategy for overproducing these enzymes. Although cell wall biosynthesis can be tightly linked to the secretory pathway by regulation of all involved processes, the effect of its modifications on protein production has not been extensively studied. In this study, we systematically studied the effect of engineering cell wall biosynthesis on the activity of cellulolytic enzyme β-glucosidase (BGL1) by comparing seventy-nine gene knockout S. cerevisiae strains and newly identified that inactivation of DFG5, YPK1, FYV5, CCW12 and KRE1 obviously improved BGL1 secretion and surface-display. Combinatorial modifications of these genes, particularly double deletion of FVY5 and CCW12, along with the use of rich medium, increased the activity of secreted and surface-displayed BGL1 by 6.13-fold and 7.99-fold, respectively. Additionally, we applied this strategy to improve the activity of the cellulolytic cellobiohydrolase and amylolytic α-amylase. Through proteomic analysis coupled with reverse engineering, we found that in addition to the secretory pathway, regulation of translation processes may also involve in improving enzyme activity by engineering cell wall biosynthesis. Our work provides new insight into the construction of a yeast cell factory for efficient production of polysaccharide degrading enzymes.

Keywords: Amylase; Cell wall biosynthesis; Cellulase; Saccharomyces cerevisiae; Secretion; Surface-display.