Improving the functionality of surface-engineered yeast cells by altering the cell wall morphology of the host strain

Abstract

The expression of functional proteins on the cell surface using glycosylphosphatidylinositol (GPI)-anchoring technology is a promising approach for constructing yeast cells with special functions. The functionality of surface-engineered yeast strains strongly depends on the amount of functional proteins displayed on their cell surface. On the other hand, since the yeast cell wall space is finite, heterologous protein carrying capacity of the cell wall is limited. Here, we report the effect of CCW12 and CCW14 knockout, which encode major nonenzymatic GPI-anchored cell wall proteins (GPI-CWPs) involved in the cell wall organization, on the heterologous protein carrying capacity of yeast cell wall. Aspergillus aculeatus β-glucosidase (BGL) was used as a reporter to evaluate the protein carrying capacity in Saccharomyces cerevisiae. No significant difference in the amount of cell wall-associated BGL and cell-surface BGL activity was observed between CCW12 and CCW14 knockout strains and their control strain. In contrast, in the CCW12 and CCW14 co-knockout strains, the amount of cell wall-associated BGL and its activity were approximately 1.4-fold higher than those of the control strain and CCW12 or CCW14 knockout strains. Electron microscopic observation revealed that the total cell wall thickness of the CCW12 and CCW14 co-knockout strains was increased compared to the parental strain, suggesting a potential increase in heterologous protein carrying capacity of the cell wall. These results indicate that the CCW12 and CCW14 co-knockout strains are a promising host for the construction of highly functional recombinant yeast strains using cell-surface display technology. Key points• CCW12 and/or CCW14 of a BGL-displaying S. cerevisiae strain were knocked out.• CCW12 and CCW14 co-disruption improved the display efficiency of BGL.• The thickness of the yeast cell wall was increased upon CCW12 and CCW14 knockout.

Direct production of 4‐hydroxybenzoic acid from cellulose using cellulase‐displaying Pichia pastoris

“…aculeatus BGL1 fused with the GCW61 under the control of the SPI1 promoter and the SPI1 secretion signal (Pp-BG-SSG61) showed cell-surface BGL activity of 3399 U/g dry cells. This was approximately 2.7-fold higher than that of the previously recorded highest level in S. cerevisiae (1250 U/g dry cells) in our previous study (Inokuma et al, 2021) measured by the same method. Although no reports have directly compared the performance of S. cerevisiae and P. pastoris as hosts for yeast cell-surface display technology, these results clearly demonstrate the potential of P. pastoris as a host for this technology.…”

The Aspartic Protease Yps3p and Cell Wall Glucanase Scw10p Are Novel Determinants That Enhance the Secretion of the Antitumor Triterpenoid GA-HLDOA in Saccharomyces cerevisiae

“…32 A thicker cell wall was also observed in yeast strains with the cell wall mannoprotein genes CCW12 and CCW14 deleted; 33 mannoprotein reduction in these strains led to increased cell wall flexibility and reduced mechanical strength. 33 Reduction of mannoprotein can also increase cell wall porosity. 34 Although a thicker cell wall was observed concomitantly with reduced mannoprotein, no direct connection was reported between a thicker cell wall and increased cell wall flexibility.…”

“…The release of β-1,3-glucan can cause the release of closely linked mannoproteins from the cell wall . A thicker cell wall was also observed in yeast strains with the cell wall mannoprotein genes CCW12 and CCW14 deleted; mannoprotein reduction in these strains led to increased cell wall flexibility and reduced mechanical strength . Reduction of mannoprotein can also increase cell wall porosity .…”

Yeast Surface Display System: Strategies for Improvement and Biotechnological Applications

“…However, one genetic defect can cause pleiotropic changes in the cell wall structure-function relationship ( Matsuoka et al, 2014 ). Inokuma et al (2021) increased the cell wall thickness of S. cerevisiae by performing the knockout of proteins CCW12 and CCW14, which allowed an increased amount of displayed β-glucosidase as compared to the parental strain.…”