(629b) Rational Approach to Yeast Whole Cell Biocatalyst Design: Compositionally Uniform, High Enzyme Density Display for Improved Cellulosic Biomass Fermentation
AIChE Annual Meeting
2020
2020 Virtual AIChE Annual Meeting
Food, Pharmaceutical & Bioengineering Division
Enhancing Metabolic Processes by Chassis Engineering and Electrocatalysis
Friday, November 20, 2020 - 8:15am to 8:30am
In this study, we engineer yeast cells to display anchor scaffolds that bind primary scaffolds, onto which cellulases are docked. The anchor scaffolds are based on the most commonly used yeast surface display platform, Aga1-Aga2, which is known to yield a mixture of non-displaying and displaying cells (i.e. inactive and active biocatalysts). Through careful evaluation of the display system, we speculated low copy number plasmid and plasmid loss as the root cause of limited enzyme density at the single cell and population level. To address this, we replaced the plasmid-based expression with genomic integration, yielding uniformly active whole cell biocatalysts. By adjusting the gene copy number, we were able to further tune the enzyme display level of these biocatalysts. More importantly, the creation of compositionally uniform yeast biocatalysts with high enzyme display levels improved the accuracy of our mathematical model, thereby providing insights into the cellulosome assembly process under the molecular crowding regime. The resulting model enabled rational design of the optimal yeast biocatalyst achieving the highest cellulose to ethanol conversion yield reported to date. This work demonstrates the possibility to rationally design whole cell biocatalysis aided by quantitative tools.