(629b) Rational Approach to Yeast Whole Cell Biocatalyst Design: Compositionally Uniform, High Enzyme Density Display for Improved Cellulosic Biomass Fermentation | AIChE

(629b) Rational Approach to Yeast Whole Cell Biocatalyst Design: Compositionally Uniform, High Enzyme Density Display for Improved Cellulosic Biomass Fermentation

Authors 

Ma, X. Y. - Presenter, University of Michigan
Wen, F., University of Michigan
Ponnandy, P., University of Michigan
As the most abundant biopolymer on Earth, cellulose represents a promising and sustainable feedstock for the production of biofuels and value-added chemicals. Due to its recalcitrant nature, microbes in nature organize cellulases into a multienzyme complex termed cellulosome for efficient cellulose hydrolysis. Inspired by this, researchers have engineered whole cell biocatalysts to display designer cellulosomes using chimeric scaffolds for directed assembly of enzymes. While much engineering effort has been dedicated to improve their performance, most approaches are largely empirical due to a limited mechanistic understanding of the cellulosome assembly process on the cell surface. Our recent study demonstrated that a rational approach can be realized through quantitative characterization of whole cell biocatalysts by revealing structure-performance relationships. The mathematical model predicts enzyme density rather than enzyme proximity as the most important parameter for catalytic performance. Given the heterogeneous nature of biological systems, we hypothesize that harnessing the full potential of cellulosomes will require a concerted effort of increasing enzyme density at the population and single cell level.

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.