(63f) Quantitative Whole-Cell Biocatalyst Characterization: Elucidating Structure-Performance Relationships in Cell-Surface Displayed Multi-Enzyme Assemblies

Authors: 
Smith, M., University of Michigan
Wen, F., University of Michigan
Gao, H., Konkuk University
Over millions of years of evolution, highly efficient cooperative enzyme assemblies have developed in nature that allow cells to perform life-sustaining complex chemical transformations using a variety of natural substrates. One of the most elegant enzyme assemblies to emerge in nature is the cellulosome – a modular, multi-protein complex of synergistic enzymes that catalyzes cellulose hydrolysis. Despite considerable interest in assembling cellulosome-inspired multi-enzyme complexes on microbial surfaces, efforts to harness the activity of these assemblies have been based on trial and error rather than rational design due to a lack of quantitative tools. This empirical approach has created a gap in our understanding of how multi-enzyme complexes assemble, and what parameters affect assembly efficiency and overall activity. To address these issues, we constructed a series of yeast whole-cell biocatalysts displaying an anchor scaffold protein (aScaf) accommodating one (aScaf1), two (aScaf2), or four (aScaf4) primary scaffold proteins (pScafs), with the pScaf binding four different cellulases. Using quantitative flow cytometry we revealed that, under saturating in vitro loading conditions, the overall assembly efficiency of these tetrafunctional multi-scaffolded enzyme assemblies (mSEAs) is limited by aScaf crowding on the yeast cell surface. By modeling aScaf surface distribution, we determined that aScaf-pScaf binding requires a minimum aScaf spacing of 9.1 nm for aScaf1 and 13.2 nm for larger aScafs. In contrast, pScaf-cellulase binding is not affected by the aScaf crowding. As a result, the lower expression of aScaf4 allowed for more efficient assembly leading to both improved enzyme proximity (10.6 enzymes per aScaf4-mSEA vs. 2.9 and 1.6 per aScaf2-mSEA and aScaf1-mSEA, respectively) and slightly increased enzyme density (1.2-fold over aScaf1-mSEA). From this quantitative characterization, we revealed that the increased enzyme proximity of aScaf4-mSEA accounted for a majority (75%) of its 1.7-fold improvement in cellulolytic activity over aScaf1-mSEA. To our knowledge, aScaf4-mSEA is the most complex cellulosomal structure displayed on yeast and achieved the highest ethanol titer reported to date from amorphous cellulose (4.8 g/L). These experimental and theoretical findings provide important insights into how multi-scaffolded enzymes assemble on yeast cell surface. More importantly, this work demonstrates that a quantitative approach to whole-cell biocatalyst characterization is possible, and this realization should help advance the field of biocatalyst engineering from trial and error to rational design.