Elucidating structure–performance relationships in whole-cell cooperative enzyme catalysis

Abstract

Cooperative enzyme catalysis in nature has long inspired the application of engineered multi-enzyme assemblies for industrial biocatalysis. Despite considerable interest, efforts to harness the activity of cell-surface displayed multi-enzyme assemblies have been based on trial and error rather than rational design due to a lack of quantitative tools. In this study, we developed a quantitative approach to whole-cell biocatalyst characterization enabling a comprehensive study of how yeast-surface displayed multi-enzyme assemblies form. Here we show that the multi-enzyme assembly efficiency is limited by molecular crowding on the yeast cell surface, and that maximizing enzyme density is the most important parameter for enhancing cellulose hydrolytic performance. Interestingly, we also observed that proximity effects are only synergistic when the average inter-enzyme distance is > ~130 nm. The findings and the quantitative approach developed in this work should help to advance the field of biocatalyst engineering from trial and error to rational design.

Yeast Intracellular Staining (yICS): Enabling High-Throughput, Quantitative Detection of Intracellular Proteins via Flow Cytometry for Pathway Engineering

“…Therefore, comparing the abundance of different proteins using yICS requires an additional fluorescence quantification step. 20 As anticipated, fixation with higher formalin concentrations resulted in both slightly increased background autofluorescence signal (Figure 3A, top) and reduced fluorescence signal for both β actin and GAPDH (Figure 3A, middle and bottom), likely due to decreased antibody binding affinity for cross-linked epitopes as has been reported elsewhere. 54 As a result, the lowest formalin concentration (i.e., 0.5%) yielded the best signal-to-noise ratio.…”

“…Note that, although actin is the most abundant protein in eukaryotic cells, the fact that β actin showed lower overall signal compared to GAPDH highlights that signal intensity is a function of several variables beyond protein abundance such as antibody affinity, antibody concentration, and antibody accessibility to its epitope. Therefore, comparing the abundance of different proteins using yICS requires an additional fluorescence quantification step . As anticipated, fixation with higher formalin concentrations resulted in both slightly increased background autofluorescence signal (Figure A, top) and reduced fluorescence signal for both β actin and GAPDH (Figure A, middle and bottom), likely due to decreased antibody binding affinity for cross-linked epitopes as has been reported elsewhere .…”

“…Due to the ease of library generation and model-eukaryote status, Saccharomyces cerevisiae remains one of the most popular organisms for pathway engineering. − Further, its compatibility with flow cytometry allows for a convenient, quantitative, and high-throughput means of protein detection as long as the protein expression can be coupled to fluorescence. While surface-displayed proteins can be conveniently labeled with fluorescent antibodies, − the vast majority of proteins that participate in cellular pathways are not surface accessible and therefore require alternative labeling strategies . The coupling of fluorescence to an intracellular protein of interest (POI) is most commonly accomplished by genetic fusion of the POI with a reporter protein .…”

“…These substrates are usually conjugated with organic dyes, which offer superior photophysical properties compared to fluorescent proteins including greater brightness and broader spectral diversity. 29 However, both the fluorescent proteins and self-labeling enzymes are relatively large in size (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33), and therefore their fusion with the POI often negatively affects protein function. 30−34 Moreover, the production of reporter protein fusions consumes additional cellular resources that can inhibit cell growth or detract from the production of pathway products.…”

Yeast Surface Display System: Strategies for Improvement and Biotechnological Applications

“…Enzyme-substrate interactions can be negatively affected if distance is not optimum, the opposite effect is detected when a favorable distance is established. Smith et al (2019) highlighted that the enzyme density is a pivotal parameter to enhance cellulose hydrolytic performance when a multi-enzyme assembly is designed. Tsai et al (2013) observed a 2-fold increase in ethanol production when cells displayed a tetravalent cellulosome instead of a divalent cellulosome.…”