Single-cell analysis of yeast surface display for designer cellulosome applications using a fluorescent protein complex

Designer cellulosomes (DCs) are precisely engineered multi-enzyme complexes aimed at lignocellulose saccharification. Achieving spontaneous designer cellulosome display on yeast cell surfaces has been a long-term objective to enhance consolidated bioprocesses with concurrent ethanol production. A "self-assembly" approach involves simultaneous scaffoldin display and docking enzyme secretion. However, challenges arise from the size and complexity of designer cellulosomes, coupled with the yeast cells' limited capacity for heterologous protein expression. A comprehensive examination of <i>Saccharomyces cerevisiae</i> as a host for DC expression remains unaddressed. We meticulously examined the capability of <i>S. cerevisiae</i> to produce and display a fluorescent protein complex, which mimics the designer cellulosome architecture and allows for convenient detection of all individual components on the cell surface, using flow cytometry and confocal microscopy. Population-wide analysis revealed a fluorescent protein complex production efficiency of approximately 10%. Single-cell analysis highlighted a clear mutual influence between the expression of scaffoldin and docking proteins, impacting cellular fitness. Newly emerging buds were identified as hotspots for scaffoldin display. The finite capacity of yeast cells to produce heterologous proteins was identified as a major bottleneck. While distributing the cellular load among multiple hosts within a synthetic yeast consortium can alleviate this burden, the use of fluorescent protein complex surface display has visualized the heterogeneity and constraints of <i>S. cerevisiae</i> as a host for designer cellulosome expression at the population and single cell level. This study provides a realistic assessment of the challenges in achieving efficient <i>S. cerevisiae</i>-based DC display for consolidated bioprocessing.IMPORTANCEEfficient and economically viable biomass conversion into fermentable sugars is a pivotal challenge in transitioning from a petroleum-based economy to a bio-economy. Drawing inspiration from nature, cellulosomes represent an exemplary solution for the effective digestion of lignocellulose. These multi-enzyme complexes can be precisely engineered to tailor their properties and transferred to the surface of yeast cells, which can subsequently ferment the sugars into bulk or fine chemicals. Achieving this transfer successfully necessitates a comprehensive understanding of how yeast cells can recombinantly produce and attach such multi-component complexes to their surface. This study employs a fluorescent surrogate to provide novel insights into the capabilities of yeast cells at both the single-cell and population levels.