Emma Cremelie

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 Saccharomyces cerevisiae as a host for DC expression remains unaddressed. We meticulously examined the capability of S. cerevisiae 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 S. cerevisiae 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 S. cerevisiae -based DC display for consolidated bioprocessing. IMPORTANCE: Efficient 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.

Summary: SPAED is an accessible tool for the accurate segmentation of protein domains that applies hierarchical clustering to the predicted aligned error (PAE) matrix obtained from AlphaFold predictions. It leverages information contained in the PAE matrix to better identify domain-linker boundaries and detect disordered regions. On a dataset of 376 bacteriophage endolysins (proteins that degrade the bacterial cell wall), SPAED achieves a mean intersect-over-union score of 96% and a domain-boundary-distance score of 89% compared to 94% and 70%, respectively, for the state-of-the-art tool Chainsaw. Availability and Implementation: SPAED is available on the web at http://spaed.ca and available for download at https://github.com/Rousseau-Team/spaed . Contact: Elsa Rousseau - elsa.rousseau@ift.ulaval.ca , Roberto Vázquez - rvazqf@gmail.com

Phage lysins, bacteriophage-encoded enzymes tasked with degrading their host’s cell wall, are increasingly investigated and engineered as novel antibacterials across diverse applications. Their rapid action, tuneable specificity, and low likelihood of resistance development make them particularly interesting. Despite numerous application-focused lysin studies, the art of their recombinant production remains relatively undiscussed. Here, we provide an overview of the available expression systems for phage lysin production and discuss key considerations guiding the choice of a suitable recombinant host. We systematically surveyed recent literature to evaluate the hosts used in the lysin field and cover various recombinant systems, including the well-known bacterial host Escherichia coli or yeast Saccharomyces cerevisiae, as well as plant, mammalian, and cell-free systems. Careful analysis of the limited studies expressing lysins in various hosts suggests a host-dependent effect on activity. Nonetheless, the multitude of available expression systems should be further leveraged to accommodate the growing interest in phage lysins and their expanding range of applications.