Engineered microorganisms and methods of making and using same
Inventors
Dantas, Gautam • KWAK, Suryang • Virgin, Herbert
Assignees
Washington University in St Louis WUSTL • Vir Biotechnology Inc
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Publication Number
US-12247196-B2
Publication Date
2025-03-11
Expiration Date
Abstract
The present disclosure provides for engineered microorganisms and methods of making and using same. The engineered microorganisms as described herein can have a surface display and can be useful as therapeutic agents (e.g., sponges) and biosensors.
Core Innovation
The invention relates to engineered Saccharomyces boulardii cells that present a binding agent on the cell surface. Each engineered cell comprises a genome-integrated display cassette that expresses, in operable linkage, a signal peptide, a binding agent with binding affinity to a target microorganism, and an anchor comprising a cell wall anchor protein or a fragment thereof.
The engineered display cassette is integrated at named intergenic sites within the Saccharomyces boulardii genome, selected from IS1n, IS2n, IS3n, IS4n, IS5n, and IS6n. The anchor is selected from Cwp2p, Sed1p, Ccw12p, Flo1p, and Flo428, and the binding agent is selected based on the target microorganism to be bound.
The document further describes engineered yeast biosensors in which reporter outputs can be produced. It provides examples including CLM-1 displayed on S. boulardii for binding of murine norovirus (MNOV), and it reports that the engineered S. boulardii is non-colonizing, with clearance within days after stopping administration.
Claims Coverage
The partial content provides one independent claim (clm-00001). This claim covers an engineered Saccharomyces boulardii cell with a genome-integrated surface-display cassette that includes a signal peptide, a pathogen-targeting binding agent, and a specified cell wall anchor, integrated at one of several named intergenic sites, with enumerated target microorganism-binding agent pairings.
Genome-integrated surface display cassette on engineered Saccharomyces boulardii
An engineered Saccharomyces boulardii cell comprising a genome-integrated display cassette encoding a signal peptide, a binding agent with binding affinity to a target microorganism, and an anchor comprising a cell wall anchor protein or a fragment thereof, wherein the signal peptide is operably linked to the binding agent and the binding agent is operably linked to the anchor.
Integration at named intergenic sites in Saccharomyces boulardii genome
The genome integrated display cassette is integrated at any one of IS1n, IS2n, IS3n, IS4n, IS5n, and IS6n in the Saccharomyces boulardii genome.
Specific binding-agent pairings for listed target microorganisms
The target microorganism is norovirus and the binding agent is CLM-1; the target microorganism is Clostridium difficile or Clostridium perfringens and the binding agent is a cell binding domain of ΦCP26F; the target microorganism is Listeria monocytogenes and the binding agent is cell binding domain 500 of endolysin Ply500 or cell wall binding domain of PlyP35; the target microorganism is Streptococcus pyogenes and the binding agent is PlyC binding domain of endolysin PlyC; the target microorganism is methicillin-resistant Staphylococcus aureus (MRSA) or Staphylococcus aureus and the binding agent is LysGH15B, a cell wall binding domain of endolysin LysGH15, LysSA97, or ZW88; or the target microorganism is Campylobacter coli and the binding agent is a cell wall binding domain of phiCcoIBB35.
Across the independent claim, the protected subject matter centers on engineered Saccharomyces boulardii cells having a genome-integrated, operably linked surface-display cassette integrated at specified intergenic sites, with binding agent selection tied to enumerated target microorganisms.
Stated Advantages
The engineered S. boulardii is non-colonizing, with clearance within days after stopping administration.
Documented Applications
Using CLM-1-displaying S. boulardii to bind murine norovirus (MNOV) and reporting in vivo changes in MNOV shedding in mice when administering CLM-1-displaying yeast.
Testing engineered S. boulardii stability under perturbations including streptomycin and stomach neutralization.
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