Engineering Orthogonal Quorum-Sensing Circuits Using LuxR-Type Systems in Yeast Consortia

Abstract Engineered microbial communities hold significant biotechnological potential because their collective metabolism can produce functions beyond those achievable by individual strains. However, multicellular synthetic gene circuits require orthogonal communication systems that enable precise, programmable signaling between cells. Quorum sensing (QS), where cells both produce and detect small diffusible signal molecules, offers a natural framework for such intercellular communication. However, the construction of complex multicellular circuits for applications such as biobased production is currently hampered by the limited number of orthogonal QS channels available in yeast. Here, we expand the QS toolkit in Saccharomyces cerevisiae by characterizing four LuxR-type biosensors based on EsaR, LasR, TraR, and RpaR, alongside the previously established LuxR biosensor. We functionally expressed acyl-CoA-dependent HSL synthases in yeast, producing a diverse range of aliphatic and aromatic HSL signals. The regulators LuxR and RpaR were compatible with in vivo ligand production by synthases MesI and RpaI, respectively, enabling the construction of two orthogonal QS systems. Co-culture experiments of two engineered strains, each expressing one synthase and the regulator from the other QS system, demonstrated simultaneous orthogonal QS-dependent intercellular signaling, with 3.9-fold and 6.4-fold induction relative to monocultures. Together, these results establish a modular and extensible platform for orthogonal intercellular communication in yeast, enabling the construction of multicellular synthetic gene circuits.