Damgaard Jensen, E. | bioRxiv Science

bioRxiv2026

Engineering orthogonal quorum sensing circuits using LuxR-type systems in yeast consortia

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. LuxR and RpaR, were compatible with in vivo ligand production and established as orthogonal QS signaling pair with synthases MesI and RpaI, respectively. Co-culture experiments demonstrated 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.

synthetic biology↗

bioRxiv2024

Heat pre-treatment reduces multiplicity of plasmid transformations in yeast during electroporation, without diminishing the transformation efficiency

High-throughput DNA transformation techniques are invaluable when creating high-diversity mutant libraries, and the success rate of any protein engineering endeavors is directly dependent on both the size and diversity of the mutant library that is to be screened. It is also widely accepted that in both bacteria and yeast there is an inverse correlation between the DNA transformation efficiency and the likelihood of transforming multiple DNA molecules into each cell. However, most successful high-throughput mutant screening efforts require high quality libraries, i.e., libraries comprised of cells with a clear phenotype-to-genotype relationship (one genotype/cell). Thus, DNA transformation methods with a high multiplicity of transformation are highly undesirable and detrimental to most mutant screening assays. Here we describe a simple, robust, and highly efficient yeast plasmid DNA transformation methodology, using a dual heat-shock and electroporation approach (HEEL) that generates more than 2 x 107 plasmid-transformed yeast cells per reaction, while simultaneously increasing the fraction of mono-transformed cells from 20% to more than 70% of the transformed population. By also using an automated yeast genotyping workflow coupled with a dual-barcoding approach, consisting of a SNP and high-diversity barcode (10N), we can consistently identify and enumerate unique plasmid genotypes within a heterogeneous population merely through Sanger sequencing. We demonstrate here that the size and quality of a transformed library no longer need to be inversely correlated when transforming large mutant DNA libraries in yeast using highly efficient DNA electroporation methods. SignificanceWith the recent expansion of artificial intelligence in the field of synthetic biology there has never been a greater need for high-quality data and reliable measurements of phenotype-to-genotype relationships. However, one major obstacle to creating accurate computer-based models is the current abundance of low-quality phenotypic measurements originating from numerous high-throughput, but low-resolution assays. Rather than increasing the quantity of measurements, new studies should aim to generate as accurate measurements as possible. The HEEL methodology presented here aims to address this issue by minimizing the problem of multi-plasmid uptake during high-throughput yeast DNA transformations, which leads to the creation of heterogeneous cellular genotypes. HEEL should enable highly accurate phenotype-to-genotype measurements going forward, which could be used to construct better computer-based models.

microbiology↗