21EBTA: Engineering Biology with Synthetic Genomes (EBSynerGy)

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The yeast synthetic genome project provides engineerable yeasts (Sc2.0) with chromosomal loxP sites to allow rapid strain optimization using Synthetic Chromosome Recombination and Modification by LoxP-mediated Evolution (SCRaMbLE). We will develop methods for faithful chromosome consolidation to deliver the world's first synthetic eukaryote. Systematic characterization of fitness will allow detection of any negative genetic interactions during the merger of chromosomes, which will then be fixed to ensure high fitness. Given that the Sc2.0 SCRaMbLE technology is constrained by the disperse distribution of essential genes, we will relocate all essential genes onto a dedicated chromosome which will remain intact during SCRaMbLE experiments. By doing so, the synthetic yeast can undergo more extensive genome-wide directed evolution to generate highly productive host strains for many industrial biotechnology purposes. We will exemplify the utility of Sc2.0 & 3.0 strains through the optimized production of valuable statins, antibiotics, and nucleoside precursors for mRNA vaccines. These natural products are typically isolated in small quantities from native organisms which can be difficult to cultivate and genetically manipulate. These studies will be facilitated by the development of biosensors that bind the target product and generate a fluorescence output upon ligand binding. Finally, we will develop synthetic yeast strains that are specifically tailored for the production of proteins containing a broad range of non-canonical amino acids. This will be achieved by equipping Sc2.0/Sc3.0 with new PylRS/tRNA homologs that operate efficiently in yeast, and eukaryotic release factors will be engineered so that they no longer terminate protein synthesis on the UAG codon. Strain optimization using the SCRaMbLE function will allow scalable production of functionalized proteins to underpin the development of precisely engineered protein therapies, catalysts and materials.