21EBTA: Engineering Biology with Synthetic Genomes (EBSynerGy)

Yeast, Saccharomyces cerevisiae, has been used for centuries to bake bread and in brewing. Yeast is utilised across the world in many important industrial biotechnology applications, and is a model eukaryotic organism of great importance in fundamental research. Over the past decade, a large international consortium (Sc2.0) has been synthesising yeast chromosomes (16 in total). We aim to combine these chromosomes to create the world's first fully synthetic eukaryotic cell, which has a number of in-built design features to allow for its rapid optimization for new applications. For example, a genome scrambing function allows researchers to re-shuffle the genome like a deck of cards and can be applied to generate mutant strains with improved properties for industrial applications. However, genome scrambling can lead to the loss of essential genes which kills the cells. In light of this, we propose to relocate all the essential genes onto a dedicated "neochromosome" creating Sc3.0 cells. These new Sc3.0 cells will retain the essential genes during scrambling, so that more cells survive with desirable traits. We will exploit the synthetic yeast (Sc2.0/3.0), as a platform for the production of valuable natural products (e.g. antibiotics), cholesterol lowering agents (statins) and precursors required for manufacture of mRNA vaccines (e.g. Pfizer's COVID vaccine). Pathways to the target compounds will be introduced in the synthetic yeast and genome scrambling will be used to create large numbers of mutants producing the target compounds. We will also develop biosensors that can bind to the target products, triggering a fluorescent response to allow us to rapidly select the scrambled mutant cells that produce the highest levels of the target compound. In addition, we will develop novel imaging methods that can rapidly identify cells producing the desired compound. Finally, we will use the synthetic yeast to efficiently produce new generations of functionalized proteins. Nature produces proteins with an extraordinary range of functions, from enzymes that accelerate biochemical reactions needed for life to the antibodies that protect us from infectious diseases. These proteins are chains (polymers) made from only twenty building blocks called amino acids. In recent years an exciting technology has emerged called genetic code expansion (GCE) that allows us to produce proteins from more than these twenty building blocks. This technology is well developed for use in bacterial cells, but unfortunately many proteins can only be produced in higher organisms such as yeast - and here many of the important GCE tools do not operate effectively. Here we will develop yeast strains that are specifically optimized for the production of proteins with an expanded range of building blocks. These strains will underpin the development of new generations of protein therapies, catalysts and materials.

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.