14-ERASynBio - IESY - Inducible Evolution of Synthetic Yeast genomes

Induced Evolution of Synthetic Yeast genomes (IESY) will use the first synthetic eukaryote, Saccharomyces cerevisiae 2.0 (Sc2.0), as a platform for metabolic engineering and genome minimization, and more importantly for generating and understanding industrially high-value phenotypes. Synthetic chromosomes in Sc2.0 permit rapid and comprehensive genome evolution through synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE). SCRaMbLE will be exploited here to evolve strains selected for high-value phenotypes for biofuels and biotechnology, using both chemostats and batch transfer methods in the USA and Europe. Technologies for neochromosomes and orthogonal SCRaMbLE of gene classes will be developed. Evolutionary trajectories will be analysed to relate genome structure with genome function: DNA sequencing will reveal the genome sequence, rearrangements, and copy number changes in the evolved strains; chromosome conformation capture will show how massive rearrangements affect 3D structure; and deep sequencing technologies will relate sequence and structure to gene expression and isoform abundance. Computational analysis will identify the evolutionary drivers for high fitness, with the potential for further optimization. IESY builds on resources uniquely available from the international Sc2.0 consortium and will be an international resource for efficient evolution of high-value phenotypes. This project represents a new paradigm in synthetic biology in which a genome is pre-programmed to explore combinatorial diversity space to evolve new and useful function.

Our goals are specifically to generate strains with high fitness for three valuable phenotypes: growth in glucose-limiting medium, relevant for use of yeast in industrial bioreactors; growth in high ethanol concentrations, essential for yeast as route to biofuels; and production of a carotenoid (see letter, DSM) or other high-value metabolite to be selected by the IESY members.

This project will also identify the evolutionary changes in genome structure and gene expression that lead to high fitness under each condition. This will permit us to identify complementary or even synergistic rearrangements (loss or duplication of gene(s) that can be combined for greater fitness. We will be able to generalize findings from the synthetic strain to yeast strains already used for biotechnological processes.

While IESY benefits from existing Sc2.0 resources and expertise, it also generates new resources. A tRNA neochromosome is an important new resource, and an orthogonal SCRaMbLE system will provide new capabilities for engineering modular diversity. All the intermediate and final IESY strains, along with all the genomic data, will be made publicly available through the Sc2.0 UK Genome Engineering Resource (SUGER) centre.

Each condition will be represented by ten independent evolutionary trajectories, and each trajectory will be measured at 5 time points. These measurements will provide an in-depth, state-of-the-art characterization of the evolutionary snapshot: the genome sequence, the 3D genome conformation through chromosome conformation capture (3C), and the transcriptional state through RNA-seq. Advanced RNA-Seq protocols will permit measurements of alternative splicing through transcript isoform profiling (TIF-Seq). Progress can be assessed directly as the 150 strains are generated and characterized, and enhancement of target phenotypes will provide an overall measure of project success.

The proposed project will conditionally and systematically evolve synthetic yeast genomes under various industrial relevant conditions. The evolved genomes will be whole-genome sequenced to discover how and why they lead to particular phenotype, and we will also use genome conformation capture technologies to identify the relationship between genome spatial structure and function. A neochromosome will be designed and built to maintain the high fitness of synthetic yeast. A second orthogonal SCRaMBLE system will also be developed. All the information will be centralised in a computational portal. Bioethics and social sciences will also be explore around this project. This project will lead to better understanding of genome engineering, which will benefits academic researchers in various fields such as synthetic biology, bioengineering, systems biology, genetics, molecular biology and many others. It also benefits industries, as it provides an efficient way to generate a high density library of genotypes to phenotype screening, which provides a way to evolve strains towards industrial interesting conditions, such as high pH, high ethanol. This project also opens up new angle to study bioethics and social dimensions of synthetic biology.