Towards Genomes-to-Design: Building and Testing a Minimal Essential Chromosome

Synthetic biology follows engineering principles to build novel devices, pathways and circuits encoded by modular DNA parts, and more recently has turned to the engineering of entire genomes in living cells. The aim of much of the work of synthetic biology is to design and build cells to perform useful new functions they typically don't perform in nature. To further this field, it is important to engineer the workhorse living cell so that they perform their new tasks reliably and reproducibly. To this end there is significant interest in developing simplified cells built with minimal genomes. Through genome engineering and synthesis, it should be possible to create genomes that do not encode the many genes driving processes unnecessary for the cell to perform its main desired function.

The work proposed here aims to accelerate the engineering and synthesis of such minimal genomes, by being the first project to build a working chromosome from just the elements deemed essential to support the cell for its function in the lab. Cells growing with this minimal genome should theoretically be more efficient at performing engineered tasks, such as the biosynthesis of a drug molecule at high yields. Our work will therefore be able to produce specialist strains valuable for use in biotechnology.

To achieve this, we plan to use knowledge and tools gained from our work as part of the international Sc2.0 project, which is constructing an entirely synthetic genome for baker's yeast (Saccharomyces cerevisiae). We have just completed construction of one chromosome for this project, which now encodes all 334 genes normally found on its natural counterpart. In this project, we aim to replace this entire chromosome with a much smaller minimal version built-up from modules of DNA that each encode one of the genes from this chromosome deemed essential for cell growth in the lab. To do this we will use system called SCRaMbLE that is hard-coded into the DNA of our recently completed synthetic chromosome. Switching this system on leads to genes unnecessary for growth being automatically lost from the growing cells. Doing this at a large scale with our engineered yeast cells and then genome-sequencing whole populations, should provide us with a rich set of data that tells us which genes are required for growth of the yeast in the lab.

With this important new dataset in hand, we will then proceed to building our minimal synthetic chromosome and assessing its ability to replace a full chromosome in growing yeast cells. We plan to measure how cells with the minimal chromosome perform in a variety of conditions and determine whether they can grow and express genes with greater efficiency than normal yeast, now that redundant DNA has been removed. This will generate important new insights for understanding how cells consume resources efficiently and have evolved to encode many non-essential genes on their genomes. It will also give us an opportunity to produce new specialist cells for use in biotechnology, and we plan to test our minimal chromosome yeast for their ability to produce a variety of drug molecules that are valuable for industry and particularly for UK industrial collaborators.

This project is designed to take the next step in genome-scale synthetic biology. While ourselves and others have synthesised whole chromosomes of microbes and shown these can replace their natural counterparts, so far no-one has built-up a working chromosome from modular parts. Treating essential genes from a yeast chromosome as modules, here we will construct and test minimal modular chromosomes. The project is designed to demonstrate chromosome refactoring and genome minimisation, and to show how strains streamlined to function in the lab can grow and express proteins with improved efficiency and can provide specialist production strains for drug biosynthesis.

To do this, we will first assemble a circular yeast chromosome (essXI) built from modules encoding the 70 genes annotated as essential from the 334 naturally found on chromosome XI. We will add this to our recently-completed synthetic yeast strain that has a synthetic version of chromosome XI (synXI) that can be induced to SCRaMbLE in vivo. This SCRaMbLE system can automatically delete any gene that isn't required for growth and in the presence of essXI we expect to see much gene loss. Using population-based genome sequencing and qPCR, we will then analyse the essentiality of all 334 chromosome XI genes and add all those required for lab growth into essXI. This construct will then be converted into a standard yeast chromosome, called minXI, and assessed for its ability to support yeast growth through multiple characterisation experiments in different conditions.

With strains with minimised chromosomes now in hand, we plan to then quantify how genome size relates to growth efficiency and gene expression in order to aid yeast whole-cell modelling efforts. We will also exploit our new yeast strains for biotechnology, working with our industrial collaborators to demonstrate improved drug molecule biosynthesis when heterologous metabolic pathways are run in our minimised genome strains.

This project is designed to provide the foundations for the next phase of genome-scale synthetic biology, where custom genomes will one-day be built-to-design. Over the past 5 years the UK has followed a strategy of high-profile investments in synthetic biology which has been intentionally designed to fund ambitious foundational work such as that proposed here. The aim of this strategy is to aid downstream industrialisation of scientific work, by making it easier for those in industry (or entering into it) to realise new biotechnology applications that require cell engineering of increasing size and complexity. The UK strategy will allow us to exploit our world-leading research base in biological sciences to create new industries, rejuvenate the biotechnology sector, support many SMEs, and to attract inward investment and create new jobs.

This project is designed to develop new synthetic biology tools that will provide the UK with the world-leading expertise to tackle future genome-scale synthetic biology projects, and be at the forefront when it comes to the design and application of minimal cells for industrial biotechnology applications. As such, we have incorporated into the project the testing of biosynthesis pathways within the minimal strains that we develop. We plan to do this work in partnership with our industrial collaborators who already anticipate the benefit of constructing specialised strains designed for efficient drug biosynthesis. It will be of great value to industrial biotechnology if we can rationally construct minimal genomes that allow cells to grow and produce desired products while consuming less resources. This would decrease the cost of production for target molecules and the method would also enable companies to better rationally design their own in-house production strains. It is also especially relevant that this work is done in yeast, given that S. cerevisiae is the most economically important microbe on earth. In this respect, the knowledge and skills generated by this project will likely lead to significant economic impact in many areas where yeast-made products are a key revenue-earner, e.g., in biofuels, fine-chemical production and in agro biotech too.

The proposed project also offers technology development for the biosciences, which can itself pave the way for new start-up companies or for commercial successes for existing companies involved in products for biotechnology R&D. By investigating eukaryotic chromosome features and organisation we will provide the resources to yield important new findings crucial to fundamental genome and cell research. This could feed into future applications controlling gene expression and cellular functions both at the DNA level and at the genome organisation level. We expect that downstream research from this project could lead to valuable new routes to therapeutics. It is not unreasonable to suggest that medical sciences and pharma will be impacted by new knowledge of the eukaryotic genome. Additionally, a number of SMEs are appearing worldwide that also aim to use synthetic biology with yeast to make ingredients for the food, clothing, fragrance and cosmetic industries (e.g. Evolva, Bolt Threads, Ginkgo Bioworks). These new areas for biotechnology are likely to be high-earning sectors that will benefit from advances in yeast genome engineering and new knowledge of gene function.

The project will also impact on educational training. Synthetic biology is an increasingly popular subject with students, specifically due to its anticipated impact on the future and its influence within commercial biotechnology. We expect that realisation of the proposed project will attract significant international attention due to its ambitious scale. Being able to describe how whole chromosomes can be built-up from modular parts by just one research project, will likely excite and energise students and the public in general as they learn about synthetic biology.