The Sc2.0 UK Genome Engineering Resource (SUGER)

Lead Research Organisation: Imperial College London
Department Name: Dept of Bioengineering

Abstract

Sc2.0 (Synthetic Yeast) is an international flagship project to complete the first chemical synthesis and assembly of a eukaryotic genome, that of the yeast Saccharomyces cerevisiae. The project is an open-access, academic response to genome synthesis work by Craig Venter and others and will make highly-evolvable synthetic yeast strains and new genome-scale engineering methods available to all. The Sc2.0 UK Genome Engineering Resource (SUGER) is a proposed resource that not only enables UK participation in the Sc2.0 project but will also maximise the impact Sc2.0 has on biosciences research, synthetic biology and industrial biotechnology in the UK and abroad.

The Sc2.0 international consortium now includes the UK who will lead on the synthesis and assembly of Chromosome XI. The project arises from work led by Jef Boeke at Johns Hopkins University published in Nature last year (Dymond et al). This publication showed completion of two chromosome arms and highlighted many unique modifications designed into the synthetic genome. Three of these of note are (i) eliminating all TAG codons to allow future "orthogonal" encoding of unnatural amino acids, (ii) removing non-essential and destabilising elements (introns, cryptic ORFs, transposons) to streamline the genome and (iii) placing synthetic recombination sites downstream of all non-essential genes. The latter of these is an especially useful tool, as recombination leads to these genes being removed, inverted or translocated within the genome. This novel tool can be used to rapidly evolve cells down to their minimal gene number or to produce arrangements that have increased fitness in desired environments (e.g. biofuel production). Taken together, the Sc2.0 project offers a unique way to explore genome topology, nuclear structure and will also allow researchers to uncover 'context rules' for genome design. It is also a grand challenge project to push boundaries in synthetic biology and modern biosciences. The Sc2.0 website is: http://www.syntheticyeast.org

The Sc2.0 UK Genome Engineering Resource (SUGER) gives UK participation in this project and will make all DNA, strains, data and genome engineering methods and tools developed during Sc2.0 available to the wider bioscience community as a significant resource. Dr Tom Ellis and Prof Paul Freemont will direct a chromosome team at the synthetic biology centre at Imperial College London to complete design, synthesis, assembly and verification of Chromosome XI by 2016; adding this to the full international set to produce the complete Sc2.0 strain by 2017. Dr Alistair Elfick (Edinburgh) will assist in the process, which will also includes a suite of related bioinformatics software tools and detailed genome engineering protocols. Prof Steve Oliver (Cambridge) will provide world-leading expertise on yeast and direct experiments that will provide quality control and fitness data for hybrid and synthetic yeast chromosomes.

SUGER will:
1. Complete full synthesis, assembly and verification of Synthetic Yeast Chromosome XI (SynXI - 0.67Mbp) as the UK's contribution to the international Sc2.0 project.
2. Curate and distribute a Sc2.0 strain resource, a physical collection for IP-free distribution of S. cerevisiae strains containing the hybrid and fully-synthetic chromosomes of the international Sc2.0 project and the DNA building blocks that were used in construction.
3. Extend Sc2.0 resources to be valuable legacy for the wider biosciences community. We will build a SUGER website hosting Sc2.0 and genome engineering information, extend the suite of Sc2.0 software tools and protocols to be applicable for future synthetic biology projects, and run three annual genome engineering workshops for teaching genome-scale synthetic biology concepts and methods to UK researchers.

We expect high use of these resources from epigenetics, genomics and yeast researchers as well as from systems biology, synthetic biology and industrial biotech.

Technical Summary

Sc2.0 (Synthetic Yeast) is a flagship synthetic biology project to complete the chemical synthesis and assembly of the S. cerevisiae genome. To enable the UK to join the international effort to complete Sc2.0 and maximise its impact, we propose the Sc2.0 UK Genomic Engineering Resource (SUGER). The resource consists of 1) a curated and distributable collection of Sc2.0 project DNA building blocks and yeast strains containing hybrid and synthetic chromosomes (for this the UK team will construct SynXI - 0.67Mbp), and 2) an online resource of methods, tools and data for kilobase-scale synthetic biology and genome engineering work. This will include 3 summer workshops to train UK researchers in such work.

A dedicated chromosome team at Imperial College will design SynXI according to Sc2.0 design rules and outsource DNA synthesis to commercial vendors in 10kb chunks. Using genome engineering methods established for the Sc2.0 project these chunks will be assembled into 50kb megachunks and then used to serially replace native genome sequence with synthetic via recombination and selection. Progress will be verified by PCR-tag screening and whole-genome sequencing at TGAC. Completed SynXI will then be thoroughly tested for fitness by a variety of assays by a yeast specialist team at Cambridge. It will then be crossed into other Sc2.0 strains to achieve yeast with a completely synthetic genome. The project materials and data from this work and equivalent work by partner chromosome teams around the world will be collected and DNA and strains formatted into a multiwell plate collection for distribution to end-users. Project data will be placed online for end-users to browse and request strains on a dedicated SUGER website. This website will also host the synthetic biology resource, with detailed protocols and online software tools for genome engineering work. Use of these will be taught to UK PhDs/PDRAs at week-long summer workshops in Edinburgh and London by the SUGER team.

Planned Impact

SUGER is designed to provide a valuable resource for bioscience research but we also expect it will generate significant impact beyond academia.

The SUGER project falls under the remit of synthetic biology, which in the UK is now incorporated as a priority area in the strategy of the Research Councils and the TSB. The UK strategy for synthetic biology is one of investing in foundational work in order to aid downstream industrialisation of the scientific work. In the UK this will allow us to exploit our world-leading research base in biological sciences to create new industries, rejuvenate the biotechnology sector, support many SMEs, attract inward investment and create new jobs. Much of the work done to generate the Sc2.0 resource can be classified as development of foundational tools for synthetic biology. Web-based tools and workshops aim to disseminate and standardised foundational work and provide a UK skill-base for the next stage of synthetic biology work - genome engineering and kilobase-scale projects.

Industrial biotechnology and biofuels will be impacted by the proposed resource. Already the economic impact of re-engineering of key microbes such as yeast for such work is known to be significant. As reported in IEEE Spectrum Magazine "The Hunt for the Biological Transistor" (March '11): "Genetic engineering and other forms of biotechnology account for some 40 percent of the recent growth in the U.S. gross domestic product". In this respect, the knowledge generated by the proposed project together with the biological and skill resources will likely lead to major economic impact, especially given that S. cerevisiae is the most economically important microbe on earth. Several UK companies have already expressed significant written interest in using the resource in their work for metabolic engineering, bioprocessing and DNA assembly. It is clear that the resource will impact greatly on the biotechnology sector in the UK where yeast-based products are already a key revenue-earner for many fine-chemicals and agrobiotech companies (e.g. CRODA, Syngenta).

The proposed project will also offer technology development for the biosciences, which itself can pave the way to new start-up companies or for commercial successes for existing companies involved in products for biotechnology R&D. By examining eukaryotic genome features and organisation we will provide the resource to yield important new findings crucial to fundamental genome and eukaryotic cell research. This will likely feed into future applications controlling gene expression and cellular functions both at the DNA level and at the genome organisation level. We expect that research that follows from the work done in this resource 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 provide software design tools for genome engineering and synthetic biology (e.g. Genome Complier and Synbiota) and the bioinformatics tools developed in this work will greatly influence their products, especially as the project is an exemplar of data-driven biology, using existing data in the redesign of a genome.

This 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. PIs of this project play an active role in the training and education of the next generation through synthetic biology teaching at ICL and ED and may incorporate the tools and methods of this project into courses. The UK has had remarkable success in teaching synthetic biology, producing many world-class undergraduate iGEM projects, so investment in further research here in the UK is critical to retaining the best students in the country and building a successful UK-based biotechnology industry.

Publications

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Description Construction of Synthetic Yeast Chromosome XI (synXI) is done, with all of the synthetic DNA synthesised and assembly 100% complete and verified. As of the start of 2018, we are awaiting final verification by next gen sequencing to show that we have made the synthetic chromosome as planned and fixed all assembly errors. We encountered unexpected phenotypes during construction which informed us of biological functions of genomic loci that were not previously identified. We have also developed new methods to aid our screening and verification processes, such as an improved genomic DNA isolation technique, new gene regulation optimisation tools and a CRISPR-based phenotypic reversion screen, all of which have now been published in journals.

Experiments exploiting the SCRaMbLE synthetic genome recombination system built into synXI have shown that large-scale rearrangements of synthetic chromosomes can be induced to produce strains with enhanced phenotypes, such as heat tolerance, increased heterologous gene expression and improved secondary metabolite biosynthesis. A major research paper describing this was published in Nature Communications for 2018. We have leveraged this preliminary work to establish a collaboration with a major UK pharmaceutical company which has led to a funded BBSRC PhD CASE award project beginning in late 2016.

The SUGER website, syntheticyeastresource.com is online and provides progress updates, a java-based genome sequence browser, synthetic genome assembly methods and a forum for the international Sc2.0 community. Synthetic yeast strains from various groups around the world are being collected in order to populate the Sc2.0 strain resource. These will become available as sequences are published, with the Synthetic Yeast Resource website acting as a strain request portal.

The first Synthetic Genome Workshop took place in Edinburgh in July 2016, with attendees gaining practical experience with synthetic yeast strains. Methods taught included the use of SCRaMbLE to optimise metabolic pathways and the use of CRISPR to debug synthetic yeast strains. In 2018 we published a description of this course and the tools used within it for educational use.

We are on target to meet all of the stated aims of the project by 2019. Once synXI is completed, it will be a highly valuable tool for multiple areas of research, from genomic study of how the yeast chromosome functions to the exploitation of encoded genome plasticity for industrial biotechnology purposes. The Synthetic Yeast Resource website, strain repository and Synthetic Genome Workshops will ensure that UK and worldwide researchers are able to learn from, have access to and benefit from the synXI and other SC2.0 strains.
Exploitation Route Our research is to develop new state-of-the-art methods and strains for yeast biotechnology that we expect to be in use at industrial biotech companies that use yeast in their process.
Sectors Agriculture, Food and Drink,Chemicals,Environment,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

URL http://www.syntheticyeastresource.com/
 
Description The SUGER project provides a valuable resource both to academic biological research and industrial biotechnology. The synthetic yeast strains accelerate the development of novel yeast strains with genomic arrangements that are beneficial to various industrial applications, such as fine chemical and biofuel production. The website and summer school help educate others on how to best exploit this new opportunity and apply the techniques developed to future genome building projects. In summer 2016, we organised and taught the 2016 Synthetic Genome Summer School in Edinburgh with assistance from staff at Edinburgh University. Thirty participants came from industry and academia backgrounds, as well as from citizen science labs and from education and the arts. The participants successfully completed an ambitious programme of practical work in which they build libraries of ß-carotene pathways, used the Synthetic Chromosome Recombination and Modification by LoxP-mediated Evolution (SCRaMbLE) system to optimise heterologous pathways in an Sc2.0 strain with synthetic chromosomes. They also were taught how to use new CRISPR protocols to debug and repair fitness defects within synthetic chromosomal sequences. These practical aspects were complemented by a series of talks from international experts and from industry gave an excellent overview of the current state of the art in synthetic biology and genome engineering. The overall average score given for the summer school was 4.6 out of 5. The summer school was oversubscribed, receiving many more applications than available spaces, and 21 of the 22 survey respondents indicated that they would recommend the school to others.
First Year Of Impact 2016
Sector Education,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology