From genetic parts to neochromosome in yeast

This project aims to develop tools which can be used for the rapid generation and testing of engineered yeast strains which can be applied to many different industrial and healthcare needs, using the tools of synthetic biology. Synthetic biology, also known as biological engineering, is a rapidly developing discipline which aims to apply the techniques of engineering to the creation of new and useful biological systems. One of the most useful organisms for such projects is ordinary baker's yeast, Saccharomyces cerevisiae. Yeast has been used in human food and beverage technology for thousands of years, and is currently used on an enormous scale worldwide for the production of bread, alcoholic beverages, and biofuels, among other products. Because of its long history of safe use, the availability of very large scale production technologies, and its widespread use in laboratories to study the basic processes of life, yeast is an ideal starting point for many biological engineering projects. Some recent examples include the manufacture of the anti-malarial drug artemisinin in yeast, as an alternative to plant sources, as well as the manufacture of the hydrocarbon fuel farnesene, and the recent demonstration that important pain-killers such as hydrocodone can be produced in modified yeast.

Because engineering of biology is still as much an art as a science, it can be very useful to assemble and test many different variants of a system, to see which variants work best, as a starting point for further engineering. To do this kind of 'rapid prototyping' on a large scale requires the use of automated systems, which use robots to perform all of the necessary operations, from DNA assembly through strain construction to final testing. One major manufacturer of such automation is Thermo-Fisher, the industrial partner in this project. Thermo-Fisher automation systems constitute a platform which can assemble and test DNA constructs on a very large scale, to facilitate the rapid creation of new strains which can be applied to solve industrial and healthcare problems. To aid in this process, many different technologies are used. For example, a library of yeast DNA 'parts' is available in a format called 'YeastFab', which allows rapid automated assembly to join parts together in different combinations. Software tools are also required to aid in the design process and to keep track of parts and their properties. Thermo-Fisher's automation is controlled by software called 'Momentum'.

In this project, we aim to develop a suite of tools which can be used to join all of these processes together, to enable users to take full advantage of the speed and flexibility of automated assembly and testing platforms. Specifically, we will develop and test software tools which link biological design tools and DNA library curation software to the Momentum software which controls the automated systems, enabling a seamless transition from design to construction and testing. We will use these tools to increase the usefulness of the system by expanding our YeastFab library of natural yeast promoters (DNA 'control' sequences, essential for building genetic machines) and generating new synthetic promoters which can be used to control engineered systems. We will also develop and test 'neochromosome' technology which allows the construction of entire new chromosomes in yeast, allowing the assembly of the very large metabolic pathways required for the production of many biological products such as antibiotics and anti-cancer drugs. Finally, we will demonstrate the effectiveness of these technologies by assembling a number of demonstration pathways. All of the software tools developed will be made available on an open source basis for the benefit of the user community.

This project involves the development of new tools and parts to facilitate rapid development of engineered systems in Saccharomyces cerevisiae. In particular, we will develop and test software tools to link biological design software and parts library curation software with the Momentum software which controls Thermo-Fisher's widely used automation systems. This will allow users to move rapidly and seamlessly from design to high throughput assembly and testing of constructs. These tools will be made available on an open-source basis so that they are freely available to other users of similar automated platforms. We will test these tools by expanding our existing library of yeast promoters in YeastFab format, and continuing characterization of the library under different growth conditions, representative of industrial bioreactors, providing a valuable dataset for the design of new yeast strains for industrial uses. In addition, we will address the current paucity of inducible yeast promoters by developing a library of orthogonal synthetic promoters based on fusions of DNA-binding sequences with ligand-responsive domains from mammalian transcription factors, generating promoters which can be controlled by addition of small molecules. These will also be characterized under a variety of conditions. We will also further develop our 'neochromosome' technology, currently being applied to the synthetic yeast genome project (Sc2.0), to allow assembly of large metabolic pathways as new independent chromosomes. We will demonstrate these technologies, and their usefulness for rapid multi-level pathway optimization, by assembly of demonstration pathways encoding useful compounds such as anti-tuberculosis drugs. This project will result in a suite of new tools and parts which will be of great value for the rapid generation of industrially useful engineered yeast strains.

The outcomes of this project will be of great value to those working on the generation of engineered yeast strains. In particular, both academic and industrial scientists will benefit from the ability to move rapidly and seamlessly from design of novel systems, through high throughput assembly and strain construction, to high throughput testing and data collection, allowing rapid progression through the 'design, build, test, learn' cycle of biological engineering.

The most direct impact will be to laboratories and companies working on development of yeast strains for industrial purposes, such as manufacture of biological molecules for therapeutics, flavours and fragrances, renewable feedstock chemicals from biomass, and biofuels. The applicants and their colleagues at Edinburgh and elsewhere have close contacts with a number of companies working in such areas, and will contact such companies directly, via Edinburgh Research and Innovation, to discuss possible applications. This may lead to new commercial applications as well as improvements to existing processes, which may have direct economic impact relatively quickly. In addition to such existing contacts, all of the data and tools generated will be made available on an open source basis, and thus may contribute to advances in processes developed by other researchers and companies.

The ability to rapidly generate and test many variants of systems will be of great value in basic cell biology, for which S. cerevisiae is still a very useful model system, allowing rapid testing of hypotheses and generating new basic knowledge which will be of great value in future applied biological design projects. This may contribute to fundamental advances in cell biology which may ultimately lead to applications in biomedicine. The applicants have colleagues working with yeast as a model system for cell and molecular biology, and will ensure that they are informed of advances which may enable entirely new types of experiment to be performed.

The software tools and approaches developed may also be very useful to others working with automated DNA assembly, strain construction and testing systems. Such systems are increasingly important in many areas of biology, allowing new types of experiment to be performed. Such rapid high-throughput hypothesis testing has the potential to lead to new insights in many areas. All methods will be described in appropriate publications, and all software tools will be made available on an open source basis, to minimize barriers to uptake by other groups.