Genome Organisation for Optimising Synthetic Secondary Metabolism

This project will research genome organisation and in particular how changing the location and arrangement of metabolic enzyme genes within a yeast genome can alter the amount of the metabolite they produce, in this case the antibiotic penicillin. Antibiotics are just one class of complex chemicals that the diverse array of organisms on Earth has naturally evolved to produce. The production of chemicals by life is known as metabolism and the more complex high-value chemicals that specialist cells produce (e.g. in plants) are called secondary metabolites and these include most therapeutic molecules known today. Production of secondary metabolites in cells requires specific enzymes which are encoded by genes usually under strict control (regulation). As our understanding of biology improves through many fundamental research breakthroughs, scientists are now looking to re-engineer secondary metabolism to produce valuable compounds in cellular systems that are easy to work with. Microbes like brewer's yeast are perfect as they are easy to culture and so could cheaply produce high yields of valuable compounds from renewable resources like sugar.

The most promising way to perform this 'metabolic engineering' is to use what is known as a synthetic biology approach, where genes and their controllers are treated as modular components with well-defined behaviours and then combined in a rational design-based manner. So far the synthetic biology approach to metabolic engineering has been successful in producing compounds useful as anti-malarials, cosmetics and biofuels by taking genes for enzymes found in plants and exotic microbes and combining these inside industrially-used microbes such as yeast. Crucial for achieving high yields of production in these microbes is fine-control over the precise levels of enzymes in each cell.

One method of tuning enzyme levels that is currently unexplored by scientists is how the genes for these enzymes are physically arranged within a cell's genome. It is already known that gene location and orientation within a genome plays an important role in gene expression in all forms of life. Recently, it has also been established that in cells that naturally perform secondary metabolism, such as plant cells, the location of genes that make up a pathway is often tightly conserved, usually in occurring in gene 'clusters' in areas known as 'sub-telomeric regions'. Clearly, if nature and evolution are correct, then the location of where pathway genes are added to a genome must affect the enzyme levels and therefore the pathway output.

This project seeks to test the hypothesis that modifying pathway gene location within a genome can result in improved yields of a high-value secondary metabolite, in this case penicillin. The genes encoding the penicillin pathway will be added to the genome of a lab yeast strain and cells will be selected that produce the greatest amounts of penicillin. The amount produced will then be monitored in a series of experiments where the pathway genes are systematically rearranged around five different places in the genome. This will give valuable information on how the arrangement of genes in the genome affects the pathway. Finally, the pathway genes will be placed in an engineered lab strain specifically designed to shuffle parts of its genome when under an evolutionary pressure. The strain will be grown to compete against bacteria and in doing will automatically rearrange pathway genes to produce the most penicillin. This project will therefore provide an important new synthetic biology approach to metabolic engineering, and also uncover valuable new information on the fundamental science of genomes and genome evolution.

For synthetic biology and metabolic engineering projects, the localisation and arrangement of foreign genes within a genome could be crucial for achieving desired functions and optimal yields. In this project we aim to test this hypothesis via genome engineering and heterologous secondary metabolism. Using S. cerevisiae, we will add genes that encode non-ribosomal peptide (NRP) synthesis and a 4 gene pathway that converts NRPs into penicillin. The NRP synthase gene will be chromosomally integrated and the pathway genes will be on a plasmid. Penicillin biosynthesis by this yeast will be measured using a halo assay and by LC-MS. Next, the relationships between gene location, arrangement, expression levels, their regulation and penicillin pathway yield will be systematically explored by constructing and assaying 125 yeast strains where pathway genes are integrated into 5 different genomic loci in all combinations. This will be done with a set of modular integration vectors and assaying with Q-PCR, LC-MS and the halo assay. This will yield an unprecedented dataset for future work. Regulation of the pathway also incorporates a GFP-tagged transcription factor so we can explore 3D spatial co-ordination of genes with confocal microscopy of yeast nuclei. In parallel, we will also use a directed evolution strategy to optimise pathway yield through recombination-mediated gene re-arrangement. Using a novel co-culture assay we have developed, we will put pressure on yeast to produce more penicillin to outcompete B. subtilis growth. We will place pathway genes in synthetic sub-telomeric regions of yeast chromosomes XI and VI, flanking them with repeat sequences and loxPsym sites allowing for inducible recombination. Under selective pressure these cells will recombine pathway genes to produce greater penicillin yields. This method, called ACRONYM (Automatic Chromosome Rearrangement for Optimised Novel Yeast Metabolism) provides an exciting new technique for metabolic engineering.

While the proposed project touches on many research disciplines, the core work falls under the remit of synthetic biology and will have a major impact on two major developing fields within synthetic biology, namely genome engineering and metabolic engineering. In the UK synthetic biology is now incorporated as a priority area in the strategy of the Research Councils and the TSB, and the UK research strategy of investing in foundational work in synthetic biology is intended to aid downstream industrialisation. In the UK this will allow us to exploit our current research base in engineering and biological sciences to create new industries, rejuvenate the existing biotechnology sector, support many SMEs, attract inward investment and create new jobs. By design, this project will directly contribute to the growing world-class excellence in synthetic biology research in the UK, producing new methods that will greatly aim downstream industrialisation of synthetic biology and realisation of the future bioeconomy. It will impact greatly on the biotechnology sector by providing new routes towards optimising biosynthesis pathways, already a key revenue earner for many biofuels and agrobiotech companies (e.g. TMO Renewables, Syngenta). In turn it is likely to lead to the creation of new start-up companies producing high-value secondary metabolites as products. Already a number of such companies exist in the US and elsewhere with some companies, such as Amyris already worth in excess of 1 billion US dollars after less than 10 years of operations. Secondary metabolites account for the vast majority of therapeutic compounds used and developed by pharma companies in the UK and elsewhere, and new methods to optimise their production or diversify their final structure will be of great interest. In particular, it is highly-likely that the work of this project will impact on future research to generate new antibiotics which are in vital need worldwide. Methods and strains developed in this project may even lead to systems for directed evolution of new antibiotics on demand.

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 (e.g. Promega, Life Technologies, Agilent). By examining eukaryotic genome topology and organisation we will yield important new findings crucial to fundamental genome and eukaryotic cell research that will likely feed into future applications controlling gene expression and cellular functions both at the DNA level and at the genome organisation level. Additionally a number of SMEs are appearing worldwide that aim to provide software design tools for genome engineering and synthetic biology (e.g. Genome Complier, DNA2.0 and Synbiota) and the findings of this work will greatly influence their products.

This project will also impact on educational training. Synthetic biology is an increasingly popular subject with students, specifically because of its increasing repeatability, ease-of-use and due to its anticipated impact on the future. The members of this project play an active role in the training and education of the next generation through synthetic biology teaching at Imperial and intend to incorporate the findings, methods and parts from this project into courses and suitable research competitions such as iGEM (http://igem.org/Main_Page). The UK has had remarkable success in teaching synthetic biology, producing many world-class undergraduate 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.