Understanding the control of cell metabolism and growth in Saccharomyces cerevisiae through manipulation of purine nucleotide concentrations

All cells require energy to survive and to grow. Green plants get this energy from the sun and convert it into biochemicals. The cells of most other organisms, including ourselves, get their energy from nutrients (biochemicals). However, the energy requirements of a cell vary, as does the supply of the nutrients that provide that energy. For instance, rapidly growing cells consume significantly more energy than cells that have stopped growing, and cells supplied with a high level of nutrients have more energy available for use than those kept on a starvation diet. Therefore, cells need control systems capable of both sensing the quantity and quality of nutrients available, and ensuring that the chemical processes which generate and consume energy are always balanced properly. What we don't understand is what it is that cells monitor to perform this balancing act. We have some clues that they record the amount or ratio of two families of high-energy biochemicals. Thus these biochemicals act not just as sources of energy but also as signals of nutrient status. We aim to investigate their role in the nutrient control systems using cells of the brewing and baking yeast, Saccharomyces cerevisiae. The structure and ways of working of yeast cells are pretty much the same as those of human cells. This means that what we learn about yeast provides important information about how our own cells, and those of our farm animals and crop plants, work. This can help us to understand how cells malfunction to produce disease. One of the primary characteristics of tumour cells, for instance, is that they grow and divide much more rapidly than a normal healthy cell of the same tissue type. Moreover, the energy metabolism of tumour cells is different from that of normal cells; in fact, it is more like that of yeast.
This project is not only important for our understanding of disease. Yeast is also increasingly used as a living factory for the sustainable production of renewable/carbon-neutral energy sources, and of biochemicals of pharmaceutical importance. Biotechnologists are introducing new metabolic pathways into yeast for the production of fuels or chemicals. This hijacks yeast metabolism away from its normal processes, and requires it to use its energy resources in a significantly different way. Additional burdens on metabolism may also arise as a result of nutrients being supplied to the yeast, to convert into these useful products, that are very different from its normal diet. This would typically be the case in commercial biotechnology processes where cheap waste materials, from forestry, agriculture, or the food industry are employed. A detailed understanding of the mechanisms which control and balance the supply and use of energy in yeast cells will help us increase the yields and efficiencies of novel biotechnological processes like these, thereby making it more likely that they can be implemented and commercially viable.

The intracellular concentrations of the phosphorylated purine nucleotides ATP and GTP have been proposed to be important global signals for regulating the balance of catabolism and anabolism in eukaryotic cells. How the relative ratios of adenine (ATP, ADP, AMP) and guanine (GTP, GDP) nucleotides change under different nutritional conditions has been characterized in S. cerevisiae, but how these changes are sensed and translated into cellular signals for the regulation of metabolism is not understood. We will use the regulated expression of bacterial genes which encode enzymes that use ATP or GTP as substrates to develop methods for inducing controlled changes to in vivo nucleotide concentrations which are independent of nutrient supply, and for generating nucleotide analogues to interfere with the sensing of GTP/GDP levels. The consequences of the inductions on global cellular metabolism will be characterized using transcriptomics and metabolomics, thereby determining the relationship between nucleotide composition and metabolic status and signalling. To identify gene functions important for mediating the responses to the induced changes in purine nucleotide abundance, we will undertake: i) functional interaction screening using the yeast heterozygous diploid mutant collection to find suppressors/enhancers of the effects of induction; ii) a focused screening of a selected subset of the yeast homozygous deletion mutant collection to identify strains exhibiting an abnormal uncoupling of growth rate and intracellular nucleotide composition.
Knowledge of how S. cerevisiae cells control their metabolism and coordinate the metabolic processes that provide and consume energy is often transferable to human cells where the dysregulation of these processes has been implicated in a number of disease states including tumourigenesis and diabetes. It is also directly applicable to optimization of the biotechnological processes for which S. cerevisiae is being increasingly used.

The results of this study will primarily be communicated through presentations in scientific conferences and through publications in the high-impact journals. We will communicate with relevant industrial partners both directly; for instance, with our collaborators - Novacta Biosystems of Welwyn Garden City, Syngenta and Unilever - and via the meetings of the Bioprocess Research Industry Club (BRIC). In year three of the Project, we will organise a half-day meeting to explain our research to interested industrial scientists. This meeting will be held in Cambridge; the Science Parks surrounding the city contain the UK's largest concentration of small and medium-sized companies involved in the biotechnological and biomedical industries, and major companies such as GSK, Unilever and Pfizer in easy reach. Two alumni from the Oliver laboratory are employed by Novacta, and another is at Pfizer, Abingdon. However, we will also provide a video link to facilitate the participation of those who are unable to travel to Cambridge. We will ask Cambridge Enterprise (the University's Technology Transfer company, see below) to sponsor this event.

We will also communicate with the industry via the web-site of the Cambridge Systems Biology Centre and data will be available there as well as at public repositories (e.g. ArrayExpress and MetaboLights at the EBI).

Our laboratories regularly contribute to Cambridge Science Week and we will communicate this project to the wider public by that route and through summer placements for 6th Form students. Currently, SGO has regular placement plans for summer students from the John Fisher School (Peaks Hill, Purley, Surrey, CR2 6BN). We will also exploit the Cambridge Press Office and the 'Naked Scientist' radio programme to communicate our work.

Cambridge Enterprise will be responsible for protecting any Intellectual Property arising from the research. The University's technology transfer company has been very successful at facilitating the application of research both through licensing and supporting spin-out companies, including three from our own Department - Kudos, Astex, and Biotica.