Understanding the regulation of glucose sensing and transport in budding yeast using dynamic inputs

Glucose is the preferred source of energy for nearly all cells, from microbes to plants to animals, but we do not understand how cells sense and respond to changes in glucose's availability. We propose to study the sensing and uptake of glucose in budding yeast, one of the simplest organisms that has a similar structure to our own cells. Yeast has seven main transporters for glucose, and our focus is to understand why so many are necessary and how only the appropriate transporters are present at the appropriate time. To do so, we will study both populations of and individual cells and probe their behaviour in fluctuating levels of glucose to determine if particular transporters increase in number in response to either the concentration of extracellular glucose or to how that extracellular concentration changes with time. We will thus determine under which conditions each transporter is optimum and has its highest levels. Further, using mutations to disrupt sensing and mathematical modelling of our results, we will discover how the underlying biochemistry works to ensure a suitable rate of uptake of glucose through changing the types of transporters present. Our results will both address the fundamental question of how cells can regulate their import of glucose and provide quantitative measurements of the levels of the transporters that can be exploited by the biotechnology industry to, for example, increase production of valuable biological compounds.

Cells live in changing environments and understanding how cells sense and respond to dynamic signals is thus of great importance. Yet we do not know what general aspects of such signals can be sensed by cells or how such sensing is performed biochemically. By studying the response of budding yeast to glucose, we will study both questions. Indeed, understanding how cells respond to glucose is fundamental because glucose is the preferred carbon and energy source for many cells. But even in highly-studied yeast, we do not know why seven major transporters for glucose exist. We propose that some transporters respond to the dynamics of glucose rather than its level and will test this hypothesis with a systems approach. First, we will survey and quantify the levels of the seven transporters at different stages of growth and with differing initial levels of glucose using plate readers and so create an "atlas" of expression. Second, we will use fluorescence microscopy and microfluidics to measure the levels of the transporters both in controlled dynamic environments and in mutants where components are deleted from the signalling network that both senses glucose and regulates the transporters. Such time-lapse data are ideal for fitting and discriminating between mathematical models, and we will develop and experimentally verify a mathematical model of glucose sensing that captures the logic driving the regulation of the transporters. Finally, we will use the model to predict the importance of each transporter for particular and dynamic extracellular conditions and confirm these predictions through the effect of that transporter on growth using competition experiments. Our results will address both how cells respond to dynamic signals and regulate their import of glucose as well as providing quantitative characterisation and mathematical models of promoters that drive expression at different stages of growth and so are of use for biotechnology and synthetic biology.

We see two main groups of beneficiaries:

(i) industry producing customised gene expression;

(ii) the general public through increasing their awareness and understanding of the interdisciplinary nature of today's bioscience.

First, budding yeast has long been a standard organism in industry from traditional food manufacture to synthetic biology-based start-ups. Many of these applications exploit yeast's metabolic capabilities or indeed extend these capabilities using exogenous genes.

A challenge then for industry is determining suitable promoters to drive expression of the exogenous genes of interest, which are typically enzymes involved in the synthesis of industrially relevant chemicals.

Our research, with its emphasis on the characterisation, both experimentally and mathematically, of seven promoters that are expected to activate at different stages of the yeast growth curve can meet this challenge. Working with Prof.\ Susan Rosser, a colleague at the University of Edinburgh, who is collaborating with Croda, a U.K.-based speciality chemicals company and a member of the FTSE 100 Index, we will use our knowledge of HXT expression to enable budding yeast to efficiently synthesize saponins, chemicals of interest to Croda and which can be used as surfactants. Saponins can be toxic to yeast, and our results will be used to design promoters that express the enzymes for synthesizing saponins only once the diauxic phase of growth has been reached when numbers of cells, and so potential yields, are high. If these enzymes are produced too early in the growth curve, the toxicity of the saponins prohibitively limits both growth and yields.

Second, our research has the capacity to engage the public with its focus on fluorescence and time-lapse microscopy, which provide powerful visual tools for education and increasing scientific awareness.

One goal is to demonstrate the interdisciplinary approaches necessary to understand biological phenomena. Our research uses techniques from mathematics and informatics, as well as cell and molecular biology. Most high school and many undergraduates are not aware of interdisciplinary research with curricula still typically following traditional silos. Yet breakthroughs often come from those working at the edge of disciplines, and we aim to encourage both those interested in the physical sciences to consider research in biology and those already interested in biology to realise the value of training in the physical sciences and mathematics.

A second goal is to illustrate the importance of "blue skies" research, particularly that studies on yeast can inform on our own physiology. For example, growth of yeast in low glucose can prolong life (number of replications) in yeast cells, and such dietary restriction reduces ageing in many organisms, including primates. Another example is the study of cell-to-cell heterogeneity, which appears at first sight to be of only academic interest, but is now recognised to be, for example, fundamental for understanding anti-microbial resistance. Finally, taken together, these illustrations will underpin the importance of having an evolutionary perspective for comprehending and manipulating our world.

Our third goal is to show an example of the logic of cellular regulation: how a shift in extracellular glucose is sensed by the cell and processed to cause expression of the appropriate transporters for the new glucose environment, which we will illustrate in real time using microfluidics and by following gene expression with fluorescent protein markers.