Investigating the evolutionary changes in Crabtree-negative yeasts during a long-term evolution experiment : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Albany, New Zealand

Abstract

The Crabtree effect is a metabolic strategy that allows yeast to ferment in the presence of oxygen. This is of interest as not all yeasts display this strategy, and nearly 100 years after its discovery it is still unclear what the overall benefit is. Two key theories attempt to explain the emergence of this phenomenon, the make-accumulate-consume theory and the rate/yield trade-off theory. The aim of this thesis was to investigate whether a trade-off between rate and yield develops in Crabtree-negative yeasts over the course of 1500 generations in a high sugar environment. Chapter Two demonstrates that growth rate is more likely to increase than decrease while growth yield is more likely to decrease than increase in the isolate-derived populations of yeast. We find that species that started out relatively fast, changed little while the slower species had more significant gains in growth rate. With growth yield, the species with initially high yield lost more significantly than the already low yield species. This could suggest there is an overall optimum growth rate and growth yield, that the species are evolving towards. In Chapter Three, ethanol production was measured using colorimetric tests and no change was observed to support the development of the Crabtree effect in these populations after 1500 generations. In Chapter Four growth yield was investigated using flow cytometry and it was found that several yeast populations both increased in cell size and decreased in growth yield. This is an interesting observation that has been observed in several previous experimental evolution experiments. In Chapter Five, as cell size is often associated with ploidy changes, DNA content was measured using DAPI and SYTOX DNA stains, detected by flow cytometry. This did not provide any statistically significant conclusions but highlighted the importance of employing further techniques to analyse the DNA content of these populations. This thesis has illustrated the importance of studying the competitive behaviours of microorganisms in isolation, where selfish traits appear to thrive.