Cellular Processes and Mechanisms in Saccharomyces cerevisiae Influencing Anaerobic Xylose Fermentation

Abstract: In 2009 the EU approved two directives as a first initiative towards reducing greenhouse gas emissions and becoming independent of fossil fuels: the Renewable Energy Directive and the Fuel Quality Directive. As a result, the demand for biofuels will increase enormously over the next decade, both nationally and in the entire EU. This huge demand will require a more advanced type of biofuels, produced from cellulosic and lignocellulosic raw materials that do not compete with the supply of food crops. These biofuels are referred to as second generation (2G) fuels. The production of 2G bioethanol at a commercial scale requires yeast strains capable of producing ethanol at high yield and high productivity from all sugars (hexoses and pentoses) extracted from the raw material. The aim of the work presented in this thesis has been to increase the ethanol productivity of recombinant xylose-fermenting strains of the yeast Saccharomyces cerevisiae during batch fermentation of a glucose/xylose mixture. A parameter that has a big influence on productivity is cellular growth and the yeast strains currently used today grow rather poorly on xylose. Many of the signals cells use to regulate growth originate from changes in the concentrations of metabolites inside the cells. To increase our knowledge of xylose metabolism the dynamic changes in intracellular metabolite concentrations were measured during batch fermentation of a glucose/xylose mixture using LC-MS/MS. This study gave meaningful insights about important intracellular signals, biological phenomena and mechanism. The analysis of the metabolite data pointed toward limitations in the folding of proteins inside the ER, which might be the underlying cause of the slow growth on xylose. Another important factor is the regulation of expression of genes required for sugar transport and those related to fermentative metabolism. Hexokinase 2 (Hxk2p) is an important bi-functional protein that acts both as a catalytic enzyme and a global transcription factor. This protein plays a role in the regulation of the above mentioned genes and becomes inactivated in the presence of xylose. As a consequence it loses its’ regulatory function. In an effort to improve repression signals during xylose fermentation this protein was engineered to become immune towards inactivation by xylose. By combining methods for protein and genetic engineering with fermentation technology a mutation in the gene was identified which increased the catalytic activity by 64% in the presence of xylose. The new variant allowed faster glucose consumption in the presence of xylose, but had no obvious impact on xylose fermentation. These results indicate that Hxk2p does not act alone and other proteins are involved in the regulation. These proteins remain to be identified. This thesis describes the cellular processes required for balanced anaerobic microbial growth and the intracellular signals that regulate them. The aim has been to identify biochemical mechanisms that limit anaerobic growth of recombinant S. cerevisiae strains on xylose.