Engineering endogenous hexose transporters in Saccharomyces cerevisiae for efficient D-xylose transport

During the industrial production of bioethanol, the yeast Saccharomyces cerevisiae is used as preferred organism. This yeast is like no other microorganism capable of converting hexose sugars such as glucose into bioethanol. For the development of an efficient bioethanol production process from renewable raw materials such as residual plant waste, it is necessary that pentose sugars such as xylose and arabinose are also converted. Yeast, however, is not naturally capable of fermenting these pentose sugars. In the past, a suitable xylose metabolic pathway has been introduced into yeast based on the enzyme xylose isomerase that was isolated from a fungus derived from elephant stool. Although this resulted in the desired xylose fermentation, the consumption of xylose was found to be slow in the presence of glucose, which is the most important sugar in extracts from renewable raw materials. In this thesis, we established that this slow metabolism is caused by inefficient transport of xylose. In order to realize more efficient transport of xylose, we reprogrammed the glucose transporters of S. cerevisiae. To this end, a combination of evolutionary engineering and direct mutagenesis was used. This has resulted in transporters that are specific to xylose and that are no longer inhibited by high concentrations of glucose. By means of a well-balanced transport of both glucose and xylose, we have subsequently been able to develop a new generation of yeast strains that can simultaneously convert hexose and pentose sugars into bioethanol. This is an important step towards the development of a second-generation industrial bioethanol production process.