Xylose isomerase from Piromyces: Characterization and engineering for improving S. cerevisiae-based lignocellulosic bioethanol production

Lignocellulosic bioethanol has been recognized as a possible fossil fuel alternative. This so-called “second-generation” bioethanol has gained growing interest as it can be produced using agricultural waste material or side products in the food industry as opposed to “first-generation” bioethanol which is produced from food sources such as sugar canes or corn. While the fermentation processes for first-generation bioethanol are well-established, second-generation bioethanol processes are more laborious due to the recalcitrant structural properties of lignocellulose. The process includes pretreatment of biomass, hydrolysis, fermentation as well as distillation and optimizations are required in all steps. One of the optimizations for cost-effective lignocellulosic bioethanol relies on the maximal utilization of the biomass. Following glucose which is the most abundant fermentable sugar in the biomass, xylose is the second most abundant sugar. Therefore, efficient fermentation of xylose is critical. However, S. cerevisiae, the commonly used organism for the fermentation process is inherently incapable of growing on xylose. This problem is solved by incorporating a heterologous xylose isomerase which converts xylose to xylulose which can then naturally be further assimilated by S. cerevisiae producing ethanol. Although the use of xylose isomerase enables the xylose fermentation of S. cerevisiae, its activity is yet sub-optimal and therefore requires improvement. This thesis describes biochemical and structural characterization of xylose isomerase from fungal strain Piromyces. Using this information, several enzyme engineering approaches were explored which resulted in discovering improved variants. The different approaches used, properties of discovered variants and their effects on xylose fermentation are explained throughout the thesis.