Engineering of Saccharomyces cerevisiae for the production of fuel ethanol from xylose

For various reasons mankind is looking for alternatives for fossil fuels. One of these alternatives is ethanol made from plant biomass. However, the plant material when broken down by hydrolysis into its sugar monomers contains a significant amount of xylose, a 5-carbon-sugar or pentose. Contrary to the sugars with 6 carbon atoms (hexoses) wild type baker's yeast does not convert xylose into ethanol. The most widely used method for ethanol production is fermentation with Saccharomyces cerevisiae better known as baker's yeast. In order to obtain an economically viable process for fuel ethanol production, an organism is required that can also ferment the pentose fraction of the biomass hydrolysate to ethanol. Over the last three decades people have tried to introduce xylose metabolism in baker's yeast using two strategies. 1) The introduction of a bacterial xylose isomerase gene and 2) The introduction of the fungal genes coding for xylose reductase and xylitol dehydrogenase. Both strategies have been unsuccessful. The work in this thesis describes a major breakthrough in the anaerobic fermentation of xylose by bakers yeast. The breakthrough came with the discovery of the first fungal xylose isomerase, in the obligate anaerobic fungus Piromyces sp. E2. Contrary to the bacterial genes, this fungal xylose isomerase could be functionally expressed in Saccharomyces cerevisiae at high levels. The introduction of the isomerase into the genome enabled bakers yeast to grow aerobically on xylose as the sole carbon source. Eventually, using a strategy of combined metabolic and evolutionary engineering, a yeast strain was obtained with an anaerobic growth rate on xylose of 0.12 h-1 and a xylose consumption rate of 1.4 grams xylose per gram biomass per hour. The final conclusion that can be drawn from this work is that the kinetics of xylose fermentation are no longer a bottleneck in the industrial production of bioethanol with yeasts