Enzymology of xylose utilization in yeast

A recent directive of the European Union proposed that biofuels should represent 2% of the total transportation fuel consumption by 2005. In order to achieve this ambitious goal, it is clearly necessary to improve the current biotechnologies for fuel production, particularly if non-conventional feedstocks such as lignocellulose are being used as raw materials. Lignocellulose is attractive because it is renewable through the process of plant photosynthesis and available in huge quantities as wastes from forestry, agriculture and the pulp and paper industries. It is composed of the polysaccharides cellulose and hemicellulose, and lignin. A number of studies have shown that the economics of a process for lignocellulose conversion require that efficient uses for both cellulose and hemicellulose be found. Glucose and xylose are the main constituent monosaccharides (`sugars`) in cellulose and hemicellulose, respectively. While glucose can be fermented easily into alcohol, the production of ethanol from xylose remains a challenge. The classical brewer`s or baker`s yeasts are unable to utilise xylose unless engineered with tools of molecular biology to have extra metabolic capabilities. However, the engineered yeast strains often produce little ethanol, accumulating other by-products. There is a major problem leading to this shortcoming during xylose fermentation: NAD(P) cofactors are not recycled efficiently between the enzymes catalyzing the first two steps of xylose assimilation. Therefore, the development of an industrial production organism requires that the initial steps of xylose utilisation be optimised. Recent studies in the applicant`s laboratory make possible a novel approach to overcome the intrinsic limitations of current recombinant strains designed to ferment xylose. In this project, enzymes with tailored specificities will be generated by site-directed mutagenesis, and mutated genes will be introduced into the genome of the yeast Saccharomyces cerevisiae. The organism expressing the altered genes will now be able to ferment xylose with improved yield and at a reduced level of by-products. The novel strains produced by metabolic engineering will be tested under fermentation conditions in bioreactors to provide essential information about physiology and potential industrial application.

A recent directive of the European Union proposed that biofuels should represent 10% of the total vehicle fuel consumption by 2020. In order to achieve this ambitious goal, it is clearly necessary to improve the current biotechnologies for fuel production, particularly if non-conventional feedstocks such as lignocellulose are being used as raw materials. Lignocellulose is attractive because it is renewable through the process of plant photosynthesis and available in huge quantities as wastes from forestry, agriculture and the pulp and paper industries. It is composed of the polysaccharides cellulose and hemicellulose, and lignin. A number of studies have shown that the economics of a process for lignocellulose conversion require that efficient uses for both cellulose and hemicellulose be found. Glucose and xylose are the main constituent monosaccharides (`sugars`) in cellulose and hemicellulose, respectively. While glucose can be fermented easily into alcohol, the production of ethanol from xylose remains a challenge. The classical brewer`s or baker`s yeasts (Saccharomyces cerevisiae) are unable to utilise xylose unless engineered with tools of molecular biology to have extra metabolic capabilities. However, the engineered yeast strains are slow in converting xylose eand often produce little ethanol, accumulating other by- products. In this project, different bottlenecks of xylose fermentation were addressed through a multi-level engineering approach in which the biological and process-related characteristics features of the conversion were considered in a comprehensive optimisation. By-product formation was substantially reduced and hence ethanol yield improved using enzymes designed for optimum specificity. Based on advanced understanding of the metabolic network in the yeast and the role of key enzymes in it, we were able to design a process scheme for fermentation of mixtures of glucose and xylose. The best performing strains obtained in this project are suitable for conversion of lignocellulose hydrolysates, and collaborations with national and international partners have been established in this field.