Enzymology of xylose metabolism in yeast - xylose reductase

The abundance of hemicellulose on earth and the renewability of hemicellulose has attracted considerable attention in various fields of biotechnology. The aim is to make better use of the hetero-polysaccharide hemicellulose. A common feature of nearly all processes targeted toward hemicellulose utilization is that the polysaccharide must be hydrolyzed first into its constituent components, mainly monosaccharides. The pentose sugar D-xylose is the major constituent of hemicellulose hydrolysates and can be converted into ethanol by a number of yeast species. It has been calculated that efficient xylose-to-ethanol fermenations are crucial to the economy of biomass-to-ethanol processes which are being developed worldwide. Therefore, pentose fermentation continues to be a `hot` topic in biotechnology and environmental microbiology, considering the fossile resources will come to an end in this but certainly in the next century . The catabolism of D-xylose in yeast involves a two-step redox-isomerisation into D-xylulose as its first step: an intial reduction into xylitol catalysed by xylose reductase, and a subsequent oxidation at C-2 into D-xylulose catalysed by xylitol dehydrogenase. The present project focuses on the first enzyme of the pathway, xylose reductase, a member of the aldo/keto reductase enzyme superfamily. It aims at studying and elucidating the kinetic and chemical mechanism, and other structure/function relationships of the enzyme from the yeast strain Candida tenuis CBS 4435. To achieve these goals, portein engineering, kinetic studies in the steady state and transient state, and high resolution structure studies by X-ray diffraction will be used. The key goals pertain to the investigation of : the catalytic and chemical mechanism of xylose reductase, in comparison to other aldo/keto reductases the molecular determinants of conformational changes in enzyme structure which govern coenzyme binding and release, and coenzyme specificity the structure of the transition state for aldehyde reduction by xylose reductase using structure / reactivity relationships, comparative kinetic analysis of wild type and mutants, and kinetic studies with deuterated substrates and in D20 the role of the dimer structure of the enzyme for enzyme activity and stability It cannot be anticipated that the results of the proposed studies will make an immediate contribution to improve known bottlenecks in current xylose-to-ethanol processes. However, better understanding of structure/function relationships of xylose reductase will certainly be helpful in all attempts to convey the ability of efficiently fermenting xylose to microbial strains by metabolicengineering.

A recent directive of the European Union proposed that biofuels should represent a share of 5.75% in all transportation fuels used by 2010. 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. This project has focused on xylose reductase, the enzyme that catalyzes the first step of xylose utilization and is believed to be mainly responsible for the inefficient conversion of xylose by current yeast strains. The results of the project were obtained in a joint research effort of this group and the group of Prof. Wilson at University of California, Davis, have revealed how this enzyme works, how it achieves specificity for its substrate xylose, and how it distinguishes between NADH and NADPH. They allowed the production of enzyme variants with improved properties and serve as the basis for new projects.