Engineering of Saccharomyces cerevisiae for Efficient Anaerobic Alcoholic Fermentation of l -Arabinose

Abstract

For cost-effective and efficient ethanol production from lignocellulosic fractions of plant biomass, the conversion of not only major constituents, such as glucose and xylose, but also less predominant sugars, such as L-arabinose, is required. Wild-type strains of Saccharomyces cerevisiae, the organism used in industrial ethanol production, cannot ferment xylose and arabinose. Although metabolic and evolutionary engineering has enabled the efficient alcoholic fermentation of xylose under anaerobic conditions, the conversion of L-arabinose into ethanol by engineered S. cerevisiae strains has previously been demonstrated only under oxygen-limited conditions. This study reports the first case of fast and efficient anaerobic alcoholic fermentation of L-arabinose by an engineered S. cerevisiae strain. This fermentation was achieved by combining the expression of the structural genes for the L-arabinose utilization pathway of Lactobacillus plantarum, the overexpression of the S. cerevisiae genes encoding the enzymes of the nonoxidative pentose phosphate pathway, and extensive evolutionary engineering. The resulting S. cerevisiae strain exhibited high rates of arabinose consumption (0.70 g h ؊1 g [dry weight] ؊1 ) and ethanol production (0.29 g h ؊1 g [dry weight] ؊1 ) and a high ethanol yield (0.43 g g ؊1 ) during anaerobic growth on L-arabinose as the sole carbon source. In addition, efficient ethanol production from sugar mixtures containing glucose and arabinose, which is crucial for application in industrial ethanol production, was achieved.In the past decades, it has become clear that for future sustainable and cost-effective production of fuel ethanol from plant biomass, not only the readily degradable starch and sucrose fractions but also the much more resistant lignocellulosic fractions of plant biomass should be used. Although glucose and xylose are often the predominant sugars in these feedstocks, the economically efficient production of ethanol also requires the conversion of smaller carbohydrate fractions, such as L-arabinose, at high rates and yields (9, 23).Saccharomyces cerevisiae is presently the organism of choice for industrial ethanol production. Although wild-type S. cerevisiae strains rapidly ferment hexoses with high efficiency, they cannot grow on or use pentoses, such as D-xylose and L-arabinose (3). In addition to the development of pentose-consuming bacteria such as Zymomonas mobilis, Escherichia coli, and Klebsiella oxytoca as alternative biocatalysts for ethanol production (5), this situation has inspired various studies to expand the substrate range of S. cerevisiae. The combination of metabolic and evolutionary engineering with the heterologous expression of either yeast xylose reductase and xylitol dehydrogenase (14,32,34,35,41) or a fungal xylose isomerase (19)(20)(21)(22) has already enabled the anaerobic fermentation of D-xylose by S. cerevisiae. The next challenge is the fermentation of other pentoses, such as L-arabinose. Although several yeasts and fungi can utilize L-arabinose as ...

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