Bulk Segregant Analysis by High-Throughput Sequencing Reveals a Novel Xylose Utilization Gene from Saccharomyces cerevisiae

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Abstract
Fermentation of xylose is a fundamental requirement for the efficient production of ethanol from lignocellulosic biomass sources. Although they aggressively ferment hexoses, it has long been thought that native Saccharomyces cerevisiae strains cannot grow fermentatively or non-fermentatively on xylose. Population surveys have uncovered a few naturally occurring strains that are weakly xylose-positive, and some S. cerevisiae have been genetically engineered to ferment xylose, but no strain, either natural or engineered, has yet been reported to ferment xylose as efficiently as glucose. Here, we used a medium-throughput screen to identify Saccharomyces strains that can increase in optical density when xylose is presented as the sole carbon source. We identified 38 strains that have this xylose utilization phenotype, including strains of S. cerevisiae, other sensu stricto members, and hybrids between them. All the S. cerevisiae xylose-utilizing strains we identified are wine yeasts, and for those that could produce meiotic progeny, the xylose phenotype segregates as a single gene trait. We mapped this gene by Bulk Segregant Analysis (BSA) using tiling microarrays and high-throughput sequencing. The gene is a putative xylitol dehydrogenase, which we name XDH1, and is located in the subtelomeric region of the right end of chromosome XV in a region not present in the S288c reference genome. We further characterized the xylose phenotype by performing gene expression microarrays and by genetically dissecting the endogenous Saccharomyces xylose pathway. We have demonstrated that natural S. cerevisiae yeasts are capable of utilizing xylose as the sole carbon source, characterized the genetic basis for this trait as well as the endogenous xylose utilization pathway, and demonstrated the feasibility of BSA using high-throughput sequencing. Ethanol made from fermentation of lignocellulosic biomass by baker's yeast can be considered “carbon neutral” and is one alternative to fossil fuels for powering vehicles. One of the recognized requirements for cost-effective and energy-efficient cellulosic ethanol production is the need to convert the sugar xylose—a major component of cellulosic biomass—into ethanol; however, it has traditionally been thought that baker's yeast cannot ferment xylose. We sought to investigate this assumption by looking at close relatives of baker's yeast from around the world to see if any had an intrinsic ability to grow on xylose. We identified a number of yeasts, many of them used in winemaking, that grow very slowly on this sugar, and studied one in detail. We determined that in this particular yeast the ability to grow on xylose is due to the presence of a single gene, which we named XDH1. This gene is not present in the typical laboratory strains of baker's yeast, but appears to be very common in natural wine yeasts. This gene could be useful in continuing efforts to make yeasts that can efficiently ferment xylose to ethanol.