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

Fermentation of xylose is of fundamental importance for the efficient production of ethanol from lignocellulosic biomass sources. Native Saccharomyces cerevisiae strains have traditionally been thought incapable of either fermentative or non-fermentative growth on xylose despite their high efficiency ethanol production from fermentation of hexoses, although population genetic approaches have found a few non-engineered strains that show a modest xylose utilization phenotype. Genetically engineered xylose-fermenting strains of S. cerevisiae have been constructed, yet no strain, either natural or engineered, has been identified that can 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 present as the sole carbon source; we identified 38 strains that have this xylose utilization phenotype including S. cerevisiae, other sensu stricto members, and hybrids between them. All of the S. cerevisiae positive strains we identified are wine yeasts, and of those that we were able to generate viable spores from, 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 named 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 Saccharomyces 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 provided Bulk Segregant Analysis as a novel application of high throughput sequencing.

以木质纤维素生物质为原料高效生产乙醇的核心环节，是木糖（xylose）的发酵过程。长期以来，学界普遍认为天然酿酒酵母（Saccharomyces cerevisiae）菌株无法以木糖为唯一碳源进行发酵型或非发酵型生长，尽管该类酵母在己糖发酵制备乙醇时表现出极高的效率；不过群体遗传学研究已发现少数非工程化菌株可呈现轻度木糖利用表型。尽管已有研究构建了经基因工程改造的木糖发酵酿酒酵母菌株，但目前尚未有任何天然或工程化菌株能够实现与葡萄糖相当的木糖发酵效率。本研究采用中通量筛选策略，筛选出可在以木糖为唯一碳源的培养基中提升光密度（optical density）的酿酒酵母属菌株，最终鉴定得到38株具备该木糖利用表型的菌株，涵盖酿酒酵母、狭义酿酒酵母类群的其他成员，以及二者间的杂交菌株。本次鉴定获得的所有酿酒酵母阳性菌株均为葡萄酒酵母；在我们成功获取其存活孢子的菌株中，木糖利用表型以单基因性状的方式分离遗传。我们通过基于平铺式微阵列与高通量测序的集群分离分析法（Bulk Segregant Analysis, BSA）对该基因进行定位，发现其编码一种推定的木糖醇脱氢酶，我们将其命名为XDH1。该基因位于15号染色体右端的亚端粒区域，而这一区域并不存在于S288c参考基因组中。我们进一步通过基因表达微阵列分析，以及对内源酿酒酵母木糖代谢通路的遗传解析，对该木糖利用表型开展了深入表征。本研究证实天然酿酒酵母菌株可利用木糖作为唯一碳源，阐明了该性状的遗传基础及内源木糖代谢通路，并将集群分离分析法作为高通量测序的一种新颖应用进行了展示。