Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629HZ Delft, The Netherlands.
HEINEKEN Supply Chain B.V., Global Innovation and Research, Zoeterwoude, Netherlands.
PLoS Genet. 2019 Apr 4;15(4):e1007853. doi: 10.1371/journal.pgen.1007853. eCollection 2019 Apr.
Saccharomyces eubayanus is the non-S. cerevisiae parent of the lager-brewing hybrid S. pastorianus. In contrast to most S. cerevisiae and Frohberg-type S. pastorianus strains, S. eubayanus cannot utilize the α-tri-glucoside maltotriose, a major carbohydrate in brewer's wort. In Saccharomyces yeasts, utilization of maltotriose is encoded by the subtelomeric MAL gene family, and requires transporters for maltotriose uptake. While S. eubayanus strain CBS 12357T harbors four SeMALT genes which enable uptake of the α-di-glucoside maltose, it lacks maltotriose transporter genes. In S. cerevisiae, sequence identity indicates that maltotriose and maltose transporters likely evolved from a shared ancestral gene. To study the evolvability of maltotriose utilization in S. eubayanus CBS 12357T, maltotriose-assimilating mutants obtained after UV mutagenesis were subjected to laboratory evolution in carbon-limited chemostat cultures on maltotriose-enriched wort. An evolved strain showed improved maltose and maltotriose fermentation in 7 L fermenter experiments on industrial wort. Whole-genome sequencing revealed a novel mosaic SeMALT413 gene, resulting from repeated gene introgressions by non-reciprocal translocation of at least three SeMALT genes. The predicted tertiary structure of SeMalT413 was comparable to the original SeMalT transporters, but overexpression of SeMALT413 sufficed to enable growth on maltotriose, indicating gene neofunctionalization had occurred. The mosaic structure of SeMALT413 resembles the structure of S. pastorianus maltotriose-transporter gene SpMTY1, which has high sequences identity to alternatingly S. cerevisiae MALx1, S. paradoxus MALx1 and S. eubayanus SeMALT3. Evolution of the maltotriose transporter landscape in hybrid S. pastorianus lager-brewing strains is therefore likely to have involved mechanisms similar to those observed in the present study.
贝酵母（Saccharomyces eubayanus）是非酿酒酵母（Saccharomyces cerevisiae）的拉格啤酒酿造杂种酵母（Saccharomyces pastorianus）的亲本。与大多数酿酒酵母和 Frohberg 型的巴氏酵母菌株不同，贝酵母不能利用麦芽三糖（α-三葡糖苷），麦芽三糖是啤酒麦汁中的主要碳水化合物。在酿酒酵母中，利用麦芽三糖由端粒附近的 MAL 基因家族编码，并需要麦芽三糖摄取的转运蛋白。尽管贝酵母 CBS 12357T 株含有四个编码摄取α-二葡糖苷麦芽糖的 SeMALT 基因，但它缺乏麦芽三糖转运蛋白基因。在酿酒酵母中，序列同一性表明，麦芽三糖和麦芽糖转运蛋白可能是由一个共同的祖先基因进化而来的。为了研究贝酵母 CBS 12357T 中利用麦芽三糖的可进化性，在富含麦芽三糖的麦汁中进行碳限制恒化器培养的实验室进化中，对经紫外线诱变获得的能利用麦芽三糖的突变体进行了筛选。在工业麦汁的 7L 发酵罐实验中，进化后的菌株显示出对麦芽糖和麦芽三糖发酵的改善。全基因组测序揭示了一个新的镶嵌 SeMALT413 基因，它是由至少三个 SeMALT 基因的非相互易位基因内流重复产生的。SeMalT413 的预测三级结构与原始的 SeMalT 转运蛋白相似，但 SeMALT413 的过表达足以使其能够利用麦芽三糖生长，表明发生了基因新功能化。SeMALT413 的镶嵌结构类似于巴氏酵母麦芽三糖转运蛋白基因 SpMTY1 的结构，该基因与交替的酿酒酵母 MALx1、奇异酵母 MALx1 和贝酵母 SeMALT3 具有高度序列同一性。因此，杂种巴氏酵母拉格啤酒酿造菌株中麦芽三糖转运蛋白景观的进化可能涉及与本研究中观察到的类似的机制。
