Ota Taku, Kanai Keiko, Nishimura Hisami, Yoshida Satoshi, Yoshimoto Hiroyuki, Kobayashi Osamu
Kirin Company Ltd, Research Laboratories for Alcoholic Beverage Technologies, 1-17-1 Namamugi, Tsurumi-ku, Yokohama, 230-8628, Japan.
Kirin Company Ltd, Integrated Beverage Analysis Center, 1-17-1 Namamugi, Tsurumi-ku, Yokohama, 230-8628, Japan.
Yeast. 2018 Jan;35(1):129-139. doi: 10.1002/yea.3291.
Crossbreeding is an effective approach to construct novel yeast strains with preferred characteristics; however, it is difficult to crossbreed strains of brewer's yeast, especially the bottom-fermenting yeast Saccharomyces pastorianus, because of the relative inefficiency of the available methods to obtain mating-competent cells (MCCs). Here, we describe a productive method for the isolation of MCCs without artificial genetic modification. We focused on the characteristics of two mating pheromone-supersensitive mutants, Δbar1 and Δsst2, that show a growth defect in the presence of the mating pheromone. When MCCs secreting α-factor and a-factor were spotted on to a lawn of MATa Δbar1 and MATα Δsst2, a halo was observed around the respective MCCs. This plate assay was successful in identifying MCCs from bottom-fermenting yeast strains. Furthermore, by selecting for cells that caused the growth defect in pheromone-supersensitive cells on cultures plates, 40 α/α-type and six a/a-type meiotic segregants of bottom-fermenting yeast strains were successfully isolated and crossed with tester strains to verify their mating type. This method of isolation is expected to be applicable to other industrial yeast strains, including wine, sake and distiller's yeasts, and will enable MCCs without genetic modifications to be obtained. As a result, it will be a useful tool for more convenient and efficient crossbreeding of industrial yeast strains that can be applied to practical brewing. Copyright © 2017 John Wiley & Sons, Ltd.
杂交是构建具有优良特性的新型酵母菌株的有效方法；然而，由于现有方法在获得具有交配能力的细胞（MCCs）方面效率相对较低，啤酒酵母菌株，尤其是下面发酵酵母巴氏酵母的杂交很困难。在此，我们描述了一种无需人工基因改造即可分离MCCs的有效方法。我们关注了两个交配信息素超敏感突变体Δbar1和Δsst2的特性，它们在交配信息素存在时表现出生长缺陷。当将分泌α-因子和a-因子的MCCs点种在MATa Δbar1和MATα Δsst2的菌苔上时，在各自的MCCs周围观察到一个晕圈。这种平板试验成功地从下面发酵酵母菌株中鉴定出了MCCs。此外，通过在培养平板上选择能在信息素超敏感细胞中导致生长缺陷的细胞，成功分离出了40个下面发酵酵母菌株的α/α型和6个a/a型减数分裂分离子，并与测试菌株杂交以验证它们的交配型。这种分离方法有望应用于其他工业酵母菌株，包括葡萄酒酵母、清酒酵母和蒸馏酒酵母，并将能够获得未经基因改造的MCCs。因此，它将成为一种有用的工具，用于更方便、高效地对可应用于实际酿造的工业酵母菌株进行杂交。版权所有© 2017约翰威立父子有限公司。
