The genetic diversity of bottom-fermenting yeast classified as Saccharomyces pastorianus is poor because strains are restricted to a few genetically distinct groups. Crossbreeding is an effective approach to construct novel yeast strains, but it is difficult because of inefficiency to obtain mating-competent cells (MCCs) of bottom-fermenting yeast. By using mating pheromone-supersensitive mutants, we previously isolated several mating-competent meiotic segregants from two bottom-fermenting yeast strains: high isoamyl acetate-producing KY1247, and low diacetyl-producing KY2645. Here, we constructed novel non-GM hybrids carrying preferable characteristics from both parents by crossbreeding these bottom-fermenting strains for the first time. Sixteen a/a-type meiotic segregants from KY2645 and 12 α/α-type meiotic segregants from KY1247 were mixed, and cells resembling zygotes were isolated via micromanipulation. In total, 149 hybrids were obtained and verified by examining known single-nucleotide polymorphisms (SNPs) between the parental strains. A sporulation test showed that some of the hybrids were able to sporulate. Moreover, fermentation tests on a test-tube and pilot-plant scale identified two hybrids with production levels of isoamyl acetate and diacetyl that were almost the same as KY1247 and KY2645, respectively. Both of these hybrids produced satisfactory beer in terms of taste, flavor, and overall quality, comparable to that produced by the parental strains. Collectively, our results suggest that crossbreeding between bottom-fermenting yeast strains has the potential to increase the diversity of yeast strains available for brewing, and our method of isolating MCCs provides a huge advance for crossbreeding of bottom-fermenting yeast without using DNA recombination techniques.

底部发酵酵母巴斯德酵母的遗传多样性品系很差，因为品系仅限于几个遗传上不同的群体。杂交是构建新型酵母菌株的有效方法，但是由于获得底部发酵酵母的交配感受态细胞（MCC）效率低下而使之困难。通过使用交配信息素超敏感突变体，我们先前从两个底部发酵的酵母菌株（高乙酸异戊酯生产的KY1247和低二乙酰基生产的KY2645）中分离了几种具有交配能力的减数分裂分离剂。在这里，我们通过首次杂交这些底部发酵菌株，构建了两个亲本均具有较好特性的新型非转基因杂种。混合来自KY2645的16个a / a型减数分裂分离物和来自KY1247的12个α/α型减数分裂分离物，并通过显微操作分离出类似于受精卵的细胞。总共，通过检查亲本菌株之间的已知单核苷酸多态性（SNP），获得并验证了149个杂种。孢子形成测试表明某些杂种能够形成孢子。此外，在试管规模和中试规模的发酵测试中，确定了两个杂合体，其乙酸异戊酯和二乙酰基的生产水平分别与KY1247和KY2645几乎相同。这两种杂种在口味，风味和整体品质方面均能产生令人满意的啤酒，与亲本菌株所产生的啤酒相当。总的来说，我们的结果表明，底部发酵酵母菌株之间的杂交具有增加可用于酿造的酵母菌株多样性的潜力，