Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan.
Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan.
J Biosci Bioeng. 2024 Nov;138(5):369-374. doi: 10.1016/j.jbiosc.2024.07.015. Epub 2024 Aug 20.
4-Vinylguaiacol (4-VG) is one of the causative compounds for the phenolic odor characteristics of brewed liquor. In the case of Japanese sake brewing, 4-VG is formed through the decarboxylation of ferulic acid produced by rice koji enzymes from steamed rice. PAD1 (phenylacrylic acid decarborxylase gene) and FDC1 (ferulic acid decarboxylase gene) genes are essential for decarboxylation of ferulic acid in Saccharomyces cerevisiae and the single polymorphisms of both genes show a relationship with ferulic acid decarboxylation ability. While most of the Kyokai yeasts distributed by the Brewing Society of Japan have homozygous non-function fdc1 alleles, many newly isolated natural yeasts for local specialities carry the wild-type FDC1 gene. In our previous research, we found that a crossbreed strain lost a significant amount of chromosomal DNA as it underwent meiosis-like adaptation. Here, we established a breeding approach to exclude undesirable gene alleles (such as the wild-type FDC1 genes) from yeast strains of interest by backcrossing using Kyokai yeasts. Homozygous fdc1 crossbreeds were generated through the three rounds of crossing procedures, and we confirmed the reduction of 4-VG in the culture supernatant of the homozygous fdc1 hybrid strain compared to the parental strain. Importantly, this approach does not include growth selection for the mutation of interest (the fdc1 mutant in the current case). Using various yeast strains generated throughout human history, it may become possible to design and build any yeast strains according to the purpose.
4-乙烯基愈创木酚（4-VG）是导致酿造酒酚类气味特征的原因之一。在日本清酒酿造过程中，4-VG 是通过米曲酶从蒸米饭中产生的阿魏酸脱羧形成的。PAD1（苯丙烯酸脱羧酶基因）和 FDC1（阿魏酸脱羧酶基因）基因是酿酒酵母中阿魏酸脱羧所必需的，这两个基因的单核苷酸多态性与阿魏酸脱羧能力有关。虽然日本酿造协会分发的大多数清酒酵母都具有纯合无功能的 fdc1 等位基因，但许多新分离的地方特色天然酵母都携带野生型 FDC1 基因。在我们之前的研究中，我们发现杂交菌株在经历类似于减数分裂的适应过程中失去了大量染色体 DNA。在这里，我们通过使用清酒酵母回交建立了一种从感兴趣的酵母菌株中排除不良基因等位基因（如野生型 FDC1 基因）的育种方法。通过三轮杂交程序产生了纯合 fdc1 杂交种，我们确认与亲本菌株相比，纯合 fdc1 杂种菌株的培养上清液中 4-VG 减少。重要的是，这种方法不包括对感兴趣的突变（在当前情况下为 fdc1 突变）进行生长选择。通过使用人类历史上产生的各种酵母菌株，根据目的设计和构建任何酵母菌株可能成为可能。
