GENETIC-ENGINEERING OF A SAKE YEAST PRODUCING NO UREA BY SUCCESSIVE DISRUPTION OF ARGINASE GENE
GENETIC-ENGINEERING OF A SAKE YEAST PRODUCING NO UREA BY SUCCESSIVE DISRUPTION OF ARGINASE GENE
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DOI：
10.1128/aem.57.1.301-306.1991
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发表时间：
1991-01-01
影响因子：
4.4
通讯作者：
TAKAHASHI, K
中科院分区：
文献类型：
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作者：
KITAMOTO, K;ODA, K;TAKAHASHI, K
据报道，尿素是氨基甲酸乙酯 (ECA) 的主要前体，而氨基甲酸乙酯被怀疑是葡萄酒和清酒中的致癌物质。 为了最大限度地减少尿素的产生，从二倍体清酒酵母 Kyokai no. 构建了精氨酸酶缺陷突变体 (DELTA-car1/DELTA-car1)。 9，通过连续破坏CAR1基因的两个拷贝。 首先，用质粒pCAT2(DELTA-car1 SMR1)转化酵母菌株，并在甲磺隆甲基平板上分离出CAR1基因杂合的菌株。 随后，通过用质粒 pCAT1 (DELTA-car1 G418r) 转化来破坏另一个 CAR1 基因，并在 G418 板上分离所得的 car1 突变体。 对突变体总细胞裂解物的精氨酸酶测定表明，G418 平板上分离的 70% 转化体没有可检测到的酶活性，这可能是由于 CAR1 基因的两个拷贝被破坏所致。 进一步的基因组 Southern 分析证实了这一结果。 我们可以用 DELTA-car1/DELTA-car1 纯合突变体酿造不含尿素的清酒。 更有趣的是，即使在 30 摄氏度下储存 5 个月，所得清酒中也未检测到 ECA。 这项分子生物学研究表明，清酒中的 ECA 主要来源于精氨酸酶产生的尿素。
Urea is reported to be a main precursor of ethyl carbamate (ECA), which is suspected to be a carcinogen, in wine and sake. In order to minimize production of urea, arginase-deficient mutants (DELTA-car1/DELTA-car1) were constructed from a diploid sake yeast, Kyokai no. 9, by successive disruption of the two copies of the CAR1 gene. First, the yeast strain was transformed with plasmid pCAT2 (DELTA-car1 SMR1), and strains heterozygous for CAR1 gene were isolated on sulfometuron methyl plates. Successively, the other CAR1 gene was disrupted by transformation with plasmid pCAT1 (DELTA-car1 G418r) and the resulting car1 mutants were isolated on a G418 plate. Arginase assay of the total cell lysate of the mutants showed that 70% of transformants isolated on G418 plates had no detectable enzyme activity, possibly as a result of the disruption of the two copies of the CAR1 gene. Further genomic Southern analysis confirmed this result. We could brew sake containing no urea with the DELTA-car1/DELTA-car1 homozygous mutant. It is of additional interest that no ECA was detected in the resulting sake, even after storage for 5 months at 30-degrees-C. This molecular biological study suggests that ECA in sake originates mainly from urea that is produced by the arginase.