Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow 119071, Russia.
Institute of Bioengineering, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow 119071, Russia.
J Microbiol Methods. 2023 Nov;214:106840. doi: 10.1016/j.mimet.2023.106840. Epub 2023 Oct 10.
A common problem in engineering industrial yeasts, and wine yeasts in particular, is the lack or scarcity of selective markers for introducing desired genetic changes. Almost all such markers, which are usually auxotrophic mutations, would reduce the growth characteristics of yeast strains. However, a potentially useful marker could be the CAR1 gene encoding arginase, the deletion of which reduces the accumulation of the carcinogen ethyl carbamate in wine, making such a deletion beneficial for wine production and maintainable in wine yeast strains. Here we demonstrate the use of the CAR1 gene as a selective marker. First, we observe that complete deletion of CAR1 in a triploid wine strain of Saccharomyces cerevisiae causes strong growth inhibition on a medium containing arginine as the only nitrogen source. Then, we show that strains with CAR1 deletion can be reliably transformed using CAR1 as a plasmid marker. Thus, the CAR1 gene can be used as a convenient selective marker in genetic engineering of wine yeasts, in particular using CRISPR/Cas9 technology.
在工程工业酵母中，特别是在葡萄酒酵母中，一个常见的问题是缺乏或稀缺用于引入所需遗传变化的选择性标记物。几乎所有这些标记物通常都是营养缺陷型突变，会降低酵母菌株的生长特性。然而，一种潜在有用的标记物可以是编码精氨酸酶的 CAR1 基因，该基因的缺失会减少葡萄酒中致癌物质乙酰胺的积累，因此这种缺失有利于葡萄酒生产，并可在葡萄酒酵母菌株中维持。在这里，我们展示了将 CAR1 基因用作选择性标记物的用途。首先，我们观察到在酿酒酵母的三倍体葡萄酒菌株中完全缺失 CAR1 会导致在仅含有精氨酸作为唯一氮源的培养基上强烈的生长抑制。然后，我们表明可以使用 CAR1 作为质粒标记物可靠地转化具有 CAR1 缺失的菌株。因此，CAR1 基因可用于葡萄酒酵母的遗传工程，特别是使用 CRISPR/Cas9 技术。
