Department of Microbiology and Biochemistry, Hochschule Geisenheim University, Von-Lade-Strasse 1, 65366 Geisenheim, Germany.
ARC Centre of Excellence in Synthetic Biology, Department of Molecular Sciences, Macquarie University, Sydney, NSW 2113, Australia.
Int J Mol Sci. 2021 Feb 16;22(4):1943. doi: 10.3390/ijms22041943.
Apiculate yeasts belonging to the genus are commonly isolated from viticultural settings and often dominate the initial stages of grape must fermentations. Although considered spoilage yeasts, they are now increasingly becoming the focus of research, with several whole-genome sequencing studies published in recent years. However, tools for their molecular genetic manipulation are still lacking. Here, we report the development of a tool for the genetic modification of . This was employed for the disruption of the gene, which encodes a putative alcohol acetyltransferase involved in acetate ester formation. We generated a synthetic marker gene consisting of the promoter controlling a hygromycin resistance open reading frame (ORF). This new marker gene was used in disruption cassettes containing long-flanking (1000 bp) homology regions to the target locus. By increasing the antibiotic concentration, transformants were obtained in which both alleles of the putative gene were deleted in a diploid . strain. Phenotypic characterisation including fermentation in Müller-Thurgau must showed that the null mutant produced significantly less acetate ester, particularly ethyl acetate. This study marks the first steps in the development of gene modification tools and paves the road for functional gene analyses of this yeast.
属于的尖状酵母通常从葡萄栽培环境中分离出来，并且经常在葡萄汁发酵的初始阶段占主导地位。尽管被认为是变质酵母，但近年来，随着几项全基因组测序研究的发表，它们正越来越成为研究的焦点。然而，它们的分子遗传操作工具仍然缺乏。在这里，我们报告了一种用于的遗传修饰的工具的开发。这被用于破坏编码参与乙酸酯形成的假定醇乙酰转移酶的基因。我们生成了一个由控制潮霉素抗性开放阅读框（ORF）的启动子组成的合成标记基因。这个新的标记基因被用于包含靶位点长同源区（1000bp）的敲除盒。通过增加抗生素浓度，在二倍体中获得了两个等位基因均被缺失的突变体。在 Müller-Thurgau 葡萄汁中的发酵表型特征表明，缺失突变体产生的乙酸酯，特别是乙酸乙酯显著减少。这项研究标志着基因修饰工具开发的第一步，并为该酵母的功能基因分析铺平了道路。
