The so-called nonconventional yeasts are becoming increasingly attractive in food and industrial biotechnology. Among them, Zygosaccharomyces rouxii is known to be halotolerant, osmotolerant, petite negative, and poorly Crabtree positive. These traits and the high fermentative vigour make this species very appealing for industrial and food applications. Nevertheless, the biotechnological exploitation of Z. rouxii has been biased by the low availability of genetic engineering tools and the recalcitrance of this yeast towards the most conventional transformation procedures. Centromeric and episomal Z. rouxii plasmids have been successfully constructed with prototrophic markers, which limited their usage to auxotrophic strains, mainly derived from the Z. rouxii haploid type strain Centraalbureau voor Schimmelcultures (CBS) 732T . By contrast, the majority of industrially promising Z. rouxii yeasts are prototrophic and allodiploid/aneuploid strains. In order to expand the genetic tools for manipulating these strains, we developed two centromeric and two episomal vectors harbouring KanMXR and ClonNATR as dominant drug resistance markers, respectively. We also constructed the plasmid pGRCRE that allows the Cre recombinase-mediated marker recycling during multiple gene deletions. As proof of concept, pGRCRE was successfully used to rescue the kanMX-loxP module in Z. rouxii ATCC 42981 G418-resistant mutants previously constructed by replacing the MATαP expression locus with the loxP-kanMX-loxP cassette.

中文翻译：

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一套带有抗生素抗性标记和Cre重组酶的质粒，用于非常规酵母鲁氏酵母（Zygosaccharomyces rouxii）的基因工程。

所谓的非常规酵母在食品和工业生物技术中正变得越来越有吸引力。其中，众所周知，鲁氏酵母属具有耐卤代水平，耐渗透性，娇小阴性和Crabtree阳性差。这些特性和高发酵力使该物种对工业和食品应用非常有吸引力。然而，由于遗传工程工具的低可用性以及该酵母对最常规转化程序的顽固性，对鲁氏沼虾的生物技术开发一直存在偏见。已经使用原养型标记成功构建了着丝粒和附加型Z. rouxii质粒，这将其用途仅限于营养缺陷型菌株，主要来自Z. rouxii单倍体型菌株Schimmelalcultures（CBS）732T。相比之下，工业上有前途的Z. rouxii酵母大多数是原养型和异源二倍体/非整倍体菌株。为了扩展用于操纵这些菌株的遗传工具，我们开发了两个着丝粒载体和两个附加型载体，分别带有KanMXR和ClonNATR作为主要的耐药标记。我们还构建了质粒pGRCRE，该质粒在多个基因缺失过程中允许Cre重组酶介导的标记物回收。作为概念的证明，pGRCRE已成功用于抢救鲁氏弧菌ATCC 42981 G418抗性突变体中的kanMX-loxP模块，该突变体以前是用loxP-kanMX-loxP盒取代MATαP表达基因座而构建的。我们开发了两个着丝粒载体和两个附加型载体，分别带有KanMXR和ClonNATR作为主要的耐药标记。我们还构建了质粒pGRCRE，该质粒在多个基因缺失过程中允许Cre重组酶介导的标记物回收。作为概念的证明，pGRCRE已成功用于抢救鲁氏弧菌ATCC 42981 G418抗性突变体中的kanMX-loxP模块，该突变体以前是用loxP-kanMX-loxP盒取代MATαP表达基因座而构建的。我们开发了两个着丝粒载体和两个附加型载体，分别带有KanMXR和ClonNATR作为主要的耐药标记。我们还构建了质粒pGRCRE，该质粒在多个基因缺失过程中允许Cre重组酶介导的标记物回收。作为概念的证明，pGRCRE已成功用于抢救鲁氏弧菌ATCC 42981 G418抗性突变体中的kanMX-loxP模块，该突变体以前是用loxP-kanMX-loxP盒取代MATαP表达基因座而构建的。