Shandong Provincial Key Laboratory of Synthetic Biology, Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No.189 Songling Road, Laoshan District, Qingdao, 266101, Shandong Province, China.
University of Chinese Academy of Sciences, Beijing, 100039, China.
Appl Microbiol Biotechnol. 2019 Jan;103(2):963-971. doi: 10.1007/s00253-018-9496-1. Epub 2018 Dec 4.
Selectable marker recycling is a basic technique in bioengineering. However, this technique is usually unavailable in non-model microorganisms. In this study, we proposed a simple and efficient method for selectable marker recycling in the astaxanthin-synthesizing yeast Xanthophyllomyces dendrorhous. This method was based on a Cre-loxP system, in which the transient expression of the Cre recombinase was controlled by a genetically unstable vector independent of episomal plasmids and inducible promoters. The selectable markers in single-gene locus and multigene loci were removed along with the loss of the Cre vector with a ratio of 100% and 29%, respectively. The significance of the method was highlighted by the finding that stable autotrophic mutants were not readily obtained in X. dendrorhous. Comparative studies in X. dendrorhous and the non-homologous end joining dominant yeast Yarrowia lipolytica suggested that the method could be universally used in homologous recombination dominant yeasts.
可选择标记回收是生物工程中的一项基本技术。然而，这种技术通常在非模式微生物中不可用。在这项研究中，我们提出了一种在虾青素合成酵母红发夫酵母中进行可选择标记回收的简单而有效的方法。该方法基于 Cre-loxP 系统，其中 Cre 重组酶的瞬时表达由遗传不稳定的载体控制，该载体独立于附加体质粒和诱导型启动子。单基因座和多基因座中的可选择标记与 Cre 载体一起丢失，分别为 100%和 29%。该方法的意义在于，红发夫酵母中不易获得稳定的自养突变体。在红发夫酵母和非同源末端连接主导酵母解脂耶氏酵母中的比较研究表明，该方法可普遍用于同源重组主导酵母。
