The unconventional yeast Yarrowia lipolytica is extensively applied in bioproduction fields owing to its excellent metabolite and protein production ability. Nonetheless, utilization of this promising host is still restricted by limited availability of precise and effective gene integration tools. In this study, a novel and efficient genetic tool was developed for targeted, repeated, and markerless gene integration based on Cre/lox site-specific recombination system. The developed tool required only a single selection marker and could completely excise all of the unnecessary sequences. A total of three plasmids were created and seven rounds of marker-free gene integration were examined in Y. lipolytica. All the integration efficiencies remained above 90%, and analysis of protein production and growth characteristics of the engineered strains confirmed that genome modification via the novel genetic tool was feasible. Further work also confirmed the genetic tool was effective for integration of other genes, loci, and strains. Thus, this study significantly promotes the application of Cre/lox system and presents a powerful tool for genome engineering in Y. lipolytica.

中文翻译：

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一种新颖，有效的基于Cre / lox的遗传工具，用于重复，靶向和无标记的基因整合

非常规酵母解脂耶氏酵母因其优异的代谢产物和蛋白质生产能力而广泛应用于生物生产领域。但是，这种有希望的宿主的利用仍然受到精确和有效的基因整合工具的有限可用性的限制。在这项研究中，基于Cre / lox位点特异性重组系统，针对靶向，重复和无标记基因整合开发了一种新颖而有效的遗传工具。开发的工具只需要一个选择标记，就可以完全切除所有不必要的序列。共创建了三个质粒，并在解脂耶氏酵母中检测了七轮无标记的基因整合。所有集成效率均保持在90％以上，对工程菌株的蛋白质生产和生长特性进行分析，证实了通过新型遗传工具进行基因组修饰是可行的。进一步的工作还证实了该遗传工具对于整合其他基因，基因座和菌株有效。因此，这项研究显着促进了Cre / lox系统的应用，并为解脂耶氏酵母中的基因组工程提供了强大的工具。