Leplat Christophe, Nicaud Jean-Marc, Rossignol Tristan
INRA, UMR1319 Micalis, F-78352 Jouy-en-Josas, France AgroParisTech, UMR Micalis, F-78352 Jouy-en-Josas, France.
INRA, UMR1319 Micalis, F-78352 Jouy-en-Josas, France AgroParisTech, UMR Micalis, F-78352 Jouy-en-Josas, France
FEMS Yeast Res. 2015 Sep;15(6). doi: 10.1093/femsyr/fov052. Epub 2015 Jun 22.
As a microorganism of major biotechnological importance, the oleaginous yeast Yarrowia lipolytica is subjected to intensive genetic engineering and functional genomic analysis. Future advancements in this area, however, require a system that will generate a large collection of mutants for high-throughput screening. Here, we report a rapid and efficient method for high-throughput transformation of Y. lipolytica in 96-well plates. We developed plasmids and strains for the large-scale screening of overexpression mutant strains, using Gateway® vectors that were adapted for specific locus integration in Y. lipolytica. As an example, a collection of mutants that overexpressed the alkaline extracellular protease (AEP) was obtained in a single transformation experiment. The platform strain that we developed to receive the overexpression cassette was designed to constitutively express a fluorescent protein as a convenient growth reporter for screening in non-translucid media. An example of growth comparison in skim milk-based medium between AEP overexpression and deletion mutants is provided.
作为一种具有重要生物技术意义的微生物，解脂耶氏酵母受到了深入的基因工程和功能基因组分析。然而，该领域未来的进展需要一个能够产生大量突变体用于高通量筛选的系统。在此，我们报告了一种在96孔板中对解脂耶氏酵母进行高通量转化的快速有效方法。我们开发了用于大规模筛选过表达突变菌株的质粒和菌株，使用了适用于解脂耶氏酵母特定基因座整合的Gateway®载体。例如，在一次转化实验中获得了一组过表达碱性细胞外蛋白酶（AEP）的突变体。我们开发的用于接收过表达盒的平台菌株被设计为组成型表达一种荧光蛋白，作为在非透明培养基中筛选的便捷生长报告基因。提供了在基于脱脂乳的培养基中AEP过表达和缺失突变体之间生长比较的示例。
