The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet 220, DK-2800 Kongens Lyngby, Denmark.
Biotechnol J. 2018 Sep;13(9):e1700543. doi: 10.1002/biot.201700543. Epub 2018 Feb 14.
The oleaginous yeast Yarrowia lipolytica is an emerging host for production of fatty acid-derived chemicals. To enable rapid iterative metabolic engineering of this yeast, there is a need for well-characterized genetic parts and convenient and reliable methods for their incorporation into yeast. Here, the EasyCloneYALI genetic toolbox, which allows streamlined strain construction with high genome editing efficiencies in Y. lipolytica via the CRISPR/Cas9 technology is presented. The toolbox allows marker-free integration of gene expression vectors into characterized genome sites as well as marker-free deletion of genes with the help of CRISPR/Cas9. Genome editing efficiencies above 80% were achieved with transformation protocols using non-replicating DNA repair fragments (such as DNA oligos). Furthermore, the toolbox includes a set of integrative gene expression vectors with prototrophic markers conferring resistance to hygromycin and nourseothricin.
产油酵母解脂耶氏酵母是一种新兴的脂肪酸衍生化学品生产宿主。为了能够快速进行代谢工程的反复迭代，需要对这种酵母进行良好的基因元件的特征描述，并需要方便可靠的方法将这些元件整合到酵母中。这里介绍了 EasyCloneYALI 遗传工具包，该工具包通过 CRISPR/Cas9 技术可实现 Y. lipolytica 中高通量的基因编辑效率和简化的菌株构建。该工具包允许通过 CRISPR/Cas9 无标记地将基因表达载体整合到特征基因组位点，以及无标记地删除基因。使用非复制 DNA 修复片段（如 DNA 寡核苷酸）的转化方案实现了超过 80%的基因组编辑效率。此外，该工具包还包括一组带有原养型标记的整合型基因表达载体，这些标记赋予对潮霉素和诺赛菌素的抗性。
