The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet Building 220, DK-2800 Kgs. Lyngby, Denmark.
Ajinomoto-Genetika Research Institute, Moscow, Russian Federation.
FEMS Yeast Res. 2021 May 11;21(4). doi: 10.1093/femsyr/foab027.
Biotechnological production requires genetically stable recombinant strains. To ensure genomic stability, recombinant DNA is commonly integrated into the genome of the host strain. Multiple genetic tools have been developed for genomic integration into baker's yeast Saccharomyces cerevisiae. Previously, we had developed a vector toolkit EasyClone-MarkerFree for stable integration into eleven sites on chromosomes X, XI, and XII of S. cerevisiae. The markerless integration was enabled by CRISPR-Cas9 system. In this study, we have expanded the kit with eight additional intergenic integration sites located on different chromosomes. The integration efficiency into the new sites was above 80%. The expression level of green fluorescence protein (gfp) for all eight sites was similar or above XI-2 site from the original EasyClone-MarkerFree toolkit. The cellular growth was not affected by the integration into any of the new eight locations. The eight-vector expansion kit is available from AddGene.
生物技术生产需要遗传稳定的重组菌株。为了确保基因组稳定性，重组 DNA 通常被整合到宿主菌株的基因组中。已经开发了多种用于基因组整合到酿酒酵母 Saccharomyces cerevisiae 中的遗传工具。以前，我们开发了一个用于稳定整合到酿酒酵母 X、XI 和 XII 染色体上十一个位点的载体工具包 EasyClone-MarkerFree。无标记整合是通过 CRISPR-Cas9 系统实现的。在这项研究中，我们用位于不同染色体上的另外八个基因间整合位点扩展了试剂盒。新位点的整合效率超过 80%。所有八个位点的绿色荧光蛋白 (gfp) 的表达水平与原始 EasyClone-MarkerFree 工具包中的 XI-2 位点相似或更高。整合到任何新的八个位置都不会影响细胞生长。这个由八个载体组成的扩展工具包可从 Addgene 获取。
