Frontier Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, China.
Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin University, Tianjin, 300072, China.
Sci China Life Sci. 2021 Dec;64(12):2114-2128. doi: 10.1007/s11427-020-1885-x. Epub 2021 Mar 2.
Genomic variants libraries are conducive to obtain dominant strains with desirable phenotypic traits. The non-homologous end joining (NHEJ), which enables foreign DNA fragments to be randomly integrated into different chromosomal sites, shows prominent capability in genomic libraries construction. In this study, we established an efficient NHEJ-mediated genomic library technology in Yarrowia lipolytica through regulation of NHEJ repair process, employment of defective Ura marker and optimization of iterative transformations, which enhanced genes integration efficiency by 4.67, 22.74 and 1.87 times, respectively. We further applied this technology to create high lycopene producing strains by multi-integration of heterologous genes of CrtE, CrtB and CrtI, with 23.8 times higher production than rDNA integration through homologous recombination (HR). The NHEJ-mediated genomic library technology also achieved random and scattered integration of loxP and vox sites, with the copy number up to 65 and 53, respectively, creating potential for further application of recombinase mediated genome rearrangement in Y. lipolytica. This work provides a high-efficient NHEJ-mediated genomic library technology, which enables random and scattered genomic integration of multiple heterologous fragments and rapid generation of diverse strains with superior phenotypes within 96 h. This novel technology also lays an excellent foundation for the development of other genetic technologies in Y. lipolytica.
基因组变异文库有利于获得具有理想表型特征的优势菌株。非同源末端连接（NHEJ）能够使外源 DNA 片段随机整合到不同的染色体位点，在基因组文库构建中表现出突出的能力。在本研究中，我们通过调控 NHEJ 修复过程、使用有缺陷的 Ura 标记以及优化迭代转化，在解脂耶氏酵母中建立了一种高效的 NHEJ 介导的基因组文库技术，分别将基因整合效率提高了 4.67、22.74 和 1.87 倍。我们进一步应用该技术通过异源基因 CrtE、CrtB 和 CrtI 的多整合，创建了高番茄红素生产菌株，其产量比通过同源重组（HR）整合 rDNA 高 23.8 倍。NHEJ 介导的基因组文库技术还实现了loxP 和 vox 位点的随机和分散整合，拷贝数分别高达 65 和 53，为进一步应用重组酶介导的基因组重排在解脂耶氏酵母中创造了潜力。这项工作提供了一种高效的 NHEJ 介导的基因组文库技术，能够在 96 小时内随机和分散地整合多个异源片段，并快速生成具有优越表型的多种菌株。这项新技术也为解脂耶氏酵母中其他遗传技术的发展奠定了良好的基础。
