Improvement of the CRISPR-Cas9 mediated gene disruption and large DNA fragment deletion based on a chimeric promoter in Acremonium chrysogenum
Improvement of the CRISPR-Cas9 mediated gene disruption and large DNA fragment deletion based on a chimeric promoter in Acremonium chrysogenum
复制标题
基于嵌合启动子的 CRISPR-Cas9 介导的基因破坏和大 DNA 片段缺失的改进
DOI：
10.1016/j.fgb.2019.103279
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发表时间：
2020-01-01
影响因子：
3
通讯作者：
Liu, Gang
中科院分区：
文献类型：
--
作者：
Chen, Chang;Liu, Jiajia;Liu, Gang
在头孢菌素C （CPC）的工业生产中，已应用了黄顶孢菌。然而，由于黄芽孢杆菌（A. chrysogenum），特别是CPC高产菌株C10的遗传操作困难，对CPC生物合成调控和改良菌株的理解仍存在一些障碍。本研究基于U6/tRNA嵌合启动子构建了改进的CRISPR-Cas9系统。利用该系统对C10进行了高效率的单基因裂解。此外，当提供同源定向修复模板（供体dna）时，双位点同时被靶向。基于该系统，成功地高效删除了黄色化合物山梨霉素生物合成所需的31.5 kb大片段DNA。此外，敲除山梨霉素类生物合成基因后，CPC的产量显著提高。该研究为黄芽孢杆菌的基因编辑和品系改良提供了有力的工具。
Acremonium chrysogenum has been employed in the industrial production of cephalosporin C (CPC). However, there are still some impediments to understanding the regulation of CPC biosynthesis and improving strains due to the difficulty of genetic manipulation in A. chrysogenum, especially in the CPC high-producing strain C10. Here, an improved CRISPR-Cas9 system was constructed based on an U6/tRNA chimeric promoter. Using this system, high efficiency for single gene disruption was achieved in C10. In addition, double loci were simultaneously targeted when supplying with the homology-directed repair templates (donor DNAs). Based on this system, large DNA fragments up to 31.5 kb for the yellow compound sorbicillinoid biosynthesis were successfully deleted with high efficiency. Furthermore, CPC production was significantly enhanced when the sorbicillinoid biosynthetic genes were knocked out. This study provides a powerful tool for gene editing and strain improvement in A. chrysogenum.