Li Mingyue, Chen Jiuzhou, Wang Yu, Liu Jiao, Huang Jingwen, Chen Ning, Zheng Ping, Sun Jibin
College of Biotechnology, Tianjin University of Science and Technology, Tianjin, China.
Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China.
Front Bioeng Biotechnol. 2020 Apr 24;8:357. doi: 10.3389/fbioe.2020.00357. eCollection 2020.
is an important workhorse for industrial production of diversiform bioproducts. Multiplex control of metabolic pathway genes is crucial for maximizing biosynthesis of desired products. However, few tools for simultaneously regulating multiple genes in have been reported. Here, a CRISPR-dCpf1-based multiplex gene repression system was developed for . This system successfully repressed two fluorescent reporter genes simultaneously by expressing a dCpf1 (E1006A, D917A) and a designed single crRNA array. To demonstrate applications of this CRISPR-dCpf1 system in metabolic engineering, we applied this system to repress four genes involved in lysine biosynthesis (, , , and ) with a single array, which increased the lysine titer and yield for over 4.0-fold. Quantitative PCR demonstrated that transcription of all the four endogenous target genes were repressed by over 90%. Thus, the CRISPR-dCpf1 system is a simple and effective technique for multiplex gene repression in and holds promise for metabolic engineering of to produce valuable chemicals and fuels.
是工业生产多种生物产品的重要主力。代谢途径基因的多重控制对于最大化所需产品的生物合成至关重要。然而，很少有报道用于在……中同时调控多个基因的工具。在此，开发了一种基于CRISPR-dCpf1的多重基因抑制系统用于……。该系统通过表达dCpf1（E1006A，D917A）和设计的单个crRNA阵列成功同时抑制了两个荧光报告基因。为了证明这种CRISPR-dCpf1系统在代谢工程中的应用，我们将该系统应用于用单个阵列抑制赖氨酸生物合成中涉及的四个基因（……、……、……和……），这使赖氨酸滴度和产量提高了4.0倍以上。定量PCR表明所有四个内源性靶基因的转录被抑制了90%以上。因此，CRISPR-dCpf1系统是一种用于在……中进行多重基因抑制的简单有效技术，有望用于……的代谢工程以生产有价值的化学品和燃料。
