Cleto Sara, Jensen Jaide Vk, Wendisch Volker F, Lu Timothy K
Department of Electrical Engineering & Computer Science and Department of Biological Engineering, Massachusetts Institute of Technology , 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.
MIT Synthetic Biology Center , 500 Technology Square, Cambridge, Massachusetts 02139, United States.
ACS Synth Biol. 2016 May 20;5(5):375-85. doi: 10.1021/acssynbio.5b00216. Epub 2016 Feb 16.
Corynebacterium glutamicum is an important organism for the industrial production of amino acids. Metabolic pathways in this organism are usually engineered by conventional methods such as homologous recombination, which depends on rare double-crossover events. To facilitate the mapping of gene expression levels to metabolic outputs, we applied CRISPR interference (CRISPRi) technology using deactivated Cas9 (dCas9) to repress genes in C. glutamicum. We then determined the effects of target repression on amino acid titers. Single-guide RNAs directing dCas9 to specific targets reduced expression of pgi and pck up to 98%, and of pyk up to 97%, resulting in titer enhancement ratios of l-lysine and l-glutamate production comparable to levels achieved by gene deletion. This approach for C. glutamicum metabolic engineering, which only requires 3 days, indicates that CRISPRi can be used for quick and efficient metabolic pathway remodeling without the need for gene deletions or mutations and subsequent selection.
谷氨酸棒杆菌是用于氨基酸工业化生产的重要微生物。该微生物中的代谢途径通常通过同源重组等传统方法进行改造，同源重组依赖于罕见的双交换事件。为了便于将基因表达水平与代谢输出进行关联，我们应用了使用失活Cas9（dCas9）的CRISPR干扰（CRISPRi）技术来抑制谷氨酸棒杆菌中的基因。然后我们确定了靶向抑制对氨基酸滴度的影响。引导dCas9靶向特定靶点的单向导RNA（single-guide RNA）使pgi和pck的表达降低了98%，pyk的表达降低了97%，导致L-赖氨酸和L-谷氨酸产量的滴度提高比率与基因缺失所达到的水平相当。这种用于谷氨酸棒杆菌代谢工程的方法仅需3天，表明CRISPRi可用于快速高效地重塑代谢途径，而无需进行基因缺失或突变以及后续筛选。
