One major mission of microbial breeding is high-level production of desired metabolites. Overproduction of intermediate metabolites in core pathways is challenging as it may impair cell growth and viability. Here we report that aconitic acid, an intermediate metabolite in tricarboxylic acid (TCA) cycle, can be overproduced by an engineered CRISPR interference (CRISPRi) system in Escherichia coli. This CRISPRi system was designed to simultaneously target pyruvate kinase (PK) and isocitrate dehydrogenase (IDH), two enzymes in glycolytic pathway and TCA cycle, respectively. Reverse transcription and quantitative PCR and enzyme activity assays showed that this engineered CRISPRi system significantly repressed the genes encoding IDH and PK, resulting in simultaneous reduction in the activities of IDH and PK. In shake-flask and fed-batch cultivation, this CRISPRi strain produced 60-fold (362.80 ± 22.05 mg/L) and 15-fold (623.80 ± 20.05 mg/L) of aconitic acid relative to the control strain, respectively. In addition, this two-target CRISPRi strain maintained low levels of acetate and lactate, two problematic byproducts. This work demonstrates that CRISPRi system can improve aconitic acid production by coordinating glycolysis and TCA cycle. This study provides insights for high-level production of the intermediate metabolites in central pathways.

中文翻译：

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CRISPR干扰引导下的葡萄糖途径调节，以增强大肠杆菌中乌头酸的产生。

微生物育种的一项主要任务是高水平生产所需的代谢产物。核心途径中中间代谢物的过量生产具有挑战性，因为它可能会损害细胞生长和生存能力。在这里我们报告乌头酸是三羧酸（TCA）循环中的一种中间代谢产物，可通过大肠杆菌中的工程CRISPR干扰（CRISPRi）系统过量生产。该CRISPRi系统设计为同时靶向丙酮酸激酶（PK）和异柠檬酸脱氢酶（IDH），这两种酶分别位于糖酵解途径和TCA循环中。逆转录，定量PCR和酶活性测定表明，这种经过工程改造的CRISPRi系统显着抑制了编码IDH和PK的基因，从而导致IDH和PK的活性同时降低。在摇瓶和分批补料栽培中，该CRISPRi菌株产生的乌头酸分别是对照菌株的60倍（362.80±22.05 mg / L）和15倍（623.80±20.05 mg / L）。此外，这种具有两个目标的CRISPRi菌株保持了低水平的乙酸盐和乳酸（两种有问题的副产物）。这项工作表明，CRISPRi系统可以通过协调糖酵解和TCA循环来改善乌头酸的产生。这项研究为在中央途径中高水平生产中间代谢产物提供了见识。这项工作表明，CRISPRi系统可以通过协调糖酵解和TCA循环来改善乌头酸的产生。这项研究为在中央途径中高水平生产中间代谢产物提供了见识。这项工作表明，CRISPRi系统可以通过协调糖酵解和TCA循环来改善乌头酸的产生。这项研究为在中央途径中高水平生产中间代谢产物提供了见识。