CRISPR interference-guided modulation of glucose pathways to boost aconitic acid production in Escherichia coli
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Microbial Cell Factories Open Access
RESEARCH
CRISPR interference‑guided modulation of glucose pathways to boost aconitic acid production in Escherichia coli Qingyang Li1, Peng Zhao2, Hang Yin1, Zhaonan Liu2, Haifeng Zhao1 and Pingfang Tian2*
Abstract Background: 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. Results: 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. Conclusions: 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. Keywords: Aconitic acid, CRISPR interference, Glucose metabolism, Isocitrate dehydrogenase, Pyruvate kinase Background Aconitic acid was first identified in Aconitum napellus and thereby named after this plant. In nature, aconitic acid exists as two isomers (trans- and cis-) and is one of plentiful organic acids in sugar cane [1]. In addition, aconitic acid is rich in Pseudomonas spp. [2] and sugarcontaining plants such as wheat (Triticum aestivum) [3] and maize (Zea mays) [4]. Specially, aconitic acid is an intermediate metabolite in tricarboxylic acid (TCA) cycle and thus is of paramount importance for cell viability. In addition to the participation in core metabolisms and its *Correspondence: [email protected] 2 College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China Full list of author information is available at the end of the article
use as a food additive, trans-aconitic acid has nematicidal [5] and antiedematogenic activities [6], suggesting its potentials in plant protection and therapeutic development. Apart from its versatile bio-functions, aconitic acid can be converted to itaconic acid, which is the feedstock for manufacturing of acrylic plastics, acrylate latexes, super-absorbents, and anti-scaling agents [7]. Aconitic acid can be chemically synthesized through dehydration of citric acid in the presence of concentrated sulf
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