Abstract
BackgroundOne 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.ResultsHere 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.ConclusionsThis 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.
Highlights
One major mission of microbial breeding is high-level production of desired metabolites
Performance of CRISPR interference (CRISPRi) system In E. coli, biosynthesis of aconitic acid relies on a panel of enzymes
To clarify the influences of their expression on aconitic acid production, three genes icdA, pykA and pykF native to E. coli were chosen as the targets of CRISPRi system (Fig. 1)
Summary
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. Aconitic acid is an intermediate metabolite in tricarboxylic acid (TCA) cycle and is of paramount importance for cell viability. Li et al Microb Cell Fact (2020) 19:174 is acidified by sulfuric acid. This route brings about troublesome lactones of isocitric acid and alloisocitric acid, which are unwanted byproducts [9], as they entangle downstream separation and increase the production cost of aconitic acid. It is highly desirable to develop a novel method for the production of aconitic acid. Bio-production has emerged as an alternative to conventional chemical synthesis, and it requires moderate instead of stringent reaction conditions
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