Abstract
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.
Funder
National Key Research and Development Program of China
National Natural Science Foundation of China
National High-tech Research and Development Program
Publisher
Springer Science and Business Media LLC
Subject
Applied Microbiology and Biotechnology,Bioengineering,Biotechnology
Cited by
10 articles.
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