A CRISPRi mediated self-inducible system for dynamic regulation of TCA cycle and improvement of itaconic acid production in Escherichia coli

Itaconic acid (ITA), an effective alternative fossil fuel, derives from the bypass pathway of the tricarboxylic acid (TCA) cycle. Therefore, the imbalance of metabolic flux between TCA cycle and ITA biosynthetic pathway seriously limits the production of ITA. The optimization of flux distribution between biomass and production has the potential to the productivity of ITA. Based on the previously constructed strain Escherichia coli MG1655 Delta 1SAS-3 (ITA titer: 1.87 g/L), a CRISPRi-mediated self-inducible system (CiMS), which contained a responsive module based on the ITA biosensor YpItcR/Pccl and a regulative CRISPRi-mediated interferential module, was developed to regulate the flux of the TCA cycle and to enhance the capacity of the strain to produce ITA. First, a higher ITA-yielding strain, Delta 4-Prmd-SAS-3 (ITA titer: 3.20 g/L), derived from Delta 1-SAS-3, was constructed by replacing the promoter PJ23100, for the expression of ITA synthesis genes, with Prmd and knocking out the three bypass genes poxB, pflB, and ldhA. Subsequently, the CiMS was used to inhibit the expression of key genes icd, pykA, and sucCD to dynamically balance the metabolic flux between TCA cycle and ITA biosynthetic pathway during the ITA production stage. The constructed strain Delta 4-Prmd-SAS-3 under the dynamic regulation of the CiMS, showed a 23% increase in the ITA titer, which reached 3.93 g/L. This study indicated that CiMS was a practical strategy to dynamically and precisely regulated the metabolic flux in microbial cell factories.

Automated summary

This study developed a CRISPRi-mediated self-inducible system (CiMS) to dynamically regulate the metabolic flux and enhance the production capacity of itaconic acid (ITA) in Escherichia coli. By optimizing the flux distribution, the ITA titer was increased by 23%. The CiMS strategy demonstrated its effectiveness in regulating metabolic flux in microbial cell factories.