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

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 MG1655 Δ1-SAS-3 (ITA titer: 1.87 g/L), a CRISPRi-mediated self-inducible system (CiMS), which contained a responsive module based on the ITA biosensor ItcR/P 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, Δ4-P -SAS-3 (ITA titer: 3.20 g/L), derived from Δ1-SAS-3, was constructed by replacing the promoter P , for the expression of ITA synthesis genes, with P and knocking out the three bypass genes , , and . Subsequently, the CiMS was used to inhibit the expression of key genes , , and to dynamically balance the metabolic flux between TCA cycle and ITA biosynthetic pathway during the ITA production stage. The constructed strain Δ4-P -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.