Engineering a CRISPRi Circuit for Autonomous Control of Metabolic Flux in Escherichia coli.

Cong Gao, Liang Guo, Guipeng Hu, Jia Liu, Xiulai Chen, Xiaoxia Xia, Liming Liu

ACS synthetic biology 2021 Oct 15

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Building autonomous switches is an effective approach for rewiring metabolic flux during microbial synthesis of chemicals. However, current autonomous switches largely rely on metabolite-responsive biosensors or quorum-sensing circuits. In this study, a stationary phase promoter (SPP) and a protein degradation tag (PDT) were combined with the CRISPR interference (CRISPRi) system to construct an autonomous repression system that could shut down multiple-gene expression depending on the cellular physiological state. With this autonomous CRISPRi system to regulate one target gene, a fermenter-scale titer of shikimic acid reached 21 g/L, which was the highest titer ever reported by Escherichia coli in a minimal medium without any chemical inducers. With three target genes repressed, 26 g/L glutaric acid could be achieved with decreased byproduct accumulation. These results highlight the applicability of the autonomous CRISPRi system for microbial production of value-added chemicals.

Citation

Cong Gao, Liang Guo, Guipeng Hu, Jia Liu, Xiulai Chen, Xiaoxia Xia, Liming Liu. Engineering a CRISPRi Circuit for Autonomous Control of Metabolic Flux in Escherichia coli. ACS synthetic biology. 2021 Oct 15;10(10):2661-2671