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Abstract
Establishing microbial cell factories using metabolic engineering strategies or synthetic biology tools has enabled fast and cost-effective production of a series of valuable natural and synthetic compounds. To further improve the productivities of microbial cell factories, fine-tuning the gene expression, through either intelligently enhancing the target production or effectively inhibiting the competitive pathways with the assistance of genetically-encoded biosensors, has been demonstrated to be efficient in constructing highly efficient microbial cell factories. Herein, we explored the biosensor-assisted fine-tuning of gene expression in microbial cell factories to improve the biosynthesis of target compounds, and the further development and optimization of biosensors with desired dynamic performance and expanded substrate scopes. With the understanding of the replication mechanism for ColE1-derived plasmid origins, we established the dynamic pathway regulation at gene copy level to fine-tune the gene expression through dynamically controlling the plasmid replication with the p-coumaric acid responsive transcriptional repressor PadR derived from Bacillus subtilis 168, which enabled a significantly improved p-coumaric acid production in Escherichia coli. Then, we targeted the central metabolism of E. coli using CRISPRi with the mismatched sgRNA arrays to finely rewire the carbon flux to the production of p-coumaric acid or butyric acid. The PadR and the butyric acid responsive regulator HpdR were applied to screen the high-producers of p-coumaric acid and butyric acid in a high-throughput manner, respectively. To further improve the applicability of the transcriptional factors-based biosensors, we broadened the substrate scope of the phenolic acid-responsive regulator PadR and enabled the recognition of seven aromatic compounds with different structures. Our work demonstrated the enormous potential of utilizing transcriptional factors-based biosensors in constructing highly efficient microbial cell factories, and the newly developed regulator variants in our study that harbor altered substrate scopes would also be particularly beneficial for future applications in metabolic engineering and synthetic biology.