Efficient production of salicylic acid through CmeR-PcmeO biosensor-assisted multiplexing pathway optimization in Escherichia coli

Abstract Recent studies have demonstrated that the tolerance of strains to high-concentration products is a feasible strategy for enhancing breakthroughs in the biomanufacturing of various industrial chemicals. In this study, an engineered Escherichia coli strain W3110 with limited ability to produce salicylic acid (SA) was adaptively evolved to acquire high-tolerance of SA. To rapidly isolate SA high-tolerance variation cells, a high-throughput screening method of SA higher producer was established assisted by a a CmeR-PcmeO biosensor. Ultimately, we identified an adaptive evolved strain with salicylic acid (SA) tolerance increasing from 0.9 g/L to 2.1 g/L, and the SA yield was enhanced from 283 mg/L to 588.1 mg/L. Subsequently, the designed sensor in conjunction with a multi-pathway sgRNA array were employed to dynamically regulate the other three derivatives of branched-chain acids, thereby achieving a balance between biomass growth and the rapid production of salicylic acid (SA) in the adaptive evolved strain,, resulting in a maximum SA yield of 1477.8 mg/L, whereas the yield of SA was only 1138.2 mg/L in the control strain W3110K-2 modified with the same metabolic engineering strategy. Through comprehensive whole-genome analysis, we preliminarily validated that the adaptive mutation gene ducA* and Group C2 genes (ymdA*, ymdB*, clsC*, csgB*, csgA*, and csgC*) significantly enhanced the strain's tolerance to elevated salicylic acid concentrations, as well as its efficiency in salicylic acid production and rapid substrate utilization. Notably, the adaptively evolved strain W3110K-4 exhibited a remarkable resistance to phages, which shown an excellent candidate for the microbial fermentation of SA on an industrial scale.