Combining biosensor and metabolic network optimization strategies for enhanced l-threonine production in Escherichia coli

L-threonine is an integral nutrient for mammals, often used in animal feeds to enhance growth and reduce breeding costs. Developing L-threonine engineered strains that meet industrial production specifications has significant economic value. Here, we developed a biosensor that monitors L-threonine concentration to assist in high-throughput screening to capture high-yielding L-threonine mutants. Among them, the P_cysK promoter and CysB protein were used to construct a primary L-threonine biosensor, and then the CysB^T102A mutant was obtained through directed evolution resulting in a 5.6-fold increase in the fluorescence responsiveness of biosensor over the 0-4 g/L L-threonine concentration range. In addition, the metabolic network of mutant was further optimized through multi-omics analysis and in silico simulation. Ultimately, the THRM13 strain produced 163.2 g/L L-threonine, with a yield of 0.603 g/g glucose in a 5 L bioreactor. The biosensor constructed here could be employed for iterative upgrading of subsequent strains, and these engineering strategies described provide guidance for other chemical overproducers.