Combining biosensor and metabolic network optimization strategies for enhanced <sc>l</sc>-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 PcysK promoter and CysB protein were used to construct a primary l-threonine biosensor, and then the Cys(BT102A) 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.