Engineering of L-threonine and L-proline biosensors by directed evolution of transcriptional regulator SerR and application for high-throughput screening.

Amino acids are important bio-based products with a multi-billion-dollar market. The development of efficient high-throughput screening technologies utilizing biosensors is essential for the rapid identification of high-performance amino acid producers. However, there remains a pressing need for biosensors that specifically target certain critical amino acids, such as L-threonine and L-proline. In this study, a novel transcriptional regulator-based biosensor for L-threonine and L-proline was successfully developed, inspired by our new finding that SerE can export L-proline in addition to the previously known L-threonine and L-serine. Through directed evolution of SerR (the corresponding transcriptional regulator of SerE), the mutant SerR^F104I which can recognize both L-threonine and L-proline as effectors and effectively distinguish strains with varying production levels was identified. Subsequently, the SerR^F104I-based biosensor was employed for high-throughput screening of the superior enzyme mutants of L-homoserine dehydrogenase and γ-glutamyl kinase, which are critical enzymes in the biosynthesis of L-threonine and L-proline, respectively. A total of 25 and 13 novel mutants that increased the titers of L-threonine and L-proline by over 10% were successfully identified. Notably, six of the newly identified mutants exhibited similarities to the most effective mutants reported to date, indicating the promising application potential of the SerR^F104I-based biosensor. This study illustrates an effective strategy for the development of transcriptional regulator-based biosensors for amino acids and other chemical compounds.