Utilizing a high-throughput visualization screening technology to develop a genetically encoded biosensor for...

5-Aminolevulinic acid (5-ALA) is a non-protein amino acid widely used in agriculture, animal husbandry and medicine. Currently, microbial cell factories are a promising production pathway, but the lack of high-throughput fermentation strain screening tools often hinders the exploration of engineering strategies to increase cell factory yields. Here, mutant AC¹⁰³³H was screened from libraries of saturating mutants after response-specific engineering of the transcription factor AsnC of L-asparagine (Asn). Based on mutant AC^103-3H, a whole-cell biosensor EAC^103-3H with a specific response to 5-ALA was constructed, which has a linear dynamic detection range of 1-12 mM and a detection limit of 0.094 mM, and can be used for in situ screening of potential high-producing 5-ALA strains. With its support, overexpression of the C5 pathway genes using promoter engineering assistance resulted in a 4.78-fold enhancement of 5-ALA production in the engineered E. coli. This study provides an efficient strain screening tool for exploring approaches to improve the 5-ALA productivity of engineered strains.