[Construction and application of an l-valine-responsive biosensor in Corynebacterium glutamicum]

l- valine has extensive market demand, and its current industrial production primarily relies on microbial fermentation. With the continuous combination and accumulation of metabolic engineering strategies, rational modification of microbial strains for l- valine metabolism has encountered bottlenecks, leading to limitations in yield improvement. As a complementary strategy, irrational modification approaches are widely applied in the biological field to enhance the synthesis efficiency of target products. However, irrational modification faces challenges such as the vast scale of mutant libraries and the time-consuming, low-efficiency nature of traditional screening methods for high-yield strains. Therefore, there is an urgent need for a tool that enables rapid and efficient screening of target products to shorten the cycle of irrational strain modification. In this study, we started with an l- valine-producing strain, Corynebacterium glutamicum SX-4, which was previously constructed via rational metabolic engineering. We developed a biosensor that exhibited a positive correlation with intracellular l- valine concentration. The response accuracy and dynamic range of the sensor were optimized by incorporating ribosome-binding sites of varying strengths. Furthermore, we employed atmospheric and room temperature plasma mutagenesis to construct a comprehensive mutant library for random, non-rational strain modification. High-throughput screening of this library was performed via droplet microfluidics. The selected mutant strain, A23, achieved an l- valine titer of 21.36 g/L in shake-flask fermentation after 48 h, which represented a 46.5% increase over that of the starting strain. This integrated strategy provides a valuable framework and reference for irrational modification studies aimed at producing other amino acids.