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

内容摘要

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. l- 缬氨酸具有广泛市场需求，目前工业生产 l- 缬氨酸方法主要是微生物发酵法。随着代谢工程策略的不断组合叠加，理性改造菌株代谢 l- 缬氨酸遇到瓶颈导致产量提升受限;非理性改造策略作为提升目标产物合成效率的一种补充策略被广泛应用于生物领域，然而非理性改造面临菌株突变文库庞大、传统高产菌株筛选方式耗时长、效率低等困境，因此需要一种快速高效筛选目标产物的工具缩短非理性改造菌株的周期。本研究基于一株经理性代谢工程改造的谷氨酸棒状杆菌 l-缬氨酸生产菌SX-4，构建了一种与 l-缬氨酸浓度呈正相关的生物传感器。通过更换不同强度的核糖体结合位点(ribosome binding site, RBS)序列优化响应精准度、拓宽响应范围;进一步利用常压室温等离子体诱变(atmospheric and room temperature plasma, ARTP)技术构建大量诱变菌株文库，并利用液滴微流控技术实现了 l-缬氨酸高产菌株的高通量筛选。所获得的菌株A23于摇瓶培养48 h， l-缬氨酸产量为21.36 g/L，相较于出发菌株提升了46.5%。本研究为其他氨基酸非理性改造研究提供了理论基础与借鉴。.