Bio-manufacturing leverages renewable resources to produce value-added chemicals as an alternative to unstainable fossil-based economy. Achieving maximum efficiency in bio-production requires the optimal spatial-temporal switch. However, the lack of versatile and functional quorum sensing (QS) components limits the broader application of this approach. To address this challenge, QS components with diverse properties were engineered through combinatorial strategies including element selection and promoter replacement. This process increased the dynamic range by 8.82-fold and threshold by 3.03-fold. Subsequently, aiming to develop regulatory elements with different dynamic range with high threshold, a LuxR mutant library was constructed via random mutation. High-throughput screening was then employed to identify mutants with decreased sensitivity to acyl-homoserine lactone. Notably, the triple mutation V36E/H89L/P97L reduced the sensitivity of LuxR by as much as 266-fold. As a proof-of-concept, iso-butylamine biosynthesis was tested by re-directing pyruvate catabolism. The optimal QS-based strain IB21 produced 15.4 g/L iso-butylamine in fed-batch fermentation, marking a 2.96-fold increase over the statically-optimized strain IB04. Finally, its final titer reached 44.23 g/L in a 7 L bioreactor, representing the highest reported for de novo synthesis. In summary, this study enriches the genetic toolbox of synthetic QS system, which facilitates the self-regulated enhancement of value-added chemical production.
