Programming Low-Molecular-Weight Hyaluronic Acid Biosynthesis in <i>Bacillus amyloliquefaciens</i> Via an Autoinducible Molecular Switch

Dynamic metabolic regulation facilitates real-time sensing of pathway states and precise redirection of metabolic flux, thereby enhancing the biosynthesis of diverse value-added compounds. Here, we designed an autoinducible molecular switch in <i>Bacillus amyloliquefaciens</i> by integrating a <i>Pantoea alhagi</i>-derived PaSspB-ssrA* proteolytic system with the DegU-RapG-PhrG quorum-sensing (QS) module. The OFF module employs the novel PaSspB-ssrA* system for targeted degradation of ssrA*-tagged proteins with 86% efficiency, and the ON module utilizes a DegU-activated <i>P</i>_<i>ispA</i> promoter for cell density-dependent gene expression. To improve hyaluronic acid (HA) production and reduce byproduct accumulation, this system was applied by fusing ssrA* tags into the glycolysis-critical genes <i>pfkA</i> and <i>fruA</i> to attenuate glycolytic flux, together with QS-regulated coexpression of PaSspB and SthHL in engineered Δ<i>ldh</i>/Δ<i>wbpA</i>/Δ<i>nagB</i> strains, achieving 13.5 ± 0.19 g/L of low-molecular-weight HA (2.89 × 10⁴ Da). The autonomous, inducer-free platform enables cost-effective HA biosynthesis in <i>B. amyloliquefaciens</i>, with broad applicability for synthesizing high-value bioproducts.