Construction of an intelligent dynamic system for alternating biosynthesis of pyruvate and its diol derivative (AIPAD)

Natural microbes prioritize cell growth, not metabolic engineering production. Pyruvate, as a cross-linked metabolite of native metabolic network, could be catalyzed by pathway enzymes to generate various precursors for growth needs. Pyruvate derivatives show high physiological and biochemical value. The common biosynthesis method for these derivatives was to intercept pyruvate by building the artificial production pathways within microbes, thereby triggering competitive allocation of pyruvate in growth and production. However, the conventional and mechanical introduction of these pathways disrupted the stability of native metabolic network of the microbes. The resulting microbes were unable to maximize the resources to the derivatives production. In response to this challenge, this study constructed an intelligent dynamic system for alternating biosynthesis of pyruvate and its diol derivative (AIPAD) by coupling the transcription factors (TFs) PdhR and BmoR biosensors, aiming to achieve the efficient biosynthesis of R,R-2,3-butanediol (R,R-2,3-BDO), a high-value diol derivative of pyruvate. In this system, pyruvate-regulated PdhR biosensor could dynamically regulate the overexpression of the enzymes for the conversion of pyruvate-to-R,R-2,3-BDO, while R,R-2,3-BDO-regulated BmoR could dynamically regulate the overexpression of the enzymes that competed pyruvate for growth or by-product biosynthesis. This system allowed the microbes to rationally allocate the resources between growth and production as needed, resulting in a continuous production of R,R-2,3-BDO throughout the fermentation cycle. Further, the performance of PdhR and BmoR was repurposed to increase the start-up speed and accuracy of the system, and the chassis was preferred and engineered to increase the compatibility with R,R-2,3-BDO production pathway, ultimately achieving 124 g/L R,R-2,3-BDO production in a 3-L bioreactor. This study created an AIPAD system to generate a long-term production manner, providing the guidance for constructing the intelligent microbial cell factory and exploring the large-scale production mode.