(326c) Establishing a Feedback Control System for Efficient Biosynthesis of Natural Products

Genetic regulation tools have been examined for their ability to facilitate sophisticated dynamic control of biosynthesis in microbial cell factory, enhancing the production performance of significant valuable compounds. However, little is known about whether such toolkits could be employed to balance the metabolic fluxes between cell growth and downstream synthesis, thereby achieving efficient biosynthesis of natural products. To answer this question, we established a feedback control system that responds to central metabolite pyruvate to dynamically regulate the distribution of metabolic fluxes. In this study, we firstly characterized the dynamic performance of pyruvate-responsive feedback system by screening multiple PdhR homologs derived from various microorganisms. Due to differences in critical residues of regulators and promoters, different dynamic performances were observed. Assisted by computational analysis, we further explored the reason causing these differences, helping to optimize the PdhR feedback system through site-directed mutagenesis. Subsequently, we integrated antisense RNA upon the optimal engineered pyruvate-responsive feedback system to achieve simultaneous feedback and feedforward regulation. As a proof of concept, we employed our feedback system to improve the biosynthesis of the disaccharide trehalose. Specifically, we constructed a bifunctional regulation system to dynamically inhibit the carbon flux to cell growth and competitive pathways, and meanwhile to dynamically activate downstream trehalose synthesis pathways, leading to enhanced production of trehalose. In summary, the established central metabolism-responsive feedback system underlined the necessity of metabolic flux distribution and validated its applicability in the biosynthesis of natural products.