(67b) Temperature- and Pyruvate-Driven Layered Circuits for Chemical Production in Escherichia coli

Microbial engineering and genetically encoded bioswitch provide excellent and alternative platforms for reprogramming metabolic networks and producing high-value chemicals. However, the synthesis of biochemicals, especially those bio-based compounds associated with the central pathway or multiple bypasses, is often restricted by robust primary metabolism and incompatible metabolic homeostasis. Here, the programmable and bifunctional temperature- and pyruvate-responsive biosensor is firstly developed to establish efficient biosynthesis platforms for pyruvate derivatives. Especially, variants V110M, E147G, I167V, and L185P of regulator CI⁸⁵⁷ individually exhibit improvements of 8.62%, 11.35%, 10.58%, and 37.08% in thermostability by forming a ring oscillation and greater rigidity structures. Then, different genetic layered circuits, named TPCL and TPPLC, are designed to autonomously regulate the bioproduct pathway and improve the microbial phenotype in a pathway-independent manner. As a proof-of-concept, precise fine-tuning between the tricarboxylic acid (TCA) cycle and the iso-butylamine pathway results in the highest titer of iso-butylamine (17.05 g L^-1) than previously reported, and the yield can reach 81.5% (0.66 g g^-1 glucose) of the theoretical yield. In addition, an increase in butyrate production of 3.52-fold is generated by controlling the acetyl-CoA catabolism. Taken together, this study provides versatile and efficient toolkits for redistributing metabolic bypasses or synthesizing complicated biochemicals.