Engineering the Reductive TCA Pathway to Dynamically Regulate the Biosynthesis of Adipic Acid in

Adipic acid is a versatile aliphatic dicarboxylic acid. It is applied mainly in the polymerization of nylon-6,6, which accounts for 50.8% of the global consumption market of adipic acid. The microbial production of adipic acid avoids the usage of petroleum resources and the emission of harmful nitrogen oxides that are generated by traditional chemical synthetic approaches. However, in the fermentation process, the low theoretical yield and the usage of expensive inducers hinders the large-scale industrial production of adipic acid. To overcome these challenges, we established an oxygen-dependent dynamic regulation (ODDR) system to control the expression of key genes (sucD, pyc, mdh, and frdABCD) that could be induced to enhance the metabolic flux of the reductive TCA pathway under anaerobic conditions. Coupling of the constitutively expressed adipic acid synthetic pathway not only avoids the use of inducers but also increases the theoretical yield by nearly 50%. After the gene combination and operon structure were optimized, the reaction catalyzed by frdABCD was found to be the rate-limiting step. Further optimizing the relative expression levels of sucD, pyc, and frdABCD improved the titer of adipic acid 41.62-fold compared to the control strain Mad1415, demonstrating the superior performance of our ODDR system.