Engineering Precursor and Cofactor Metabolism in Escherichia Coli for Enhanced Adipic Acid Production From Glucose

The reverse adipate degradation pathway (RADP) for adipic acid synthesis has garnered significant interest. However, the limited efficiency of existing pathways and their dependence on plasmids have hindered the practical application of microbial cell factories. In this study, the efficiency of the adipic acid synthetic pathway was enhanced by substituting and combinatorially expressing RADP enzymes. To obviate the need for chemical inducers and antibiotics, we integrated the reconstructed pathway genes into the genome of a succinic acid-producing strain Escherichia coli FMME N-26 and increased the copy number of rate-limiting enzymes. The supply of two critical precursors for adipic acid synthesis, acetyl-CoA and succinyl-CoA, was enhanced by deleting tdcD and overexpressing cat1. Additionally, cofactor metabolism was balanced through the overexpression of the udhA and dppD genes. Following these modifications, the adipic acid fermentation process was optimized in a 5 L bioreactor, resulting in a titer of 4.97 g/L after 72 h of fed-batch fermentation. This study lays a theoretical foundation and provides a technical basis for constructing cell factories to produce adipic acid and other dicarboxylic acids.