Overproduction of Phenolic Compounds in Pseudomonas putida KT2440 Through Endogen Deregulation of the Shikimate Pathway

Metabolic engineering of the shikimate pathway offers a promising strategy for enhancing the production of aromatic compounds in microbial hosts. However, feedback inhibition of key enzymes, such as the 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHP synthase), often limits the yield of target products. In this study, we focused on the DAHP synthase (AroF-I) from Pseudomonas putida. Through computational modeling and experimental validation, we identified specific amino-acid residues responsible for tyrosine-mediated feedback inhibition. By targeted mutagenesis, we engineered DAHP synthase variants that exhibit reduced sensitivity to feedback inhibition. The introduction of these engineered enzymes into a metabolically engineered Pseudomonas putida strain resulted in significantly increased production of p-coumaric acid. Our findings provide valuable insights into the regulation of the shikimate pathway and demonstrate the potential of protein engineering to improve microbial production of aromatic compounds.