Production of trans-2,3-dihydro-3-hydroxyanthranilic acid by engineered Pseudomonas chlororaphis GP72

Trans-2,3-dihydro-3-hydroxyanthranilic acid (DHHA) is a cyclic β-amino acid that can be used for the synthesis of chiral materials and nonnatural peptides. The aim of this study was to accumulate DHHA by engineering Pseudomonas chlororaphis GP72, a nonpathogenic strain that produces phenazine-1-carboxylic acid and 2-hydroxyphenazine. First, the phzF deletion mutant DA1 was constructed, which produced 1.91 g/L DHHA. Moreover, rpeA and pykF were disrupted and then ppsA and tktA were co-expressed in strain DA1. The resulting strain DA4 increased DHHA concentration to 4.98 g/L, which is 2.6-fold than that of DA1. The effects of the addition of glucose, glycerol, L-tryptophan, and Fe³⁺on DHHA production were also investigated. Strain DA4 produced 7.48 g/L of DHHA in the culture medium in the presence of 12 g/L glucose and 3 mM Fe³⁺, which was 1.5-fold higher than the strain in the original fermentation conditions. These results indicate the potential of P. chlororaphis GP72 as a DHHA producer.