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Trans-2,3-dihydro-3-hydroxyanthranilic acid (DHHA) is a cyclic β-amino acid used for the synthesis of non-natural peptides and chiral materials. And it is an intermediate product of phenazine production in  Pseudomonas spp . Lzh-T5 is a  P. chlororaphis  strain isolated from tomato rhizosphere found in China. It can synthesize three antifungal phenazine compounds. Disruption the  phzF  gene of  P. chlororaphis  Lzh-T5 results in DHHA accumulation. Several strategies were used to improve production of DHHA: enhancing the shikimate pathway by overexpression, knocking out negative regulatory genes, and adding metal ions to the medium. In this study, three regulatory genes ( psrA ,  pykF,  and  rpeA ) were disrupted in the genome of  P. chlororaphis  Lzh-T5, yielding 5.52 g/L of DHHA. When six key genes selected from the shikimate, pentose phosphate, and gluconeogenesis pathways were overexpressed, the yield of DHHA increased to 7.89 g/L. Lastly, a different concentration of Fe 3+  was added to the medium for DHHA fermentation. This genetically engineered strain increased the DHHA production to 10.45 g/L. According to our result,  P. chlororaphis  Lzh-T5 could be modified as a microbial factory to produce DHHA. This study laid a good foundation for the future industrial production and application of DHHA.

Genetic engineering of Pseudomonas chlororaphis Lzh-T5 to enhance production of trans-2,3-dihydro-3-hydroxyanthranilic acid