Genetic Engineering of Pseudomonas Chlororaphis Lzh-T5 to Enhance Production of Trans-2,3-Dihydro-3-Hydroxyanthranilic Acid

Abstract

<jats:title>Abstract</jats:title> <jats:p><jats:bold>Background:</jats:bold> 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. 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. <jats:bold>Results:</jats:bold> Disrupting 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 4.55 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 6.89g/L. Fe 3+ was added to the medium for DHHA fermentation. This genetically engineered strain increased the DHHA production to 10.45g/L. <jats:bold>Conclusions:</jats:bold> P. chlororaphis Lzh-T5 could be modified as a microbial factory to produce DHHA by inactivating phzF , disrupting negative regulatory genes, overexpressing key genes, and adding metal ions to medium for fermentation.</jats:p>