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

Kaiquan Liu,Ling Li,Wei Wang,Ruiming Wang,Yujie Huang,Piwu Li,Wentao Yao

doi:10.1038/s41598-021-94674-8

Abstract

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 Fe3+ 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.

Highlights

Trans 2,3-dihydro-3-hydroxyanthranilic acid (DHHA) can be used in cycloaddition reactions as an enantiomerically pure building block, for biosynthesis of unnatural peptides, and for preparation of various useful intermediate acid derivatives of benzoic acid, such as 3-hydroxyanthranilic acid and anthranilic acid, which are important aromatic compounds[1,2,3,4]
All Escherichia coli strains were cultured in LB medium at 37 °C, and all P. chlororaphis strains were cultured in KB medium at 30 °C
After 48 h of culture, the P. chlororaphis LDA-1 strain on an agar plate turns milky; the colony color of Lzh-T5 remains orange. This suggests that the phenazine derivatives could not be synthesized in LDA-1

Summary

Introduction

Trans 2,3-dihydro-3-hydroxyanthranilic acid (DHHA) can be used in cycloaddition reactions as an enantiomerically pure building block, for biosynthesis of unnatural peptides, and for preparation of various useful intermediate acid derivatives of benzoic acid, such as 3-hydroxyanthranilic acid and anthranilic acid, which are important aromatic compounds[1,2,3,4]. These compounds are widely used in chemicals, food, cosmetics, and pharmaceuticals. Lzh-T5 is a P. chlororaphis strain isolated from the tomato rhizosphere located in China It has a phenazine biosynthesis cluster phzABCDEFG, and could produce phenazine-1-carboxylic acid (PCA) and other phenazine derivatives[15]. A final DHHA yield of 10.45 g/L was obtained

Methods

Results

Conclusion