Metabolic engineering of Pseudomonas chlororaphis P3 for high-level and directed production of phenazine-1,6-dicarboxylic acid from crude glycerol

Abstract

Phenazine-1,6-dicarboxylic acid (PDC) is a precursor of complex substituted phenazines used as pesticides and pharmaceuticals. The PDC biosynthesis exists the low production and the high proportion of by-products phenazine-1-carboxylic acid (PCA) derivatives in Pseudomonas P3△A. Herein, PDC production were improved by systematic metabolic engineering and synthetic regulation. The directed PDC biosynthesis was achieved by introducing the isozymes of PhzF', and PCA derivatives was barely detectable. Subsequently, a high-level PDC-producing strain P3FK2E-aF'EC was obtained by co-overexpression of aroE, phzE, phzC, and aphzF' in a multi-knockout strain. Through scale-up culture, the highest PDC production and proportion reached 6,447.05 mg/L and 99.68 %, with the productivity of 89.54 mg/L·h using KB. Economically, PDC production achieved 5,584.35 mg/L accounting for 99.43 % with the highest productivity of 108.32 mg/L·h from crude glycerol. This study first achieved the directed high-level production of PDC from renewable energy, and presented a potential biosynthesis platform for PDC derivatives in Pseudomonas.

Keywords: Crude glycerol; Directed biosynthesis; Phenazine-1,6-dicarboxylic acid; Pseudomonas chlororaphis; Systems metabolic engineering.