Engineering the central biosynthetic and secondary metabolic pathways of Pseudomonas aeruginosa strain PA1201 to improve phenazine-1-carboxylic acid production

The secondary metabolite phenazine-1-carboxylic acid (PCA) is an important component of the newly registered biopesticide Shenqinmycin. We used a combined method involving gene, promoter, and protein engineering to modify the central biosynthetic and secondary metabolic pathways in the PCA-producing Pseudomonas aeruginosa strain PA1201. The PCA yield of the resulting strain PA-IV was increased 54.6-fold via the following strategies: (1) blocking PCA conversion and enhancing PCA efflux pumping; (2) increasing metabolic flux towards the PCA biosynthetic pathway through the over-production of two DAHP synthases and blocking the synthesis of 21 secondary metabolites; (3) increasing the PCA precursor supply through the engineering of five chorismate-utilizing enzymes; (4) engineering the promoters of two PCA biosynthetic gene clusters. Strain PA-IV produced 9882mg/L PCA in fed-batch fermenation, which is twice as much as that produced by the current industrial strain. Strain PA-IV was also genetically stable and comparable to Escherichia coli in cytotoxicity. •The secondary metabolite phenazine-1-carboxylic acid (PCA) is active against a wide range of fungal plant pathogens, and was developed as a new biopesticide.•The PCA-producing strain PA1201 was modified via a combined strategy involving gene deletion and replacement, promoter engineering and protein engineering.•A total of 478 genes responsible for virulence factor production, secondary metabolite production, PCA biosynthesis and efflux pumping were engineered.•The genetically stable strain PA-IV exhibited a 54.6-fold PCA yield increase over PA1201, producing 9882mg/L of PCA in fed-batch fermentation.