Biosynthetic pathway construction and production enhancement of phenazine-1-carboxylic acid derivatives in Pseudomonas chlororaphis H18

BACKGROUND: Phenazines represent a promising class of natural products with diverse biological activities, including antitumor, antiparasitic and antibacterial properties. Among them, phenazine-1-carboxylic acid (PCA)-a registered biopesticide-stands out for its broad-spectrum antifungal efficacy, low environmental toxicity and potential as a lead structure for developing anti-tumor and antituberculosis agents. However, the chemical synthesis of PCA derivatives is often hindered by harsh reaction conditions, toxic byproducts and poor selectivity, limiting their sustainable production. In contrast, biosynthesis emerges as a green and scalable alternative, yet efficient microbial platforms for structurally diverse PCA derivatives remain underdeveloped. RESULTS: To address this gap, we engineered Pseudomonas chlororaphis H18 into a versatile cell factory for PCA derivative biosynthesis. We first characterized the substrate promiscuity of the S-adenosylmethionine (SAM)-dependent methyltransferase Pcm2, demonstrating its capacity to catalyze methylation and ethylation of many aromatic carboxylic acids. This catalytic versatility broadens the scope of PCA derivatives that can be accessed through biosynthesis. By integrating the pcm2 gene into the P. chlororaphis H18 genome, we established de novo pathways for two key derivatives: phenazine-1-carboxymethyl (PCM) and 2-hydroxy-phenazine-1-carboxymethyl (2-OH-PCM) for the first time, with PCM having been reported to exhibit superior fungicidal activity against the tobacco brown spot pathogen, Alternaria alternata, compared to PCA. To enhance titers of PCM and 2-OH-PCM, we implemented a systematic metabolic engineering strategy comprising: (1) increasing intracellular SAM supply, (2) overexpressing key genes, (3) knocking out the negative regulators and (4) disrupting competitive pathway. The engineered strain H18A4 produced 124.6 mg/L PCM and 130.8 mg/L 2-OH-PCM in KB medium. To further increase production of PCM and prevent PCA hydroxylation, we disrupted the phzO gene to block conversion to hydroxylated derivatives, increased pcm2 gene copy number and optimized the fermentation medium. The resulting engineered strain H18A6 produced 1.37 g/L PCM in fed-batch fermentation-a marked advance toward scalable, bio-based production of this high-value compound. CONCLUSIONS: This study fills a technical gap by establishing a novel biosynthetic route for diverse PCA derivatives, overcoming the limitations of chemical synthesis and expanding the scope of sustainable natural product production. And the integrated metabolic engineering strategy (spanning enzyme characterization, pathway construction, metabolic flux optimization and process refinement) validated herein offers a reusable template for engineering microbial cell factories for complex high-value natural products. Ultimately, this study contributes to the green production of functionally complex phenazine-based compounds for agricultural and pharmaceutical applications.