Heterologous Production of Phenazines in the Biocontrol Agent Lysobacter enzymogenes C3

Lysobacter enzymogenes, an environmental bacterium, holds promise as a biocontrol agent due to its ability to produce bioactive compounds effective against plant pathogens, such as fungi, oomycetes, and Gram-positive bacteria. However, it lacks activity against Gram-negative bacteria. To address this, we applied new genetic tools to manipulate the phenazine biosynthetic gene cluster (LaPhz) from L. antibioticus, converting L. enzymogenes to a robust producer of phenazine antibiotics. Through transcriptomics, we identified potent promoters and constructed the first ΦC31-mediated site-specific recombination system for Lysobacter. Engineered strains C3-cophz and C3-phz retained the ability to produce antifungal/antioomycete and anti-Gram-positive compounds while also synthesizing the well-known phenazine antibiotics such as phenazine dicarboxylic acid and phenazine carboxylic acid, along with new derivatives 1,6-dimethoxyphenazine and 1-hydroxy-6-methoxyphenazine-N10-oxide. These strains demonstrated potent activity against Gram-negative bacteria, showing promise for the development of versatile biopesticides. The new tools will facilitate the exploration of silent biosynthetic gene clusters in Lysobacter genomes.