Controlling the Production of Pseudomonas Phenazines by Modulating the Genetic Repertoire

Microbial phenazines are getting increasing attention for antimicrobial and biotechnological applications. Phenazine production of the most well-known producer Pseudomonas aeruginosa is subject to a highly complex regulation network involving both quorum sensing and catabolite repression. These networks affect the expression of the two redundant phz gene operons responsible for phenazine-1-carboxylate (PCA) production and two specific genes phzM and phzS necessary for pyocyanin production. To decipher the specific functionality of these genes, in this study, specific phenazine gene deletion mutants of P. aeruginosa PA14 were generated and characterized in glucose and 2,3-butanediol media. Phenazine concentration and expression levels of the remaining genes were analyzed in parallel experiments. The findings suggest a strong dominance of operon phzA2-G2 resulting in a 10-fold higher expression of phz2 compared to phzA1-G1 and almost exclusive production of PCA from this operon. The genes phzM and phzS seem to exhibit antagonistic function in phenazine production. An upregulation of phzM explains the documented enhanced pyocyanin production in a 2,3-butanediol medium. Applied to a bioelectrochemical system, the altered phenazine production of the mutant strains is directly translated into current generation. Additionally, the deletion of the phenazine genes induced the activation of alternative energy pathways, which resulted in the accumulation of various fermentation products. Overall, modulating the genetic repertoire of the phenazine genes tremendously affects phenazine production levels, which are naturally kept in tight homeostasis in the P. aeruginosa wildtype. This important information can be directly utilized for ongoing efforts of heterologous biotechnological phenazine production.