Metabolic design of a platform Pseudomonas strain producing various phenazine derivatives

Phenazine derivatives, a class of nitrogen-containing heterocyclic compounds, exhibit broad-spectrum antifungal, anticancer, and antimalarial activities. Pseudomonas and Streptomyces are the primary microbial strains responsible for the synthesis of phenazine derivatives. In general, Pseudomonas strains use phenazine-1-carboxylic acid (PCA) as a precursor for enzymatic modification, while Streptomyces strains employ phenazine-1,6-dicarboxylic acid (PDC) as the precursor. Pseudomonas is considered an ideal platform for the efficient biosynthesis of various phenazine derivatives due to its rapid growth rate, ease of genetic manipulation, and well-established fermentation systems. However, the synthesis of phenazine derivatives in Pseudomonas largely relies on previously reported natural biosynthetic pathways from other microbial strains. The biosynthesis of phenazine derivatives through unknown pathways often presents significant challenges for researchers. The concept of combinatorial biosynthesis offers a promising solution to overcome these difficulties. In this study, we designed and constructed a platform Pseudomonas strain producing 15 phenazine derivatives by exchanging and combining the modifying enzymes of PCA and PDC, besides 16 constructed modification pathways. Among these, three derivatives feature novel chemical structures, while 13 represent previously unreported biosynthetic pathways. With the discovery of new phenazine modifying enzymes, they can be quickly incorporated into our platform, enabling the rapid synthesis of a wide variety of phenazine derivatives. This work demonstrates the potential of designing non-natural metabolic pathways to enable the production of diverse phenazine derivatives, thereby enhancing bacterial capacity for the synthesis of high-value phenazine compounds. This combinatorial biosynthetic approach provides a potential alternative for exploring unknown biosynthetic routes and for the development of unexplored natural biosynthetic pathways for phenazine derivatives.