Biocatalytic synthesis of alkoxylated phenazine derivatives using methyltransferases

BACKGROUND: Phenazines are important nitrogen-containing heterocycles with diverse applications in the chemical and pharmaceutical industry. Alkoxylated phenazines, in particular, exhibit promising acaricidal and fungicidal properties. Currently, chemical synthesis is the main approach for alkoxylated phenazines and derivatives production. However, these processes are associated with harsh reaction conditions, accumulation of chemical waste (e.g., organic solvents, noble metal catalysts), and environmental concerns. RESULTS: In this study, we developed biocatalytic systems for the synthesis of alkoxylated phenazines by designing enzymatic systems and de novo biosynthetic pathways. The O-methyltransferase LaphzM from Lysobacter antibioticus OH13 was shown to catalyze the alkoxylation of phenazines using SAM analogs generated in situ by halide methyltransferases (HMTs) from Burkholderia xenovorans and Arabidopsis thaliana. Using these enzymatic systems, we successfully synthesized six alkoxylated phenazine derivatives, including three novel compounds. We further established de novo biosynthetic pathways for 1-ethoxyphenazine and 2-ethoxyphenazine in Pseudomonas chlororaphis via the direct sulfurylation pathway from S. cerevisiae. To improve production, we optimized whole-cell systems, achieving 486.3 mg/L (54% yield) of 2-ethoxyphenazine, 218.1 mg/L (22.8% yield) of 2-propoxyphenazine and 378.2 mg/L (78.4% yield) of 1-ethoxyphenazine-N'10-oxide within 7 h using microbially produced 2-hydroxyphenazine (for 2-ethoxyphenazine and 2-propoxyphenazine) or 1-hydroxyphenazine (for 1-ethoxyphenazine-N'10-oxide) as the substrate, along with ethyl iodide (EtI) or propyl iodide (PrI) accordingly. CONCLUSIONS: Overall, we successfully established biocatalytic platforms for alkoxylated phenazines through enzymatic catalysis and de novo biosynthetic pathways. Methyltransferases derived from diverse species, including LaphzM and HMTs, play key roles in the SAM-analog-mediated alkylation of phenazines. This study provides a promising alternative to conventional chemical synthesis, with significant potential for the manufacturing of alkoxylated phenazine derivatives.