Investigations of the Flavin‐Dependent Monooxygenase PhzO Involved in Phenazine Biosynthesis
ABSTRACT Phenazines are bioactive secondary metabolites with antifungal, anticancer, and insecticidal properties, while hydroxylated derivatives often exhibit enhanced bioactivity. 2‐hydroxyphenazine (2‐OH‐PHZ), which is synthesised by the flavin‐dependent monooxygenase PhzO from phenazine‐1‐carboxy...
I tiakina i:
| Ngā kaituhi matua: | , , , , , , |
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| Hōputu: | Tuhinga |
| Reo: | Ingarihi |
| I whakaputaina: |
Wiley
2025-06-01
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| Rangatū: | Microbial Biotechnology |
| Ngā marau: | |
| Urunga tuihono: | https://doi.org/10.1111/1751-7915.70186 |
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| Whakarāpopototanga: | ABSTRACT Phenazines are bioactive secondary metabolites with antifungal, anticancer, and insecticidal properties, while hydroxylated derivatives often exhibit enhanced bioactivity. 2‐hydroxyphenazine (2‐OH‐PHZ), which is synthesised by the flavin‐dependent monooxygenase PhzO from phenazine‐1‐carboxylic acid (PCA), shows better bioactivity against the pathogenic fungus Gaeumannomyces graminis vars. tritici. However, the low catalytic efficiency (10%–20% conversion) of PhzO limited 2‐OH‐PHZ production. To boost PhzO activity, engineering flavin reductase (Fre)‐mediated FADH2 regeneration was applied to Pseudomonas chlororaphis LX24AE. Remarkably, this approach improved catalytic efficiency from 25% to 40% and increased the production of a novel dihydroxylated derivative. Then, it was first characterised by UPLC‐MS and NMR, and identified as 3,4‐dihydroxyphenazine‐1‐carboxylic acid (3,4‐OH‐PCA). Next, the Fre‐PhzO module through heterologous co‐expression in P. putida KT2440 demonstrated a 4.5‐fold enhancement in hydroxylation efficiency relative to the PhzO mono‐component system, which also confirmed that PhzO and flavin reductase are essential for 3,4‐OH‐PCA biosynthesis. Moreover, in vitro assays further verified that PhzO exhibits FAD‐dependent catalytic promiscuity, simultaneously generating 2‐OH‐PCA and 3,4‐OH‐PCA. Furthermore, in vitro and in vivo assays demonstrated that substrate concentration affected the distribution of products. Finally, cytotoxicity evaluation of the isolated 3,4‐OH‐PCA was performed, and it showed substantial inhibition against oesophageal cancer TE‐1 cells and human cervical cancer HeLa cells with an IC50 value of 8.55 μM and 17.69 μM, respectively. This work redefines PhzO as a promiscuous biocatalyst capable of dual hydroxylation, offering a modular platform for engineering bioactive phenazine derivatives. |
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| ISSN: | 1751-7915 |