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...
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| Format: | Article |
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Wiley
2025-06-01
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| Series: | Microbial Biotechnology |
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| Online Access: | https://doi.org/10.1111/1751-7915.70186 |
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| author | Yan‐Fang Nie Sheng‐Jie Yue Peng Huang Xue‐Hong Zhang Xiang‐Rui Hao Lian Jiang Hong‐Bo Hu |
| author_facet | Yan‐Fang Nie Sheng‐Jie Yue Peng Huang Xue‐Hong Zhang Xiang‐Rui Hao Lian Jiang Hong‐Bo Hu |
| author_sort | Yan‐Fang Nie |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-6c8012c2aba7424896cae7d4533ab08a |
| institution | Kabale University |
| issn | 1751-7915 |
| language | English |
| publishDate | 2025-06-01 |
| publisher | Wiley |
| record_format | Article |
| series | Microbial Biotechnology |
| spelling | doaj-art-6c8012c2aba7424896cae7d4533ab08a2025-08-20T03:27:43ZengWileyMicrobial Biotechnology1751-79152025-06-01186n/an/a10.1111/1751-7915.70186Investigations of the Flavin‐Dependent Monooxygenase PhzO Involved in Phenazine BiosynthesisYan‐Fang Nie0Sheng‐Jie Yue1Peng Huang2Xue‐Hong Zhang3Xiang‐Rui Hao4Lian Jiang5Hong‐Bo Hu6State Key Laboratory of Microbial Metabolism, and School of Life Sciences & Biotechnology Shanghai Jiao Tong University Shanghai ChinaState Key Laboratory of Microbial Metabolism, and School of Life Sciences & Biotechnology Shanghai Jiao Tong University Shanghai ChinaState Key Laboratory of Microbial Metabolism, and School of Life Sciences & Biotechnology Shanghai Jiao Tong University Shanghai ChinaState Key Laboratory of Microbial Metabolism, and School of Life Sciences & Biotechnology Shanghai Jiao Tong University Shanghai ChinaShanghai Jialeli Biotechnology Co., Ltd Shanghai ChinaJiangsu Good Harvest‐Weien Agrochemical Co., Ltd Qidong ChinaState Key Laboratory of Microbial Metabolism, and School of Life Sciences & Biotechnology Shanghai Jiao Tong University Shanghai ChinaABSTRACT 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.https://doi.org/10.1111/1751-7915.701863,4‐dihydroxy‐phenazine‐1‐carboxylic acid (3,4‐OH‐PCA)anticancer propertiesflavin reductase Fremonooxygenase PhzOphenazine biocatalysispromiscuous enzymatic activity |
| spellingShingle | Yan‐Fang Nie Sheng‐Jie Yue Peng Huang Xue‐Hong Zhang Xiang‐Rui Hao Lian Jiang Hong‐Bo Hu Investigations of the Flavin‐Dependent Monooxygenase PhzO Involved in Phenazine Biosynthesis Microbial Biotechnology 3,4‐dihydroxy‐phenazine‐1‐carboxylic acid (3,4‐OH‐PCA) anticancer properties flavin reductase Fre monooxygenase PhzO phenazine biocatalysis promiscuous enzymatic activity |
| title | Investigations of the Flavin‐Dependent Monooxygenase PhzO Involved in Phenazine Biosynthesis |
| title_full | Investigations of the Flavin‐Dependent Monooxygenase PhzO Involved in Phenazine Biosynthesis |
| title_fullStr | Investigations of the Flavin‐Dependent Monooxygenase PhzO Involved in Phenazine Biosynthesis |
| title_full_unstemmed | Investigations of the Flavin‐Dependent Monooxygenase PhzO Involved in Phenazine Biosynthesis |
| title_short | Investigations of the Flavin‐Dependent Monooxygenase PhzO Involved in Phenazine Biosynthesis |
| title_sort | investigations of the flavin dependent monooxygenase phzo involved in phenazine biosynthesis |
| topic | 3,4‐dihydroxy‐phenazine‐1‐carboxylic acid (3,4‐OH‐PCA) anticancer properties flavin reductase Fre monooxygenase PhzO phenazine biocatalysis promiscuous enzymatic activity |
| url | https://doi.org/10.1111/1751-7915.70186 |
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