Metarhizium spp. encode an ochratoxin cluster and a high efficiency ochratoxin-degrading amidohydrolase revealed by genomic analysis
Introduction: Ochratoxins (OTs) are worldwide regulated mycotoxins contaminating a variety of food-environment and agro-environment. Several Aspergillus and Pencillium species synthesize OTs from a six-gene biosynthetic gene cluster (BGC) to produce the highly toxic final product OTA. Although many...
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Elsevier
2025-06-01
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| Series: | Journal of Advanced Research |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2090123224003084 |
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| author | Gang Wang Wenqing Wu Nancy P. Keller Xu Guo Erfeng Li Junning Ma Fuguo Xing |
| author_facet | Gang Wang Wenqing Wu Nancy P. Keller Xu Guo Erfeng Li Junning Ma Fuguo Xing |
| author_sort | Gang Wang |
| collection | DOAJ |
| description | Introduction: Ochratoxins (OTs) are worldwide regulated mycotoxins contaminating a variety of food-environment and agro-environment. Several Aspergillus and Pencillium species synthesize OTs from a six-gene biosynthetic gene cluster (BGC) to produce the highly toxic final product OTA. Although many studies on OTA-degrading enzymes were performed, high efficiency enzymes with strong stability are extremely needed, and the OTA degrading mechanism is poorly understood. Objectives: The study aimed to explore the OT-degradation enzyme and investigate its degradation mechanisms in Metarhizium, which contain an OT biosynthetic gene cluster. Methods: Phylogenomic relationship combined with RNA expression analysis were used to explore the distribution of OT BGC in fungi. Bioactivity-guided isolation and protein mass spectrometry were conducted to trace the degrading enzymes in Metarhizium spp., and the enzymes were heterologously expressed in E. coli and verified by in vitro assays. Structure prediction and point mutation were performed to reveal the catalytic mechanism of MbAmh1. Results: Beyond Aspergillus and Pencillium species, three species of the distant phylogenetic taxon Metarhizium contain an expressed OT-like BGC but lack an otaD gene. Unexpectedly, no OT BGC products were found in some Metarhizium species. Instead, Metarhizium metabolized both OTA and OTB to their non-toxic degradation products. This activity of M. brunneum was attributed to an intracellular hydrolase MbAmh1, which was tracked by bioactivity-guided proteomic analysis combined with in vitro reaction. Recombinant MbAmh1 (5 μg/mL) completely degraded 1 μg/mL OTA within 3 min, demonstrating a strong degrading ability towards OTA. Additionally, MbAmh1 showed considerable temperature adaptability ranging from 30 to 70 °C and acidic pH stability ranging from 4.0 to 7.0. Identification of active sites supported the crucial role of metal iron for this enzymatic reaction. Conclusion: These findings reveal different patterns of OT synthesis in fungi and provide a potential OTA degrading enzyme for industrial applications. |
| format | Article |
| id | doaj-art-e00fd0ddb08b403ba7bd5e98ebdae281 |
| institution | OA Journals |
| issn | 2090-1232 |
| language | English |
| publishDate | 2025-06-01 |
| publisher | Elsevier |
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| series | Journal of Advanced Research |
| spelling | doaj-art-e00fd0ddb08b403ba7bd5e98ebdae2812025-08-20T01:55:27ZengElsevierJournal of Advanced Research2090-12322025-06-0172859510.1016/j.jare.2024.07.023Metarhizium spp. encode an ochratoxin cluster and a high efficiency ochratoxin-degrading amidohydrolase revealed by genomic analysisGang Wang0Wenqing Wu1Nancy P. Keller2Xu Guo3Erfeng Li4Junning Ma5Fuguo Xing6Key Laboratory of Agro-Products Quality and Safety Control in Storage and Transport Process, Ministry of Agriculture and Rural Affairs, Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences, Beijing 100193, PR ChinaHorticulture and Landscape College, Tianjin Agricultural University, Tianjin 300392, PR ChinaDepartment of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI 53706, USAHorticulture and Landscape College, Tianjin Agricultural University, Tianjin 300392, PR ChinaHorticulture and Landscape College, Tianjin Agricultural University, Tianjin 300392, PR ChinaKey Laboratory of Agro-Products Quality and Safety Control in Storage and Transport Process, Ministry of Agriculture and Rural Affairs, Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences, Beijing 100193, PR ChinaKey Laboratory of Agro-Products Quality and Safety Control in Storage and Transport Process, Ministry of Agriculture and Rural Affairs, Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences, Beijing 100193, PR China; Corresponding author at: No. 2 Yuanmingyuan West Road, Haidian District, Beijing 100193, PR China.Introduction: Ochratoxins (OTs) are worldwide regulated mycotoxins contaminating a variety of food-environment and agro-environment. Several Aspergillus and Pencillium species synthesize OTs from a six-gene biosynthetic gene cluster (BGC) to produce the highly toxic final product OTA. Although many studies on OTA-degrading enzymes were performed, high efficiency enzymes with strong stability are extremely needed, and the OTA degrading mechanism is poorly understood. Objectives: The study aimed to explore the OT-degradation enzyme and investigate its degradation mechanisms in Metarhizium, which contain an OT biosynthetic gene cluster. Methods: Phylogenomic relationship combined with RNA expression analysis were used to explore the distribution of OT BGC in fungi. Bioactivity-guided isolation and protein mass spectrometry were conducted to trace the degrading enzymes in Metarhizium spp., and the enzymes were heterologously expressed in E. coli and verified by in vitro assays. Structure prediction and point mutation were performed to reveal the catalytic mechanism of MbAmh1. Results: Beyond Aspergillus and Pencillium species, three species of the distant phylogenetic taxon Metarhizium contain an expressed OT-like BGC but lack an otaD gene. Unexpectedly, no OT BGC products were found in some Metarhizium species. Instead, Metarhizium metabolized both OTA and OTB to their non-toxic degradation products. This activity of M. brunneum was attributed to an intracellular hydrolase MbAmh1, which was tracked by bioactivity-guided proteomic analysis combined with in vitro reaction. Recombinant MbAmh1 (5 μg/mL) completely degraded 1 μg/mL OTA within 3 min, demonstrating a strong degrading ability towards OTA. Additionally, MbAmh1 showed considerable temperature adaptability ranging from 30 to 70 °C and acidic pH stability ranging from 4.0 to 7.0. Identification of active sites supported the crucial role of metal iron for this enzymatic reaction. Conclusion: These findings reveal different patterns of OT synthesis in fungi and provide a potential OTA degrading enzyme for industrial applications.http://www.sciencedirect.com/science/article/pii/S2090123224003084Secondary metaboliteMycotoxinOTADetoxificationOchratoxigenic fungi |
| spellingShingle | Gang Wang Wenqing Wu Nancy P. Keller Xu Guo Erfeng Li Junning Ma Fuguo Xing Metarhizium spp. encode an ochratoxin cluster and a high efficiency ochratoxin-degrading amidohydrolase revealed by genomic analysis Journal of Advanced Research Secondary metabolite Mycotoxin OTA Detoxification Ochratoxigenic fungi |
| title | Metarhizium spp. encode an ochratoxin cluster and a high efficiency ochratoxin-degrading amidohydrolase revealed by genomic analysis |
| title_full | Metarhizium spp. encode an ochratoxin cluster and a high efficiency ochratoxin-degrading amidohydrolase revealed by genomic analysis |
| title_fullStr | Metarhizium spp. encode an ochratoxin cluster and a high efficiency ochratoxin-degrading amidohydrolase revealed by genomic analysis |
| title_full_unstemmed | Metarhizium spp. encode an ochratoxin cluster and a high efficiency ochratoxin-degrading amidohydrolase revealed by genomic analysis |
| title_short | Metarhizium spp. encode an ochratoxin cluster and a high efficiency ochratoxin-degrading amidohydrolase revealed by genomic analysis |
| title_sort | metarhizium spp encode an ochratoxin cluster and a high efficiency ochratoxin degrading amidohydrolase revealed by genomic analysis |
| topic | Secondary metabolite Mycotoxin OTA Detoxification Ochratoxigenic fungi |
| url | http://www.sciencedirect.com/science/article/pii/S2090123224003084 |
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