Metarhizium spp. encode an ochratoxin cluster and a high efficiency ochratoxin-degrading amidohydrolase revealed by genomic analysis

Gang Wang; Wenqing Wu; Nancy P Keller; Xu Guo; Erfeng Li; Junning Ma; Fuguo Xing

doi:10.1016/j.jare.2024.07.023

Metarhizium spp. encode an ochratoxin cluster and a high efficiency ochratoxin-degrading amidohydrolase revealed by genomic analysis

J Adv Res. 2024 Jul 31:S2090-1232(24)00308-4. doi: 10.1016/j.jare.2024.07.023. Online ahead of print.

Authors

Gang Wang¹, Wenqing Wu², Nancy P Keller³, Xu Guo⁴, Erfeng Li⁵, Junning Ma⁶, Fuguo Xing⁷

Affiliations

¹ Key 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. Electronic address: wanggang02@caas.cn.
² Horticulture and Landscape College, Tianjin Agricultural University, Tianjin 300392, PR China. Electronic address: W18622586026@163.com.
³ Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI 53706, USA. Electronic address: npkeller@wisc.edu.
⁴ Horticulture and Landscape College, Tianjin Agricultural University, Tianjin 300392, PR China. Electronic address: guoxu525529@163.com.
⁵ Horticulture and Landscape College, Tianjin Agricultural University, Tianjin 300392, PR China. Electronic address: lef143@126.com.
⁶ Key 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. Electronic address: junning.ma@hotmail.com.
⁷ Key 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. Electronic address: xingfuguo@caas.cn.

PMID: 39089618
DOI: 10.1016/j.jare.2024.07.023

Abstract

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.

Keywords: Detoxification; Mycotoxin; OTA; Ochratoxigenic fungi; Secondary metabolite.