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

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.