Metarhizium spp. encode an ochratoxin cluster and a high efficiency ochratoxin-degrading amidohydrolase revealed by genomic analysis,Journal of Advanced Research

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Metarhizium spp. encode an ochratoxin cluster and a high efficiency ochratoxin-degrading amidohydrolase revealed by genomic analysis
Journal of Advanced Research ( IF 11.4 ) Pub Date : 2024-07-31 , DOI: 10.1016/j.jare.2024.07.023
Gang Wang ₁ , Wenqing Wu ₂ , Nancy P Keller ₃ , Xu Guo ₂ , Erfeng Li ₂ , Junning Ma ₁ , Fuguo Xing ₁

Affiliation

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.

中文翻译：

Metarhizium spp. 编码一个赭曲霉毒素簇和一个基因组分析揭示的高效赭曲霉毒素降解酰胺水解酶

介绍

赭曲霉毒素（OTs）是世界范围内受监管的霉菌毒素，会污染各种食品环境和农业环境。几种曲霉属和 Pencillium 物种从六基因生物合成基因簇（BGC）合成 OT 以产生剧毒的最终产品 OTA。尽管对 OTA 降解酶进行了大量研究，但极需要稳定性强的高效酶，而 OTA 降解机制知之甚少。

目标

该研究旨在探索 OT 降解酶并研究其在包含 OT 生物合成基因簇的 Metarhizium 中的降解机制。

方法

采用系统发育关系结合 RNA 表达分析探讨 OT BGC 在真菌中的分布。采用生物活性引导分离和蛋白质质谱法追踪 Metarhizium spp. 中的降解酶，酶在大肠杆菌中异源表达，并通过体外测定验证。进行结构预测和点突变以揭示 MbAmh1 的催化机制。

结果

除了 Aspergillus 和 Pencillium 物种之外，远距离系统发育分类单元 Metarhizium 的三个物种包含表达的 OT 样 BGC，但缺乏 otaD 基因。出乎意料的是，在某些 Metarhizium 物种中没有发现 OT BGC 产品。相反，Metarhizium 将 OTA 和 OTB 代谢成无毒的降解产物。M. brunneum 的这种活性归因于细胞内水解酶 MbAmh1，通过生物活性引导的蛋白质组学分析结合体外反应进行跟踪。重组 MbAmh1 （5 μg/mL）在 3 分钟内完全降解了 1 μg/mL OTA，表现出较强的 OTA 降解能力。此外，MbAmh1 在 30 至 70 °C 范围内表现出相当大的温度适应性，在 4.0 至 7.0 的酸性 pH 稳定性范围内。活性位点的鉴定支持金属铁在这种酶促反应中的关键作用。