Wang Gang, Wu Wenqing, Keller Nancy P, Guo Xu, Li Erfeng, Ma Junning, Xing Fuguo
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.
Horticulture and Landscape College, Tianjin Agricultural University, Tianjin 300392, PR China.
J Adv Res. 2025 Jun;72:85-95. doi: 10.1016/j.jare.2024.07.023. Epub 2024 Jul 31.
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.
The study aimed to explore the OT-degradation enzyme and investigate its degradation mechanisms in Metarhizium, which contain an OT biosynthetic gene cluster.
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.
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.
These findings reveal different patterns of OT synthesis in fungi and provide a potential OTA degrading enzyme for industrial applications.
赭曲霉毒素（OTs）是一种受全球监管的霉菌毒素，污染了各种食品环境和农业环境。几种曲霉属和青霉属物种通过一个六基因生物合成基因簇（BGC）合成OTs，以产生剧毒的最终产物OTA。尽管已经对OTA降解酶进行了许多研究，但极其需要具有强稳定性的高效酶，并且对OTA降解机制了解甚少。
本研究旨在探索OT降解酶，并研究其在含有OT生物合成基因簇的绿僵菌中的降解机制。
利用系统发育基因组关系结合RNA表达分析来探索OT BGC在真菌中的分布。进行生物活性导向分离和蛋白质质谱分析以追踪绿僵菌属中的降解酶，这些酶在大肠杆菌中进行异源表达并通过体外试验进行验证。进行结构预测和点突变以揭示MbAmh1的催化机制。
除了曲霉属和青霉属物种外，远缘系统发育分类单元绿僵菌的三个物种含有一个表达的OT样BGC，但缺少otaD基因。出乎意料的是，在一些绿僵菌物种中未发现OT BGC产物。相反，绿僵菌将OTA和OTB都代谢为无毒降解产物。球孢绿僵菌的这种活性归因于一种细胞内水解酶MbAmh1，它是通过生物活性导向的蛋白质组分析结合体外反应追踪到的。重组MbAmh1（5μg/mL）在3分钟内完全降解了1μg/mL OTA，显示出对OTA有很强的降解能力。此外，MbAmh1在30至70°C范围内表现出相当的温度适应性，在4.0至7.0的酸性pH范围内表现出稳定性。活性位点的鉴定支持了金属铁在该酶促反应中的关键作用。
这些发现揭示了真菌中OT合成的不同模式，并为工业应用提供了一种潜在的OTA降解酶。
