Oil Crops Research Institute , Chinese Academy of Agricultural Sciences , Wuhan 430061 , People's Republic of China.
Key Laboratory of Biology and Genetic Improvement of Oil Crops , Ministry of Agriculture , Wuhan 430061 , People's Republic of China.
Anal Chem. 2018 Dec 18;90(24):14331-14338. doi: 10.1021/acs.analchem.8b03703. Epub 2018 Dec 3.
Aflatoxins, highly toxic and carcinogenic to humans, are synthesized via multiple intermediates by a complex pathway in several Aspergilli, including Aspergillus flavus. Few analytical methods are available for monitoring the changes in metabolite profiles of the aflatoxin biosynthesis pathway under different growth and environmental conditions. In the present study, we developed by a D-optimal mixture design a solvent system, methanol/dichloromethane/ethyl acetate/formic acid (0.36/0.31/0.32/0.01), that was suitable for extracting the pathway metabolites. The matrix effect from dilution of cell extracts was negligible. To facilitate the identification of these metabolites, we constructed a fragmentation ion library. We further employed liquid chromatography coupled with high-resolution mass spectroscopy (UHPLC-HRMS) for simultaneous quantification of the metabolites. The limit of detection (LOD) and limit of quantitation (LOQ) were 0.002-0.016 and 0.008-0.05 μg/kg, respectively. The spiked recovery rates ranged from 81.3 to 100.3% with intraday and interday precision less than 7.6%. Using the method developed to investigate the time-course aflatoxin biosynthesis, we found that precursors, including several possible toxins (with a carcinogenic group similar to aflatoxin B), occurred together with aflatoxin, and that production increased rapidly at the early growth stage, peaked on day four, and then decreased substantially. The maximum production of aflatoxin B and aflatoxin B occurred 1 day later. Moreover, the dominant branch pathway was the one for aflatoxin B formation. We revealed that the antiaflatoxigenicity mechanism of Leclercia adecarboxylata WT16 was associated with a factor upstream of the aflatoxin biosynthesis pathway. The design strategies can be applied to characterize or detect other secondary metabolites to provide a snapshot of the dynamic changes during their biosynthesis.
黄曲霉毒素是一种对人类具有高度毒性和致癌性的物质，它是由几种曲霉（包括黄曲霉）通过一个复杂的途径，利用多种中间产物合成的。目前，几乎没有分析方法可以监测到不同生长和环境条件下，黄曲霉毒素生物合成途径中代谢产物谱的变化。本研究通过 D-最优混合设计，开发了一种适合提取途径代谢产物的溶剂系统（甲醇/二氯甲烷/乙酸乙酯/甲酸，0.36/0.31/0.32/0.01）。细胞提取物稀释的基质效应可以忽略不计。为了便于鉴定这些代谢产物，我们构建了一个碎裂离子文库。我们进一步采用高效液相色谱-高分辨率质谱（UHPLC-HRMS）对代谢产物进行同时定量。检测限（LOD）和定量限（LOQ）分别为 0.002-0.016 和 0.008-0.05μg/kg。加标回收率范围为 81.3%-100.3%，日内和日间精密度均小于 7.6%。使用所建立的方法研究了黄曲霉毒素生物合成的时程，我们发现包括几种可能的毒素（具有与黄曲霉毒素 B 相似的致癌基团）在内的前体与黄曲霉毒素一起产生，并且在早期生长阶段迅速增加，在第四天达到峰值，然后大幅下降。黄曲霉毒素 B 和黄曲霉毒素 B 的最大产量出现在一天后。此外，主要分支途径是形成黄曲霉毒素 B 的途径。我们揭示了 Leclercia adecarboxylata WT16 的抗黄曲霉毒素机制与黄曲霉毒素生物合成途径上游的一个因素有关。这些设计策略可以应用于其他次生代谢产物的特征描述或检测，以提供其生物合成过程中动态变化的快照。
