Monitoring metabolite production of aflatoxin biosynthesis by orbitrap fusion mass spectrometry and a D-optimal mixture design method

Authors: XIE, HUALI - Oil Crops Research Institute - China; WANG, XIUPIN - Oil Crops Research Institute - China; ZHANG, LIANGXIAO - Oil Crops Research Institute - China; WANG, TONG - Oil Crops Research Institute - China; ZHANG, WEN - Oil Crops Research Institute - China; JIANG, JUN - Oil Crops Research Institute - China; Chang, Perng Kuang; CHEN, ZHI-YUAN - Louisiana State University; Bhatnagar, Deepak; ZHANG, QI - Oil Crops Research Institute - China; LI, PEIWU - Oil Crops Research Institute - China

Submitted to: Analytical Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/16/2018
Publication Date: 11/16/2018
Citation: Xie, H., Wang, X., Zhang, L., Wang, T., Zhang, W., Jiang, J., Chang, P.-K., Chen, Z.-Y., Bhatnagar, D., Zhang, Q., Li, P. 2018. Monitoring metabolite production of aflatoxin biosynthesis by orbitrap fusion mass spectrometry and a D-optimal mixture design method. Analytical Chemistry. 90:14331-14338. https://doi.org/10.1021/acs.analchem.8b03703.
DOI: https://doi.org/10.1021/acs.analchem.8b03703

Interpretive Summary: The saprophytic fungus Aspergillus flavus usually lives on decaying vegetation and produces aflatoxins. We developed a method that combines advanced analytical techniques and instrumentation to simultaneously determinate intermediates and final products of the aflatoxin biosynthesis pathway. The method is ideal for selectively monitoring the complex metabolite production by A. flavus via analyses of cell extracts. We demonstrated the applicability of this approach by examining time-course metabolite changes during aflatoxin biosynthesis and by revealing the anti-aflatoxigenic mechanism of the biocontrol bacterium Leclercia adecarboxylata WT16.

Technical Abstract: 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 changes in metabolite profiles of the aflatoxin biosynthesis pathway under different growth and environmental conditions. In the present study, we developed by 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. 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 µg/kg and 0.008-0.05 µg/kg, respectively. The spiked recovery rates ranged from 81.3% to 100.3% with intra-day and inter-day precision to be less than 7.6%. Using the method developed to investigate time-course aflatoxin biosynthesis, we found that precursors, including several possible toxins (with a carcinogenic group similar to aflatoxin B1) occurred together with aflatoxin, and production increased rapidly at the early growth stage, peaked on day four and then decreased substantially. The maximum production of aflatoxin B1 and aflatoxin B2 occurred one day later. Moreover, the dominant branch pathway was the one for aflatoxin B1 formation. We revealed that the anti-aflatoxigenicity 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.