Suppression of Aflatoxin Biosynthesis in Aspergillus flavus by 2-Phenylethanol Is Associated with Stimulated Growth and Decreased Degradation of Branched-Chain Amino Acids.

Perng-Kuang Chang,Robert Li,Sui Hua,Siov Sarreal

doi:10.3390/toxins7103887

Abstract

The saprophytic soil fungus Aspergillus flavus infects crops and produces aflatoxin. Pichia anomala, which is a biocontrol yeast and produces the major volatile 2-phenylethanol (2-PE), is able to reduce growth of A. flavus and aflatoxin production when applied onto pistachio trees. High levels of 2-PE are lethal to A. flavus and other fungi. However, at low levels, the underlying mechanism of 2-PE to inhibit aflatoxin production remains unclear. In this study, we characterized the temporal transcriptome response of A. flavus to 2-PE at a subinhibitory level (1 µL/mL) using RNA-Seq technology and bioinformatics tools. The treatment during the entire 72 h experimental period resulted in 131 of the total A. flavus 13,485 genes to be significantly impacted, of which 82 genes exhibited decreased expression. They included those encoding conidiation proteins and involved in cyclopiazonic acid biosynthesis. All genes in the aflatoxin gene cluster were also significantly decreased during the first 48 h treatment. Gene Ontology (GO) analyses showed that biological processes with GO terms related to catabolism of propionate and branched-chain amino acids (valine, leucine and isoleucine) were significantly enriched in the down-regulated gene group, while those associated with ribosome biogenesis, translation, and biosynthesis of α-amino acids were over-represented among the up-regulated genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that metabolic pathways negatively impacted among the down-regulated genes parallel to those active at 30 °C, a condition conducive to aflatoxin biosynthesis. In contrast, metabolic pathways positively related to the up-regulated gene group resembled those at 37 °C, which favors rapid fungal growth and is inhibitory to aflatoxin biosynthesis. The results showed that 2-PE at a low level stimulated active growth of A. flavus but concomitantly rendered decreased activities in branched-chain amino acid degradation. Since secondary metabolism occurs after active growth has ceased, this growth stimulation resulted in suppression of expression of aflatoxin biosynthesis genes. On the other hand, increased activities in degradation pathways for branched-chain amino acids probably are required for the activation of the aflatoxin pathway by providing building blocks and energy regeneration. Metabolic flux in primary metabolism apparently has an important role in the expression of genes of secondary metabolism.

Highlights

Aspergillus flavus, which is a common plant and an opportunistic human pathogen, can produce the carcinogenic aflatoxin
We show that a subinhibitory concentration of 2-PE to mycelial growth elicits in A. flavus totally opposite changes; it promotes protein synthesis for growth as evidenced by the Gene Ontology (GO) enrichment analysis and the result that expression of about 70% (46/67) of genes encoding known or putative ribosomal proteins is increased significantly in the first 48 h (FDR corrected p-value < 0.05, data not shown), but it inhibits secondary metabolism, like the decreased expression of all genes in the aflatoxin and cyplopiazonic acid gene clusters
Our functional genomics study shows that the inhibition of aflatoxin production by the low level of

Summary

Introduction

Aspergillus flavus, which is a common plant and an opportunistic human pathogen, can produce the carcinogenic aflatoxin. Contamination of crops such as corn, cotton, peanuts, and tree nuts by aflatoxin poses a great food safety risk especially in developing countries due to ineffective farming practices and the lack of proper storage facilities. Afla-Guard® [4], to outcompete toxigenic strains in the field or spraying a yeast formulation to pistachio trees to prevent fungal growth [5]. In field tests, these biocontrol approaches have achieved greater than 80 percent reduction in aflatoxin contamination

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