Comprehensive analysis of aflatoxin B1 biosynthesis in Aspergillus flavus via transcriptome-wide m6A methylome response to cycloleucine

Abstract

Aspergillus flavus and its toxic aflatoxins secondary metabolites contaminate food and grains, posing a severe threat to human health and leading to liver cancer. Here, we demonstrated that cycloleucine blocked aflatoxin B₁ synthesis by inhibiting N⁶-methyladenosine (m⁶A) methylation modification of messenger RNA (mRNA). m⁶A Methylation Immunoprecipitation Sequencing (m⁶A MeRIP-Seq)-based comprehensive transcriptome-wide m⁶A profiling identified 102 differentially expressed genes that underwent m⁶A modification, of which 22 hypermethylated genes were downregulated and 49 hypomethylated genes were upregulated, suggesting a negative correlation between m⁶A methylation and gene expression. Notably, cycloleucine inhibited aflatoxin B₁ production via multiple targets. The m⁶A sites of several key genes involved in the aflatoxin B₁ biosynthesis pathway were significantly enriched in the coding sequence and around the stop codon, resulting in their downregulation. Furthermore, m⁶A methylation on genes related to the aflatoxin B₁ biosynthesis pathway led to reduced mRNA stability. Cycloleucine inhibition of aflatoxin B₁ production highlights its potential as an agent for removing mycotoxins in environmental pollution. ENVIRONMENTAL IMPLICATION: Aflatoxins, highly carcinogenic secondary metabolites produced by Aspergillus flavus, frequently contaminate crops such as peanut, corn, wheat and sesame leading to irreversible loss in the quality and yield of agricultural products and posing serious threats to food safety. Aflatoxins has also been linked to developmental delays and liver cancer in humans. In our study, 'monitoring aflatoxin concentrations and its bioaccumulation in organisms' has been conducted. The results demonstrated that aflatoxin production in A. flavus was completely blocked after cycloleucine treatment. Additionally, we demonstrated that inhibition of aflatoxin was linked to N⁶-methyladenosine methylation of multiple genes in aflatoxin biosynthesis pathway.

Keywords: Aflatoxin B(1); Aspergillus flavus; Cycloleucine; M(6)A methylation modification; MeRIP-Seq.