The Regulatory Mechanism Of Aflatoxin Biosynthesis By Lysine 2-hydroxyisobutyrylation In Aspergillus Flavus

Aflatoxins are a group of structurally similar toxic,mutagenic as well as carcinogenic fungal toxins,among which aflatoxin B1(AFB1)is the most toxic,and as a classⅠcarcinogen,it has brought significant harm to agricultural products around the world,and the annual loss of food and other agricultural products caused by aflatoxins is serious worldwide.Therefore,in-depth research on the mechanism of aflatoxin synthesis is of great significance for pollution prevention and control,and to ensure food safety.The 2-hydroxyisobutylation modification(Khib)is a newly discovered post-translational lysine acylation modification of proteins.It was found that this modification plays an integral role in metabolic processes such as glycolysis in animal cells,but the mechanism by which lysine2-hydroxyisobutyrylation modification occurs and its regulation of secondary metabolite synthesis in Aspergillus flavus has not been reported.In this study,we first obtained the intracellular protein 2-hydroxyisobutyrylation modification profiles of A.flavus by proteomics techniques and investigated the effect of lysine 2-hydroxyisobutyrylation modification of AflM,a key enzyme in the aflatoxin biosynthesis pathway,on the growth and toxicity production of A.flavus by point mutation.Through bioinformatics analysis and molecular biology validation,the protein associated with 2-hydroxyisobutyrylation modification was mined and identified,its subcellular localization was analyzed,and the regulatory effects of the gene on intracellular histone modification level,gene transcription level and aflatoxin biosynthesis were investigated.The main findings were as follows:(1)The presence of lysine 2-hydroxyisobutyrylation modification in Aspergillus flavus cells and the variation in the abundance of this modification correlated with aflatoxin biosynthesis.Protein blot analysis(Western Blot)showed that the protein modified by lysine2-hydroxyisobutyrylation was distributed in the range of 25-100 kDa,and the level of modification was higher in 24 h cells than in 48 h cells.A 2-hydroxyisobutyrylated modification of histone H4K8 was also detected,both indicating that this modification is conserved across species.(2)Lysine 2-hydroxyisobutyrylation modifications are involved in central metabolic pathways and aflatoxin biosynthesis processes.Protein functional annotation analysis indicates that lysine 2-hydroxyisobutyrylated modified proteins are mainly distributed in mitochondria,cytoplasm,nucleus and extracellular,and may play key roles in cellular metabolism(such as glycolysis),energy supply and gene transcription.Overall proteomic characterization showed that 7156 sites on 1473 proteins underwent2-hydroxyisobutyrylation and 4964 lysine 2-hydroxyisobutyrylation modification sites on1145 proteins.The conserved amino acids flanking modified lysine residues were glycine(G)at the-1 position,aspartate(D)at the-3 position and lysine(K)in positions-10 to-6 and +5to +10.Similarly,valine(V)was found at positions-4,-2,+2 and +4,respectively.(3)Proteomics results showed that multiple sites of AflM,a key enzyme of the aflatoxin synthesis pathway,were modified.The three-dimensional structure of AflM was simulated using Tfold,and three sites,K49,K179 and K180,were selected for point mutation to simulate the effect of the 2-hydroxyisobutyrylation modification of this enzyme on the pathogenicity of Aspergillus flavus.The results showed that both deletion and mutation of AflM enhanced aflatoxin production and that lysine 2-hydroxyisobutyrylation of K49,K179 and K180(mutating K to T to mimic the 2-hydroxyisobutyrylation modification)significantly attenuated aflatoxin biosynthesis.(4)A 2-hydroxyisobutyryltransferase Afngg1 localized in the nucleus was identified by molecular biology techniques.The deletion of this gene significantly reduced the level of lysine 2-hydroxyisobutyrylation modification of A.flavus histones H4K5 and H4K8,inhibited the mycelial growth,reduced the number of A.flavus conidia,hydrophobicity,and the ability to infect peanut and corn seeds,while the biosynthesis of AFB1 and the production of nuclei were completely inhibited.Analysis of transcriptome data before and after Afngg1 gene deletion revealed that the differentially expressed genes were mainly involved in biological processes such as chromatin remodeling,cell development,secondary metabolism and oxidative stress.