A Novel Site-Specific Integration System for Genetic Modification of Aspergillus flavus

Abstract

Aspergillus flavus is a fungus that produces aflatoxin B1, one of the most carcinogenic secondary metabolites. Understanding the regulation mechanism of aflatoxin biosynthesis in this fungus requires precise methods for genomic integration of mutant alleles. To avoid the disadvantage of DNA integration into the genome by non-homologous or ectopic recombination, we developed a novel strategy for site-specific integration of foreign DNA by using a carboxin-resistant sdh2R allele (His 249 Leu). Our results demonstrated that the transformants were generated with a high efficiency (>96%) of correct integration into the sdh2-lcus of the genome of A. flavus NRRL 3357. The advantage of this method is that introduction of the eGFP expression cassette into the sdh2-locus had little effect on fungal growth and virulence while also being rapid and efficient. This system will be a valuable tool for genetic manipulation in A. flavus. To the best of our knowledge, this is the first report on the efficient site-specific integration at the sdh2-locus in the genome of Aspergillus.

Role of Flavohemoglobins in the Development and Aflatoxin Biosynthesis of Aspergillus flavus

“…The target gene overexpression strains were constructed following a previous site-specific integration system [ 35 ]. The foreign DNA integrated into the succinate dehydrogenase (SDH) gene site via a carboxin-resistant sdh2 R allele (H249L) strategy.…”

Aflatoxins: History, Significant Milestones, Recent Data on Their Toxicity and Ways to Mitigation

“…Only precise genomic integration of mutant allele methods is required for accurate understanding of the mechanism for regulation of AF biosynthesis produced by A. flavus. The new strategy for the foreign DNA site-specific integration in the A. flavus sdh2 gene locus was evolved to prevent the disadvantage of ectopic or non-homologous recombination within integration of DNA into the genome [196].…”

“…A. flavus obtains systemic fungicide carboxin resistance as a result of a substitution of original sdh2 allele with sdh2 R . Proper integration of the A. flavus NRRL 3357 genome into the locus sdh2 resulted in the highly efficient generation (>96%) of transformants [196]. The rapidity and effectiveness of this method consist of the locus sdh2 with inserted eGFP expression cassette, which leads to the alleviation of virulence and the growth of fungi.…”

“…The rapidity and effectiveness of this method consist of the locus sdh2 with inserted eGFP expression cassette, which leads to the alleviation of virulence and the growth of fungi. This process would be a helpful instrument for genetic manipulation of A. flavus [196].…”

Regulation of Fungal Morphogenesis and Pathogenicity of Aspergillus flavus by Hexokinase AfHxk1 through Its Domain Hexokinase_2

“…Current methods to remove aflatoxins are categorized into Pre-Harvest (mainly through biological control agents) and Post-Harvest (sorting, dehulling, milling, adsorbents, acids, etc.) approaches [6,[8][9][10][11]. However, existing methods have limitations in terms of efficiency and cost.…”