Fusion expression and anti-Aspergillus flavus activity of a novel inhibitory protein DN-AflR

Int J Food Microbiol. 2019 Feb 2;290:184-192. doi: 10.1016/j.ijfoodmicro.2018.10.015. Epub 2018 Oct 18.

Abstract

The regulatory gene (aflR) encodes AflR, a positive regulator of transcriptional pathway that activates aflatoxin biosynthesis. It has been demonstrated in our laboratory that L-Asp-L-Asn (DN) extracted from Bacillus megaterium inhibited the growth of Aspergillus flavus. We fused gene encoding DN with the gene encoding specific dinuclear zinc finger cluster protein of AflR, then fusion protein competed with the AflS-AflR complex for the AflR binding site and significantly improved anti-A. flavus activity (growth of A. flavus and biosynthesis of aflatoxin B1) of DN. The fusion gene dn-aflR was cloned into pET32a and recombinant plasmid was introduced into Escherichia coli BL21. The highest expression was observed after 10 h induction and fusion protein was purified by affinity chromatography column. Compared with DN, the novel fusion protein DN-AflR significantly inhibited the growth of A. flavus and biosynthesis of aflatoxin B1 (P < 0.05). This study promoted the use of competitive inhibition of fusion proteins to reduce the expression of regulatory genes in the biosynthetic pathway of aflatoxin. Moreover, it provided more supports for deep research and industrialization of such novel anti-A. flavus bio-inhibitors and biological control of microbial contamination.

Keywords: AflR; Aspergillus flavus; Dn-aflR; Fusion expression.

MeSH terms

  • Aflatoxin B1 / antagonists & inhibitors
  • Aflatoxin B1 / biosynthesis
  • Antifungal Agents / pharmacology
  • Aspergillus flavus / drug effects
  • Aspergillus flavus / genetics*
  • Aspergillus flavus / growth & development
  • Bacillus megaterium / chemistry
  • Cloning, Molecular
  • DNA-Binding Proteins / antagonists & inhibitors*
  • Escherichia coli
  • Fungal Proteins / antagonists & inhibitors*
  • Gene Expression Regulation, Fungal
  • Microbial Sensitivity Tests
  • Peptides / pharmacology*
  • Protein Conformation
  • Recombinant Fusion Proteins / pharmacology*
  • Transcription Factors / antagonists & inhibitors*
  • Zinc Fingers

Substances

  • AFLR protein, Aspergillus
  • Antifungal Agents
  • DNA-Binding Proteins
  • Fungal Proteins
  • Peptides
  • Recombinant Fusion Proteins
  • Transcription Factors
  • Aflatoxin B1