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

Abstract

The regulatory gene (aflR) of aflatoxin encodes AflR, a positive regulator that activates transcriptional pathway of genes in aflatoxin biosynthesis. New L-Asp-L-Asn (DN) extracted from Bacillus megaterium inhibited the growth of A. flavus had been elucidated in our laboratory. The genes encoding DN and binuclear zinc finger cluster protein of AflR were fused, then fusion protein could compete with the AflS-AflR complex for the AflR binding site and significantly improve anti-A. flavus activity 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 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. 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.\n\nIMPORTANCEAflatoxin contamination has seriously influence on export of agricultural products, income of farmers and economic development. Biological methods, especially using antagonistic microorganisms to inhibit aflatoxin biosynthesis gradually become the hot spot in recent years. DN (L-Asp-L-Asn) from Bacillus megaterium, which could inhibit growth of Aspergillus flavus and synthesis of aflatoxin, has been identified. In this report, we fused the genes encoding inhibitory peptides (DN) and specific zinc finger cluster protein, and expressed the novel anti-A. flavus protein in Escherichia coli. Compared with DN, the inhibitory ability of novel protein has been improved significantly. This research showed fusion expression of anti-fungal proteins, such as DN-AflR, is a promising method to economically improve the inhibitory activity of bio-inhibitors for A. flavus.