Molecular characterization and periplasmic expression of the nlp gene of Streptomyces cyaneofuscatus UTMC 2101 in Escherichia coli

Abstract

Necrosis and ethylene-inducing peptide 1 (Nep1) proteins (NLP) are novel bioherbicides with high potential for application in integrated pest management projects. nlp genes are distributed widely among prokaryotic and eukaryotic microorganisms including members of actinomycetes. In this study, using a pair of degenerate primers, the middle fragment of the nlp gene from Streptomyces sp. UTMC 2101 was PCR-amplified, cloned and sequenced. Characterization of the 378-bp DNA fragment obtained confirmed the replacement of a tryptophan (W) with a phenylalanine (F) residue in the conserved heptapeptide motif ‘GHRHDEW’, which indicates that the necrosis-inducing Phytophtorae protein1 (NPP1) domain belongs to type II. Flanking regions of the known fragment of the nlp gene were synthesized by inverse PCR and the full nucleotide coding region of 828 bp, encoding 275 amino acid residues, was obtained. We analyzed expression of recombinant NLP in Escherichia coli by SDS-PAGE and evaluated the bioherbicide activity of the periplasmic fraction against tobacco leaves, confirming the production of soluble recombinant NLP in the periplasm. Phylogenetic identification of Streptomyces sp. UTMC 2101 showed 100 % similarity to Streptomyces cyaneofuscatus (KF381399) in the 16S rRNA gene. Thus, we report the first evidence of expression and activity of NLP from the Streptomyces genus. Our results could be useful for bioherbicide development in E. coli without the need for protein purification.