Molecular characterization of a novel subtilisin inhibitor protein produced by Streptomyces venezuelae CBS762.70

We report here on the isolation and identification of a gene coding for a novel subtilisin inhibitor (VSI) isolated from Streptomyces venezuelae CBS762.70. The vsi gene was isolated on a 5-kb chromosomal PvuII fragment as identified by DNA sequencing and inhibitor activity testing of the gene product. Primer extension studies revealed that the mRNA transcriptional start point was situated at -37 and -36 relatively to the ATG start codon assuming the presence of solely one promoter. Vsi promoter strength was about double of those of ermE-P1a and aph-P1, as tested with the mRNA production of the aphII gene preceded by the respective promoters. Translation of the vsi coding sequence revealed a 28 amino acids long signal peptide. The mature VSI protein consists of 118 amino acids of which 87% was verified by N-terminal amino acid sequence analysis. Compared with the already known Streptomyces proteinase inhibitors, VSI shows a relatively high amino acid identity in the conserved domains. Nevertheless, only a maximum amino acid identity of 56.1% was noticed and some highly conserved residues were substituted in VSI. As a consequence, VSI could be classified within a separate group of Streptomyces subtilisin inhibitors.