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Isolation and characterization of two genes encoding proteases associated with the mycelium of Streptomyces lividans 66.

ABSTRACT: A strain of Streptomyces lividans 66 deleted for a major tripeptidyl aminopeptidase (Tap) was used as a host to screen an S. lividans genomic library for clones overexpressing activity against the chromogenic substrate Ala-Pro-Ala-beta-naphthylamide. In addition to reisolation of the tap gene, clones representing another locus, slpD, were uncovered. slpD was analyzed by deletion subcloning to localize its functional sequence. Nucleotide sequence determination revealed an open reading frame encoding a 55-kDa protein exhibiting significant amino acid sequence homology to Tap, particularly around the putative active-site serine residue. No secreted protein was observed for strains harboring the slpD clone, but inspection of the predicted protein sequence revealed a putative lipoprotein signal peptide (signal peptidase II type), suggesting a mycelial location for the SlpD proteinase. In an attempt to isolate an endoprotease known to be active against some heterologous proteins, a second clone was isolated by using a longer substrate (t-butyloxycarbonyl [Boc]-APARSPA-beta-naphthylamide) containing a chemical blocking group at the amino terminus to prevent aminopeptidase cleavage. This locus, slpE, appeared to also encode a 55-kDa mycelium-associated (lipoprotein) proteinase, whose predicted protein sequences showed significant amino acid homology to Tap and SlpD, particularly around the putative active-site serine residues. Chromosomal integration and deletion analysis in both the wild-type and Tap-deficient backgrounds appeared to indicate that SlpD was essential for viability and SlpE was required for growth on minimal media.

SUBMITTER: Binnie C

PROVIDER: S-EPMC177439 | biostudies-other | 1995 Nov

REPOSITORIES: biostudies-other

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Project description:Small peptide aldehydes (SPAs) with protease inhibitory activity are naturally occurring compounds shown to be synthesized by non-ribosomal peptide synthetases (NRPS). SPAs are widely used in biotechnology and have been utilized as therapeutic agents. They are also physiologically relevant and have been postulated to regulate the development of their producing microorganisms. Previously, we identified an NRPS-like biosynthetic gene cluster (BGC) in Streptomyces lividans 66 that lacked a condensation (C) domain but included a tRNA-utilizing enzyme (tRUE) belonging to the leucyl/phenylalanyl (L/F) transferase family. This system was predicted to direct the synthesis of a novel SPA, which we named livipeptin. Using evolutionary genome mining approaches, here, we confirm the presence of L/F transferase tRUEs within the genomes of diverse Streptomyces and related organisms, including fusions with the anticipated C-minus NRPS-like protein. We then demonstrate genetic functional cooperation between the identified L/F-transferase divergent tRUE homolog with the C-minus NRPS, leading to the synthesis of a metabolic fraction with protease inhibitory activity. Semisynthetic assays in the presence of RNAse revealed that the productive interaction between the tRUE and the C-minus NRPS enzymes is indeed tRNA dependent. We expect our findings to boost the discovery of SPAs, as well as the development of protease-mediated biotechnologies, by exploiting the uncovered genetic basis for synthesizing putative acetyl-leu/phe-arginine protease inhibitors. Furthermore, these results will facilitate the purification and structural elucidation of livipeptin, which has proven difficult to chemically characterize.SignificanceThe discovery of natural products biosynthetic genes marks a significant advancement in our understanding of these metabolites, for example of their evolution, activity, and biosynthesis, but also opens biotechnological opportunities and knowledge to advance genome mining approaches. We made this possible by uncovering a new biosynthetic pathway in Streptomyces lividans 66 shown to direct the synthesis of a strong protease inhibitor, termed livipeptin, following unprecedented biosynthetic rules and genes. Thus, by shedding light on the genetic mechanisms predicted to govern the production of acetyl-leu/phe-arginine protease inhibitors, including the elusive livipeptin, this study enables novel protease-mediated biotechnologies as well as approaches for discovering protease inhibitors from genome data.

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Isolation and characterization of two genes encoding proteases associated with the mycelium of Streptomyces lividans 66.

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