Cloning and analysis of a gene from Streptomyces lividans 66 encoding a novel secreted protease exhibiting homology to subtilisin BPN′

Abstract

Amino-terminal degradation has been observed for many of the secreted heterologous proteins produced by S. lividans 66. We, therefore, set out to characterize the relevant proteinases and their genes. A tripeptide chromogenic substrate was used to identify a gene that was shown to encode a secreted protein which removed tripeptides from the amino terminus of extracellular proteins (tripeptidyl aminopeptidase, Tap; Butler et al. 1995). This activity was removed by a homologous gene deletion replacement and the ability of the S. lividans strain to remove N-terminal tripeptides was greatly reduced, but still significant. When the tap-deleted strain was used as a host for the rescreening of a S. lividans 66 genomic DNA library, a number of other genes encoding proteases with aminopeptidase activities were discovered. One clone (P5-4) produced a 45-kDa secreted protein (Ssp), which showed activity against Ala-Pro-Ala-beta-naphthylamide (APA-beta NH-Nap) substrate. Further analysis of the cloned DNA showed an open-reading frame encoding a protein larger than 45 kDa. Direct Edman degradation of the secreted protein confirmed that it was encoded within the cloned DNA and probably processed from a larger precursor. Protein sequence analysis revealed a striking homology to subtilisin BPN' in three regions around the active-site residues suggesting that the protein is a serine protease. As expected, the protease activity was inhibited by phenylmethylsulphonyl fluoride. Mutant strains with most of the ssp gene deleted exhibited reduced activity against APA-beta NH-Nap substrate compared to their non-deleted parental strains.

A novel detergent-stable solvent-tolerant serine thiol alkaline protease from Streptomyces koyangensis TN650

“…The results from sequence alignment presented in Fig. 5 showed that the typical triad catalytic residues (H45, D69, and S311) in the active site and serine protease signatures [26,44] in the peptidase S8 and S53 superfamilies were conserved in the STAP enzyme. Additionally, the alignment of the deduced amino acid sequence of STAP from S. koyagensis strain TN650 was compared to those of other known proteases from Streptomyces strains (Fig.…”

“…Two oligonucleotides were synthesized based on the high degree of sequence homology published for the protease ssp gene from S. lividans strain 66 [26] and used for the isolation and determination of the stap encoding gene sequence. The complete stap gene and its flanking regions were amplified using the upstream primer F-BM3 (5 -GACACGGCGCCCCGCGGGGCC-3 ) and downstream primer R-BM4 (5 -CCGTCCGGTCCGCCGCCCCCG-3 ) to generate an approximately 1.9 kb PCR fragment using genomic DNA from S. koyangensis strain TN650 as a template and DNA polymerase (Pyrococcus furiosus from Biotools, Madrid, Spain).…”

“…Using the protease ssp gene sequence of S. lividans strain 66 [26], two homologue primers, called F-BM3 and R-BM4, were designed and used to amplify a fragment of about 1.9 kb that could contain the stap gene. This PCR fragment was purified and cloned in a pCR-Blunt cloning vector using an E. coli HB101 host strain, thus leading to pMB2.…”

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

“…4). However, slpE deletion strains, although viable, grow slowly on complex media and failed to grow on minimal medium (containing glucose, NH 4 ϩ salts) supplemented with all 20 natural amino acids or with alternative carbon sources such as sucrose, arabinose, glycerol, or starch. Restoration of growth on minimal medium could be obtained only when a complex nitrogen source such as yeast extract was added.…”

“…Many of these proteins contained amino-terminal residues consisting of the amino acids Ala-Pro-Ala. We therefore, set out to characterize and eliminate the remaining minor aminopeptidase activities present in the S. lividans ⌬tap strain MS7. This approach resulted in the isolation and deletion of the gene encoding a subtilisin-like protease (Ssp) from S. lividans (4). The resulting deletion strain MS11 (⌬tap ⌬ssp) exhibited reduced but significant Tap activity compared with its parental strain, MS7.…”