Molecular cloning and high-level expression of a bromoperoxidase gene from Streptomyces aureofaciens Tü24

Abstract

A bromoperoxidase gene was cloned from Streptomyces aureofaciens Tu24 into Streptomyces lividans TK64 by using the promoter-probe vector pIJ486. Subcloning of DNA from the original, unstable clone allowed the gene to be localized to a 1.7-kilobase (kb) fragment of DNA. Southern blotting showed that the cloned 1.7-kb insert hybridized to a 4.3-kb fragment in an SstI digest of S. aureofaciens Tu24 total DNA. The 1.7-kb insert was shown to code for a protein with the electrophoretic properties of the subunits of the nonheme bromoperoxidase isolated from S. aureofaciens Tu24.-The protein produced by S. lividans TK64 transformed with pHM621, which contained an 8.0-kb insert, was shown to be identical to the S. aureofaciens Tu24 bromoperoxidase in terms of its electrophoretic mobility on denaturing and nondenaturing polyacrylamide gels and its NH2-terminal amino acid sequence. The bromoperoxidase was overproduced (up to 180 times) by S.lividans TK64 containing pHM621. Based on the heat stability of the S. aureofaciens Tu24 bromoperoxidase, a new and simple purification procedure with very high yields was developed.Streptomyces aureofaciens Tu24 produces the antibiotic 7-chlorotetracycline. The incorporation of chlorine during the biosynthesis of 7-chlorotetracycline is believed to be catalyzed by a chloroperoxidase (12). However, only a brominating enzyme, a bromoperoxidase, could be isolated from this strain (18). Bromoperoxidases have been isolated from other chlorometabolite-producing bacteria as well (16,17,20). The only bacterial chloroperoxidase known until now was detected in Pseudomonas pyrrocinia (2). This enzyme was detected as a bromoperoxidase by the monochlorodimedone assay developed by Hager et al. (2). Monochlorodimedone was not chlorinated by the chloroperoxidase from P. pyrrocinia; however, when indole was used as a substrate, it was chlorinated to 7-chloroindole (21), a substance which is known to be produced by P. pyrrocinia (8). Therefore, it was thought to be possible that the bromoperoxidase isolated from S. aureofaciens Tu24 could be a chloroperoxidase, too, but only if a more "natural" substrate than monochlorodimedone was used. To check this, large amounts of the enzyme are needed, and thus I decided to try to clone the bromoperoxidase gene of S. aureofaciens Tu24. This report describes the cloning of the S. aureofaciens Tu24 bromoperoxidase gene in S. lividans TK64. MATERIALS AND METHODSBacterial strains and culture conditions. S. lividans TK64 and S. lividans TK24 containing the promoter-probe plasmid vector pIJ486 (19) were kindly supplied by D. A. Hopwood, Norwich, England, and S. aureofaciens Tu24 was provided by H. Zahner, Tubingen, Federal Republic of Germany (FRG). These strains were cultured on agar plates containing 2% (wt/vol) soybean flour and 2% (wt/vol) mannitol. This medium was also used as a liquid medium when cells were grown for the production of bromoperoxidase. Other media used were yeast extract-malt extract plus 34% (wt/vol) sucrose (YEME) (1); R2YE medium (14); P medium ...

Chloroperoxidase from Streptomyces lividans: isolation and characterization of the enzyme and the corresponding gene

“…Like other Streptomyces haloperoxidases (27,33), chloroperoxidase from S. lividans was not expressed in E. coli. Therefore, subcloning was carried out in the original host.…”

“…In contrast to the untransformed strain, brominating activity could be measured in the crude (27,33,44). Therefore, a mutant of S. aureofaciens Tu24 with a deletion of its own chloroperoxidase gene (12) was transformed with pRB882.…”

“…So far, haloperoxidases have been isolated only from bacteria, which are known to produce halogenated metabolites. Thus, it was surprising that in Streptomyces lividans, which is frequently used as a cloning host, a halogenating activity had been detected during the cloning of the haloperoxidase gene from S. aureofaciens Tu24 (33), as no halogenated metabolites have been found in this strain until now. Therefore, it was of considerable interest to determine whether the haloperoxidase present in S. lividans was similar to the enzymes isolated from halometabolites-producing strains.…”

Purification, characterization and comparison of two non-haem bromoperoxidases from Streptomyces aureofaciens ATCC 10762

“…The 7-chlorotetracycline-producing strain S. aureofaciens ATCC 10762, S. aureofaciens Tu24, and S . liuidans TK64 containing the cloned bromoperoxidase gene from S. aureofaciens Tu24 on a plasmid (pHM621) (van Pee, 1988), were used. S. aureofaciens ATCC 10762 was grown in 100ml flasks containing 25 ml of complex medium (per litre of deionized water: glucose, 4 g; yeast extract, 4 g; malt extract, 10 g; KCl, 0.5 g; FeSO,.…”

Enzymology and Genetics of Halogenating Enzymes from Bacteria

“…However, there is no evidence that a metal ion is necessary for the halogenation reaction catalyzed by the prokaryotic non-haem haloperoxidases, as the addition of metal ions (Mn, Zn, Ni, Cu, Fe, V) and EDTA has no effect on enzyme activity. The chloroperoxidase gene from Pseudomonas pyrrocinia (Wolfframm, van Pee and Lingens, 1988) as well as the bromoperoxidase gene from Streptomyces aureofaciens Tu24 (van PCe, 1988) have been cloned. The chloroperoxidase gene was cloned into Escherichia coli and chloroperoxidase is extremely overproduced by E. coli.…”