The beta-lactam biosynthesis genes for isopenicillin N epimerase and deacetoxycephalosporin C synthetase are expressed from a single transcript in Streptomyces clavuligerus

Abstract

Isopenicihlin N isomerase (epimerase) has been purified from Streptomyces clavuligerus, and the amino acid sequence of the N-terminus has been determined. By using single oligonucleotide probes based on high GC codon bias ("guessmers"), the translation start codons were determined for two successive genes in the P-lactam-biosynthetic pathway and mapped within a 3.6-kilobase-pair KpnI restriction fragment. The epimerase gene (cel)) was located immediately upstream of the deacetoxycephalosporin C synthetase (expandase) gene (cefE) that was characterized previously. celD was sequenced and expressed in Escherichia coli; the resulting cell extracts contained epimerase activity. Western immunoblots demonstrated that a protein comigrated with purified S. clavuligerus epimerase at 44 kilodaltons. cel) and cejE were separated by an 81-base-pair segment. The DNA sequence upstream of the epimerase gene had a high AT content, suggestive of a promoter region. Primer extension analysis of S. clavuligerus mRNA showed that the start of transcription occurred approximately 130 base pairs upstream of the epimerase translation start site; Northern (RNA blot) analysis revealed a hybridization signal large enough to code for both epimerase and expandase, and nuclease Si protection assays showed that a single message may code for epimerase, expandase, and another ulnknown protein. When cef) and cejE were placed in an expression vector, concomitant synthesis of both epimerase and expandase occurred in E. coli.,-Lactam antibiotics are distinguished by the ,3-lactam ring plus either a five-membered thiazolidine ring (present in the penicillins) or the "expanded" six-membered dihydrothiazine structure (found in the cephalosporins). This is the main chemical difference between the core structures of these antibiotics. The pathway for these two classes of 13-lactams have common steps in their synthesis up to the production of isopenicillin N ( Fig. 1) (7, 11). The first step that commits the pathway to the production of cephalosporinlike antibiotics is the isomerization of isopenicillin N, the product of isopenicillin N synthetase (cyclase), to penicillin N (10, 12). This activity has also been referred to as epimerase and is not found in organisms (e.g., Penicillium chrysogenum) that produce hydrophobic penicillins (7).In a previous report (13) we characterized the gene (cefE) for deacetoxycephalosporin C synthetase (DAOCS; expandase) and expressed the gene in Escherichia coli. These E. coli extracts contained expandase activity. The gene was isolated from a cosmid library with a single hybridization probe based on the N-terminal amino acid sequence of the purified protein and the strict codon bias observed for streptomycetes (24). We have also shown by differential hybridization to these cosmids that genes for the cyclase and expandase enzymes are closely linked in S. clavuligerus (13,21). This potential for linkage of all the genes for the biosynthesis of cephalosporins led us to characterize the molecular organization of the epimerase ...

Homology of pyridoxal‐5′‐phosphate‐dependent aminotransferases with the cobC (cobalamin synthesis), nifS (nitrogen fixation), pabC (p‐aminobenzoate synthesis) and malY (abolishing endogenous induction of the maltose system) gene products

“…However, the highest 2 score in a search with the n i p profile, i.e. 5.4, was attributed to pyridoxal-P-dependent isopenicillin-N-epimerase (Table 2) which catalyzes the racemization of its substrate (Kovacevic et al, 1990). Perhaps, the nifs protein is a racemase.…”

Genome sequencing and analysis of the filamentous fungus Penicillium chrysogenum

“…These may include identified orthologs of 4-coumarateCoA ligase. Surprisingly, several orthologs of bacterial 25 and fungal 26 isopenicillinN epimerase were identified. The predicted protein sequence of Pc12g11540 shares 40% homology with S. clavuligerus isopenicillinN epimerase, although it probably functions as an aminotransferase.…”

Streptomyces clavuligerus relA-null mutants overproduce clavulanic acid and cephamycin C: negative regulation of secondary metabolism by (p)ppGpp

“…Transcription of cefD, encoding the isopenicillin N epimerase for cephamycin C biosynthesis (Kovacevic et al, 1990), and of ceaS2, encoding the carboxyethylarginine synthase for clavulanic acid biosynthesis, was assessed by high-resolution S1 nuclease protection analysis. In parallel, transcription of the regulatory genes ccaR (Pérez-Llarena et al, 1997) and bldG (Bignell et al, 2005) (both involved in the regulation of cephamycin C and clavulanic acid production), and of claR (Pérez-Redondo et al, 1998, Paradkar & Jensen, 1998 (involved in regulating clavulanic acid biosynthesis), was determined, as was that of relA.…”