Cloning, characterization, and expression in Escherichia coli of the Streptomyces clavuligerus gene encoding deacetoxycephalosporin C synthetase

Abstract

Biosynthesis of cephalosporin antibiotics involves an expansion of the five-membered thiazolidine ring of penicillin N to the six-membered dihydrothiazine ring of deacetoxycephalosporin C by a deacetoxycephalosporin C synthetase (DAOCS) enzyme activity. Hydroxylation of deacetoxycephalosporin C to form deacetylcephalosporin C by a deacetylcephalosporin C synthetase (DACS) activity is the next step in biosynthesis of cephalosporins. In Cephalosporium acremonium, both of these catalytic activities are exhibited by a bifunctional enzyme, DAOCS-DACS, encoded by a single gene, cefEF. In Streptomyces clavuligerus, separable enzymes, DAOCS (expandase) and DACS (hydroxylase), catalyze these respective reactions. We have cloned, sequenced, and expressed in E. coli an S. clavuligerus gene, designated cefE, which encodes DAOCS but not DACS. The deduced amino acid sequence of DAOCS from S. clavuligerus (calculated Mr of 34,519) shows marked similarity (approximately 57%) to the deduced sequence of DAOCS-DACS from C. acremonium; however, the latter sequence is longer by 21 amino acid residues.

Cloning and location of a gene governing lysine epsilon-aminotransferase, an enzyme initiating beta-lactam biosynthesis in Streptomyces spp

“…Three hybridizing lambda clones (F, G, and H [not shown]) were isolated. The insert DNA from clone F hybridized to clone E DNA, and results of further restriction mapping of the putative cefE DNA were consistent with published maps of the region (8,9). This indicated that the synthetic cejE probe had indeed hybridized with the cefE gene in our library and showed that the putative lat gene was approximately midway between pcbC and cefE (Fig.…”

“…1). To position lat relative to the DAOCS gene, cefE, the S. clavuligerus genomic library was probed with a 33-mer synthetic oligonucleotide, the sequence of which was that of the 5' terminal 11 codons in the published sequence of the DAOCS gene (9). Three hybridizing lambda clones (F, G, and H [not shown]) were isolated.…”

“…Strains of E. coli were grown in Luria broth (LB) or on LB gelled with 1.5% agar; E. coli DH5a was transformed by the CaCl2 method (13 (10). A 33-mer oligonucleotide used in isolating putative cefE-containing sequences was a synthetic product based on the N-terminal amino acid sequence of DAOCS (9). Both pBL1 and the synthetic oligonucleotide were generously supplied by Susan Jensen, University of Alberta.…”

“…Kovacevic et al (9) provided evidence of gene clustering when they used pcbC DNA encoding IPN synthetase from S. lipmanii as a probe to isolate cefE encoding deacetoxycephalosporin synthetase (DAOCS) from a genomic library of S. clavuligerus and to locate adjacent genes. Also, a DNA fragment containing both cefD (isopenicillin N epimerase) and cefE was cloned from S. clavuligerus and shown to direct the synthesis of a polygenic mRNA of about 10 kb (8), indicating that 1-lactam genes are organized in at least one operon.…”

Molecular characterization of the acyl-coenzyme A:isopenicillin N acyltransferase gene (penDE) from Penicillium chrysogenum and Aspergillus nidulans and activity of recombinant enzyme in Escherichia coli

“…The NdeI-BamHI fragment from pOW1067 containing penDE was subsequently subcloned into an E. coli expression vector containing the k PL promoter, creating pOW233. E. coli L201 cells containing this plasmid were induced for protein production (12,21). After 6 h, protein-containing granules were observed by phasecontrast microscopy.…”