Localization of the lysine epsilon-aminotransferase (lat) and delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (pcbAB) genes from Streptomyces clavuligerus and production of lysine epsilon-aminotransferase activity in Escherichia coli

Abstract

Lysine e-aminotransferase (LAT) in the ,I-lactam-producing actinomycetes is considered to be the first step in the antibiotic biosynthetic pathway. putative N and C termini of S. clavuligerus pcbAB suggests that the coding region occupies 12 kbp and codes for a polypeptide related in size to the fungal ACV synthetases, the molecular characterization of the I8-lactam biosynthetic cluster between pcbC and cefE (-25 kbp) is nearly complete.Many naturally occurring P-lactam antibiotics (isopenicillin N, cephalosporin C, and cephamycins) contain a side chain derived from a-aminoadipic acid (a-AAA). When the producer is a filamentous fungus (Penicillium chrysogenum, Aspergillus nidulans, Cephalosporium acremonium), this amino acid is formed as an obligate intermediate in the synthesis of lysine. However, in procaroytes, lysine is synthesized via the diaminopimelic acid pathway without the production of a-AAA (for reviews, see references 23 and 36). Whitney et al. (37) established that in the P-lactamproducing Streptomyces spp., ot-AAA is derived from the breakdown of lysine. Although the catabolism of lysine in bacteria is diverse, conversion of lysine to a-AAA by removal of the E-amino group was tentatively linked in Streptomyces lipmanii to an aminotransferase (16) which was later conclusively shown to be present in Nocardia (previously Streptomyces) lactamdurans (15). Recently, Madduri et al. (21) reported that in these actinomycetes, L-lysine-e-aminotransferase (LAT) is specific to P-lactam (cephamycin C) producers and provides the precursor for antibiotic synthesis; the pathway via cadaverine is obligatory for lysine catabolism (Fig. 1). Moreover, a gene governing LAT production was found exclusively in Streptomyces spp. producing P-lactams (22) MATERIALS AND METHODS Bacterial strains, growth conditions, DNA manipulations, and DNA sequence determination. E. coli strains were grown in TY broth (18) supplemented with either 5 ,ug of tetracycline per ml or 80 jig of ampicillin per ml under standard conditions, except as noted.Restriction endonuclease digestion, ligation, and determination of DNA restriction fragments by agarose or acrylamide gel electrophoresis followed established procedures (29). DNA fragments were subcloned into pUC or M13 vectors for further analysis. DNA sequencing was performed by the dideoxy method with TAQ-TRACK (Promega Biotec) and followed the supplier's recommendations. Fragments were sequenced either directly from the plasmid or after subcloning into M13 bacteriophage vectors, using forward and reverse sequencing primers for pUC and M13 templates, as well as several custom DNA oligonucleotide primers. Custom DNA oligonucleotide primers were synthesized by using an Applied Biosystems DNA Synthesizer (model 380A) according to the manufacturer's instructions.Computing. DNA sequences were analyzed with the GCG

Precursor flux control through targeted chromosomal insertion of the lysine epsilon-aminotransferase (lat) gene in cephamycin C biosynthesis

“…The presumed pcbAB gene encoding ACV synthetase in S. clavuligerus is located adjacent to lat and is separated by a 125-bp noncoding region of DNA (28). Analysis of the DNA sequence in this region showed that an inverted repeat (28), corresponding to a potential transcriptional terminator, separates the two genes.…”

Genome-minimized Streptomyces host for the heterologous expression of secondary metabolism

“…We, therefore, chose to examine heterologous expression of the gene cluster for cephamycin C biosynthesis in S. avermitilis as a model for NRPS function in this host. The full set of genes encoding cephamycin C biosynthesis has been located within a 35-kb continuous region in the S. clavuligerus chromosome (15)(16)(17)(18). We isolated two cosmid clones containing the cephamycin C biosynthetic gene cluster from an S. clavuligerus genomic library.…”

pcd Mutants of Streptomyces clavuligerus Still Produce Cephamycin C

“…In bacterial cephamycin producers, ␣-aminoadipic acid in turn arises from lysine via a two-step process catalyzed by lysine-6-aminotransferase (LAT) and piperideine-6-carboxylate dehydrogenase (PCD), and so LAT and PCD activities are considered the first two steps of cephamycin biosynthesis. While lat genes have been found in the cephamycin gene clusters of all actinomycetes that have been studied (5,24), the pcd gene has been more elusive.…”