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

Abstract

In actinomycetes that produce P-lactam antibiotics of the cephem type, lysine r-aminotransferase is the initial enzyme in the conversion of lysine to a-aminoadipic acid. We used a two-stage process ("chromosome walking") to screen a lambda library of Streptomyces clavuligerus genomic DNA for fragments that expressed lysine r-aminotransferase activity in S. lividans. Restriction analysis of the cloned DNA confirmed the location of the putative lat gene within the cluster of , -lactam biosynthesis genes, roughly midway between pcbC, the structural gene for isopenicillin N synthetase, and the putative cefE gene encoding deacetoxycephalosporin C synthetase.Streptomyces clavuligerus is a gram-positive mycelial procaryote that produces a number of P-lactam-containing metabolites. Among these are antibiotics of the penicillin and cephalosporin types which are synthesized through a series of reactions involving condensation of ot-aminoadipate, cysteine, and valine to the tripeptide 8-(L-a-aminoadipyl)-L-cysteinyl-D-valine (ACV), cyclization of ACV to isopenicillin N (IPN), and modification of this initial penicillin to a variety of end products (reviewed in reference 2).In recent years, some P-lactam biosynthesis genes have been cloned from several organisms (1,15). 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.Previously, we showed that lysine r-aminotransferase (LAT) activity and a-aminoadipate synthesis occur only in 3-lactam-producing actinomycetes (12). Here we report that LAT is governed by DNA located within the cluster of ,B-lactam biosynthesis genes in S. clavuligerus. MATERIALS AND METHODSOrganisms and growth conditions. The Streptomyces and Escherichia coli strains used in this study are listed in Table 1. Streptomyces spp. were grown at 30°C on either MYM agar (17) or R5 agar (3) modified by omitting sucrose and substituting maltose for glucose; mycelium was propagated in either tryptic soya broth (Difco Laboratories) or YEME medium (3). Streptomyces protoplasts were prepared and transformed by standard procedures (3); for protoplast regeneration R5 medium was used. 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).

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

“…2). pDQ302, an E. coli-Streptomyces shuttle vector which includes a 4.7-kb SstIEcoRI fragment from S. clavuligerus, contains the lat gene (and has been shown to express LAT activity in S. lividans and E. coli) (17) and was used as a template for PCR (Fig. 2).…”

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

“…The gene for LAT (lat), previously shown to be closely linked to other genes of the P-lactam biosynthetic pathway in S. clavuligerus (22), was characterized at the molecular level A.…”

“…Also, a previously reported ORF (6) located just upstream of pcbC in S. clavuligerus is similar to the C terminus of the fungal ACVS (13). Since P. chrysogenum pcbAB is approximately 12 kbp (5, 34) and lat maps approximately 12 kbp from pcbC (22), we believe that pcbAB has been precisely located in S. clavuligerus and that ACVS is closely related in size to the fungal protein. Furthermore, the direction of transcription for pcbAB is toward pcbC in S. clavuligerus.…”

“…Moreover, a gene governing LAT production was found exclusively in Streptomyces spp. producing P-lactams (22) and mapped within the P-lactam biosynthetic gene cluster of Streptomyces clavuligerus (18,24,33 Gene expression in E. coli. For LAT production, E. coli JM109 cells containing pOW407 were grown overnight at 25°C, diluted 20-fold, induced at 42°C for 6 h, and examined by phase-contrast microscopy for the appearance of granules.…”

Structure of the ask-asd operon and formation of aspartokinase subunits in the cephamycin producer ‘Amycolatopsis lactamdurans’ The GenBank accession number for the sequence reported in this paper is AJ298904.

“…In mycobacteria, corynebacteria, Amycolatopsis and bacilli the ask and asd genes are clustered in an operon (Kalinowski et al, 1990 ;Chen et al, 1993 ;Cirillo et al, 1994 ;Zhang et al, 1999), although that is not the case in Streptomyces akiyoshiensis (Le et al, 1996). In the β-lactam-producing actinomycetes Streptomyces clavuligerus and ' Nocardia lactamdurans ' (reclassified recently as ' Amycolatopsis lactamdurans ' ; Barreiro et al, 2000) -lysine is converted additionally to piperidine-6-carboxylate (P6C) by the enzyme lysine-6-aminotransferase (Madduri et al, 1991 ;Coque et al, 1991) and then oxidized to α-aminoadipic acid (α-AAA) by the P6C dehydrogenase (Fuente et al, 1997 ;Pe! rez-Llarena et al, 1998).…”