Juan F. Martin, Santiago Guti6rrez, Francisco J. Fermindez, Javier Velasco, Francisco Fierro,. Ana T. Marcos & Katarina Kosalkova. Area of Microbiology ...
AntonievanLeeuwenhoek65: 227-243, 1994. Q 1994KluwerAcademicPublishers.Printedin the Netherlands.
227
Expression of genes and processing of enzymes for the biosynthesis of penicillins and cephalosporins Juan F. Martin, Santiago Guti6rrez, Francisco J. Fermindez, Javier Velasco, Francisco Fierro, Ana T. Marcos & Katarina Kosalkova Area of Microbiology, Department of Ecology, Genetics and Microbiology, Faculty of Biology, University of Ledn, 24071 Le6n, Spain
Key words: Penicillin and cephalosporin biosynthesis, pcbAB genes, pcbC genes, penDE genes, ceflEFgenes, cefG genes, transcription analysis, protein processing, regulation
Abstract
The genes pcbAB, pcbC and penDE encoding the enzymes (c~-aminoadipyl-cysteinyl-valinesynthetase, isopenicillin N synthase and isopenicillin N acyltransferase, respectively) involved in the biosynthesis of penicillin have been cloned from Penicillium chrysogenum and Aspergillus nidulans. They are clustered in chromosome I (10.4 Mb) of P. chrysogenum, in chromosome II of Penicillium notatum (9.6 Mb) and in chromosome VI (3.0 Mb) of A. nidulans. Each gene is expressed as a single transcript from separate promoters. Enzyme regulation studies and gene expression analysis have provided useful information to understand the control of genes involved in penicillin biosynthesis. The enzyme isopenicillin N acyltransferase encoded by the penDE gene is synthesized as a 40 kDa protein that is (self)processed into two subunits of 29 and 1 l kDa. Both subunits appear to be required for acylCoA 6-APA acyltransferase activity. The isopenicillin N acyltransferase was shown to be located in microbodies, whereas the isopenicillin N synthase has been reported to be present in vesicles of the Golgi body and in the cell wall. A mutant in the carboxyl-terminal region of the isopenicillin N acyltransferase lacking the three final amino acids of the enzymes was not properly located in the microbodies and failed to synthesize penicillin in vivo. In C. acremonium the genes involved in cephalosporin biosynthesis are separated in at least two clusters. Cluster I (pcbAB-pcbC) encodes the first two enzymes (~-aminoadipyl-cysteinyl valine synthetase and isopenicillin N synthase) of the cephalosporin pathway which are very similar to those involved in penicillin biosynthesis. Cluster II (ceJEF-cefG), encodes the last three enzymatic activities (deacetoxycephalosporin C synthetase/hydroxylaseand deacetylcephalosporin C acetyltransferase) of the cephalosporin pathway. It is unknown, at this time, if the cey'D gene encoding isopenicillin epimerase is linked to any of these two clusters. Methionine stimulates cephalosporin biosynthesis in cultures of three different strains of A. chrysogenum. Methionine increases the levels of enzymes (isopenicillin N synthase and deacetylcephalosporin C acetyltransferase) expressed from genes (pcbC and cefG respectively) which are separated in the two different clusters of cephalosporin biosynthesis genes. This result suggests that both clusters of genes have regulatory elements which are activated by methionine. Methioninesupplemented cells showed higher levels of transcripts of the pcbAB, pcbC, cejEF genes and to a lesser extent of cefG than cells grown in absence of methionine. The levels of the cefG transcript were very low as compared to those ofpcbAB, pcbC and cefEE The induction by methionine of transcription of the four cephalosporin biosynthesis genes and the known effect of this amino acid on differentiation ofA. chrysogenum indicates that methionine exerts a pleiotropic effect that regulates coordinately cephalosporin biosynthesis and differentiation.
228 Introduction Penicillins and cephalosporins are synthesized by the condensation of the three precursor amino acids L-saminoadipic acid, L-cysteine and L-valine. Pools of these three amino acids specific for/3-1actarn antibiotic biosynthesis (separate from the main pool of each amino acid) have been proposed (Htinlinger & Kubicek 1989b) but the evidence in support of this proposal is very scanty. ~-Aminoadipic acid-requiring lysine auxotrophs are blocked in penicillin biosynthesis, thus confirming the role of this precursor amino acid in the biosynthesis of fl-lactams (Somerson et al. 1961; Luengo et al. 1980). However, the production of fllactam antibiotics in prototrophic strains is not usually stimulated by addition of exogenous o~-aminoadipic acid, cysteine or valine. Only when the cultures were limited by the availability of a nitrogen source was penicillin biosynthesis stimulated by the addition of ~-aminoadipic acid (Revilla et al. 1986; H6nlinger & Kubicek 1989a). The three amino acids are activated and linked together to form the tripeptide 6-(L-o~-aminoadipyl)L-cysteinyl-D-valine (ACV) by the enzyme ACV synthetase, a multifunctional enzyme that carries out the activation of the three amino acids with ATP (presumably as aminoacyl adenylates) and the epimerization of L- to D-valine before forming the tripeptide. The ACV synthetase is encoded by the pcbAB gene, an unusually large gene that was cloned from P. chrysogenum by Diez et al. (1990) and Smith et al. (1990) and from Acremonium chrysogenum by Guti6rrez et al. (1991). The deduced molecular weight of the ACV synthetases of P chrysogenum and A. chrysogenum were 425971 and 414791 respectively. The ACV tripeptide is cyclized by the enzyme isopenicillin N synthase (cyclase) to form isopenicillin N (Fig. 1), an intermediate that shows a weak penicillin activity. The gene pcbC encoding isopenicillin N synthase was the first cloned gene of the penicillin and cephalosporin pathways (Samson et al. 1985; Barredo et al. 1989) and a very similar gene has been also cloned from A. nidulans (Ram6n et al. 1987) and from six different bacteria which produce fl-lactam antibiotics (see reviews by Martin et al. (1992) and Aharonowitz et al. (1993)). In the last step of the penicillin biosynthetic pathway, the c~-aminoadipyl side chain of isopenicillin N is removed and exchanged by a phenylacetyl or phenoxyacetyl side chain in the penicillin G or penicillin V fermentations, respectively. The biochemistry of this
reaction is complex since five-related enzyme activities are encoded by the same gene (penDE) (Alvarez et al. 1993). The penDE gene was cloned from P. chrysogenum (Barredo et al. 1989) and A. nidulans (Montenegro et al, 1990). This gene is absent from the cephalosporin producer A. chrysogenum (Gutirrrez et al. 1991a). The first two genes of the penicillin pathway contain no introns and are very similar to those found in a variety of/3-1actam producing bacteria whereas the last gene (penDE) contains three introns and appears to be of fungal origin (Aharonowitz et al. 1993). The first two steps of the cephalosporin pathway in A. chrysogenum are identical to those of the penicillin pathway in P. chrysogenum. In cephalosporin producers isopenicillin N is later isomerized to penicillin N by the enzyme isopenicillin N epimerase encoded by the ceJD gene. Although the ceflD gene has been cloned from Streptomyces clavuligerus (Kovacevick et al. 1990) and Nocardia lactamdurans (Coque et al. 1993b), repeated attempts to clone the corresponding gene from A. chrysogenum have failed. The five-membered ring of penicillin N is expanded to a six-membered dihydrothiazine ring of deacetoxycephalosporin C by the deacetoxycephalosporin C synthase (so-called expandase) and the deacetoxycephalosporin C is later hydroxylated at the C3 ~ exocyclic methyl group to give deacetylcephalosporin C by the enzyme deacetoxycephalosporin C hydroxylase. In A. chrysogenum both the deacetoxycephalosporin C synthase and hydroxylase activities are encoded by a single gene ceflEF whereas in S. clavuligerus and N. lactamdurans they are encoded by two separate genes (ceflE and cefl?) which strongly ressemble each other (see reviews by Martfn (1992) and Aharonowtiz et al. (1993)). In the final step of cephalosporin biosynthesis, the intermediate deacetylcephalosporin C is acetylated to cephalosporin C by the enzyme deacetylcephalosporin C acetyltransferase (DAC-AT) that uses acetyl-CoA as donor of the acetyl group. This enzyme is encoded by the cefG gene (Gutirrrez et al. 1992; Matsuda et al. 1992). Very similar genes occur in cephamycin-producing bacteria (Coque et al. 1993a). None of the cephalosporin biosynthetic genes except the last one (cefG) contain introns. The cefG gene contains two introns and is in fact very similar to the met2 of filamentous fungi, what suggest that it has evolved separatedly from the other genes of the pathway. Indeed, no counterpart of the cefG is known in the cephamycin pro-
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ducing bacteria which do not appear to form significative amounts o f cephalosporin C, but instead convert the deacetylcephalosporin C intermediate into different cephamycins by adding either a carbamoyl group or an aromatic acyl residue by the action of the so-called late enzymes o f the pathway (Martfn & Liras 1989).
Results
Organization and expression of the penicillin gene cluster The three genes of the penicillin biosynthetic pathway
pcbAB (D/ez et al. 1990; Smith et al. 1990), pcbC (Carr et al, 1986; Barredo et al. 1989a) and penDE (Barredo et al. 1989b; Tobin et al. 1991) ofP. chryso-
230
genum have been cloned and shown to be linked in a single cluster (Diez et al. 1989; Dfez et al. 1990; Martin 1992). Similarly, the penicillin biosynthetic genes of A. nidulans were found to be linked in a single cluster (MacCabe et al. 1990; Montenegro et al. 1990, 1992). When the cluster of penicillin biosynthetic genes of P. chrysogenum was introduced in Neurospora crassa or Aspergillus niger (two species which do not form penicillin in nature) small levels of penicillin were synthesized (Smith et al. 1990b). This result indicates that all genetic information required for penicillin biosynthesis is contained in the cloned R chrysogenum DNA fragment that carries the penicillin gene cluster. However, the low level of penicillin produced in A. niger and N. crassa suggest that these fungi lack activator proteins or enhancer sequences that may not be present in the cloned DNA fragment. The gene encoding ACVS was designated pcbAB because two genetic loci were supposed to be responsible for synthesis of the c~-aminoadipyl-cysteine(AC) dipeptide and the ACV tripeptide. However, genetic evidence indicates that a single gene encodes all the activities needed to form ACV. Diez et al. (1990) cloned the pcbAB gene encoding ACVS by complementation of the ACVS defective mutant npe5 and npe 10. The pcbAB gene restored penicillin production to mutant npe5 but not to npelO (a deletion mutant that lacks also the pcbC and penDE genes). Transcriptional mapping studies revealed a long transcript of about 11.5 kb. The approximate transcription initiation and termination sites were identified and the entire DNA region covering them sequenced. An unusually large open reading frame (ORF) was found containing 11,376 bp that encodes a protein of 3792 amino acids with a deduced molecular weight of 425,971. Similar findings were reported independently from a different P. chrysogenum strain (Smith et al. 1990a; Smith et al. 1990b). No introns were found in the P. chrysogenum gene (or other fungal pcbAB genes). A conspicuous feature of the ACVS protein sequence is the presence of three repeated regions, or domains, each containing more than 500 amino acids. They show extensive sequence similarity with the Bacillus brevis peptide synthetases, gramicidin S synthetase I and tyrocidin synthetase I, and two other enzymes that carry out ATP-pyrophosphate exchange reactions, luciferase and 4-coumerate-CoA ligase (Turgay et al. 1992; Aharonowitz et al. 1993). All of these enzymes activate carboxyl groups present in amino acids, hydroxy acids, or simple acids. A multienzyme thiotemplate mechanism has been proposed for
the ACVS reaction (Dfez et al. 1990; Gutirrrez et al. 1991; Coque et al. 1991; Aharonowitz et al. 1993). 'The three domains of ACVS probalby specify, therefore, the amino acid-activating centers. The P. chrysogenum pcbAB gene is separated from the two remaining genes needed for penicillin production, pcbC and penDE, by about 1100 bp and is transcribed in opposite orientation to them (Dfez et al. 1989) (Fig. 1). Transcriptional mapping studies showed the presence of one long transcript of about 11.5 kb, which hybridized with several probes internal to the pcbAB gene, and two small transcripts of 1.15 kb, which hybridized with the pcbC or thepenDE gene, respectively (Fig. 2). Probes internal to the pcbAB gene hybridized with an 11.5 kb transcript and also with bands of smaller size (Fig. 2). Two hypothesis may explain this result. Either the large 11.5 kb transcript is broken down during the isolation procedure or transcription of the pcbAB may terminate prematurily, thus forming incomplete proteins. Proteins of smaller size than that of ACVS that react with anti-ACVS antibodies are often found in P. chrysogenum cultures (J. Velasco & J.F. Martfn, unpublished). These proteins are probably unable to synthesize the complete ACV molecule. Similar pcbAB genes encoding ACV synthetases have been cloned fromA, chrysogenum (Gutirrrez et al. 1991a), A. nidulans (MacCabe et al. 1991; Montenegro et al. 1993) and Nocardia lactamdurans (Coque et al. 1991a). A 24 kb region ofA. chrysogenum C10 DNA was cloned by hybridization with the pcbAB and pcbC genes of P. chrysogenum. A functional c~aminoadipyl-cysteinyl-valine (ACV) synthetase was encoded by a 15.6 kb EcoRI-BamHI DNA fragment, as shown by complementation of the ACV synthetasedeficient mutant npe5 of R chrysogenum. The pcbAB was found to be closely linked to the pcbC gene forming a cluster of early cephalosporin biosynthetic genes (Gutirrrez et al. 1991b) (Fig. 3). A 3.2 kb BamHI fragment of this region complemented the mutation in the structuralpcbC gene oftheA, chrysogenum N2 mutant, resulting in cephalosporin production. Two transcripts of 11.4 and 1.15 kb were found by Northern hybridization ofA. chrysogenum RNA with probes internal to the pcbAB and pcbC genes, respectively (Fig. 4). The A. nidulanspcbAB gene encodes a protein of 3770 amino acids with a predicted molecular weight of 422,486. The pcbAB and pcbC genes are tightly linked in A. nidulans (as in A. chrysogenum) and are transcribed from an intergenic region of 872 bp in A. nidulans (as compared to 1233 bp in A. chrysogenum).
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The isopenicillin N synthase (pcbC) gene The gene encoding isopenicillin N synthase was first cloned from A. chrysogenum (Samson et al. 1985) and P. chrysogenum (Carret al. 1986; Barredo et al. 1989a). The pcbC gene does not contain introns and is expressed in E. coli. The pcbC genes of both fungi encode enzymes of Mr about 37,900 which agrees with the reported value of 39000-t-1000 for the purified protein from P. chrysogenum (Ramos et al. 1985). The pcbC gene of A. nidulans is also very similar to those of P. chrysogenum and A. chrysogenum. The deduced protein (Mr 37480; 331 amino acids) shows extensive homology to the pcbC genes of other fungi. In addition to the three fungal pcbC genes, other isopenicillin N synthase genes have been cloned from several cephamycin producing actinomycetes (Strep-
tomyces clavuligerus, S. griseus, S. jumonjinensis, S. lipmanii, Nocardia lactamdurans, Flavobacterium sp., and Lysobacter lactamgenus (see revies by Martfn (1992) and Aharonowitz et al. (1993)). In both P. chrysogenum and A. chrysogenum the pcbC genes are transcribed as a 1.1 kb transcript (Figs. 2 and 4) and the encoded protein does not seem to be posttranslationally cleaved since the amino terminal end of the proteins correspond to the amino acid sequence deduced from the gene.
Expression of the penDE gene and processing of the isopenicillin N acyltransferase P. chrysogenum and A. nidulans produce penicillins with acyl side chains formed from a variety of intracellular or exogenously supplied carboxylic acids. When exogenous phenylacetic acid or phenoxyacetic acid is provided, penicillins with phenylacetyl or phenoxyacetyl side chains, e.g. penicillin G and V are produced (Fig. 1). The biosynthesis of penicillin G and V occurs by replacement of the L-c~-aminoadipyl side chain o f l P N with the phenylacetyl moiety of phenylacetyl-CoA (or the phenoxyacetyl moiety of phenoxyacetylCoA, respectively) by the isopenicillin N:acyl-CoA acyltransferase (IAT). This enzyme occurs in penicillinproducing strains but it is absent in C. acremonium and other cephalosporin producers (Alvarez et al. 1987) due to the lack of the corresponding gene in this fungus (Gutirrrez et al. 1991a). This is a good example of the restricted distribution of secondary metabolites among related fungi due to the absence of a particular gene in certain genera (Martin & Gutirrrez 1992). The nature of the last enzymatic step of penicillin biosynthesis and the role of 6-APA as a putative intermediate in penicillin biosynthesis has remained obscure for many years (see reviews by Queener &
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(A) Biosynthetic pathway of cephalosporin C and cephamycin C. (B) Physical map of a 30 kb region of N. lactamdurans DNA carrying 13 genes related to cephamycin biosynthesis and resistance, lat, lysine-6-aminotransferase; pcbAB, unusually large gene encoding c~-aminoadipyl-cysteinyl-valine synthetase; pcbC, isopenicillin N synthase; ceJD, isopenicillin N epimerase; ceJE, deacetoxycephalosporin C synthase (expandase); cmcT, transmembrane protein; ORF7-ORF10, late genes involved in the conversion of deacetoxycephalosporin C to cephamycin C; bla,/3-1actamase;pbp, penicillin-binding protein. ORF12, short gene of unknown function. (C) Maps of the clusters of early cephalosporin biosynthetic genes (pcbAB-pcbC) and late genes (cej'
