THE JOURNAL OF BI~L~CICAL CHEMISTRY (0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 27, Issue of September 25, pp. 16358-16365, 1990 Printed in U.S A

The Cluster of Penicillin Biosynthetic Genes IDENTIFICATION AND CHARACTERIZATION OF THE pcbAB GENE ENCODING THE o(-AMINOADIPYL- CYSTEINYL-VALINE SYNTHETASE AND LINKAGE TO THE p&C! AND penDE GENES*

(Received for publication, April 2, 1990)

Bruno Diez& Santiago Guti&rez$, Jose L. BarredoSll, Piet van Solingen , Lucia H. M. van der Voort II, and Juan F. Martin+ From the $Section of Microbiology, Department of Ecology, Genetics and Microbiology, University of Ledn, 24071 Ledn, Spain and the IIGist Brocades, Wateringseweg 1, P. 0. Box 1, 2600 Ma. De&, Holland, The Netherlands

Penicillium chrysogenum DNA fragments cloned in EMBLS or cosmid vectors from the upstream region of the gcbC-penDE cluster carry a gene (pcbAB) that complemented the deficiency of a-aminoadipyl-cystei- nyl-valine synthetase of mutants npe5 and npel0, and restored penicillin production to mutant npe5. A pro- tein of about 250 kDa was observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels of cell- free extracts of complemented strains that was absent in the npe5 and npel0 mutants but exists in the paren- tal strain from which the mutants were obtained. Tran- scriptional mapping studies showed the presence of one long transcript of about 11.5 kilobases that hybridized with several probes internal to the pcbAB gene, and two small transcripts of 1 .15 kilobases that hybridized with the pcbC or the penDE gene, respectively. The transcription initiation and termination regions of the pcbAB gene were mapped by hybridization with sev- eral small probes. The region has been completely se- quenced. It includes an open reading frame of 11,376 nucleotides that encodes a protein with a deduced M, of 425,97 1. Three repeated dominia were found in the cy-aminoadipyl-cysteinyl-valine synthetase which have high homology with the gramicidin synthetase I and tyrocidine synthetase I. The pcbAB is linked to the pcbC and penDE genes and is transcribed in the oppo- site orientation to them.

Penicillins, cephalosporins, and cephamycins are /3-lactam antibiotics formed by condensation of L-a-aminoadipic acid (an intermediate in the lysine biosynthetic pathway in fungi), L-cysteine and t-valine (see review by Martin and Liras, 1). The three amino acids are linked together to form the tripep- tide 6 (L-a-aminoadipyl)-L-cysteinyl-D-valine (ACV)’ which is the first common intermediate of penicillins and cephalo- sporin (Fig. 1) (2). ACV is then oxidatively cyclized by removal

* This work was supported by a grant from Gist-Brocades, The Netherlands. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact.

The nuckotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) 505604.

$ Recipient of a fellowship from the Diputacibn de Le6n, Spain. ?I Supported by a fellowship from the Ministry of Education and

Science of Spain. ’ The abbreviations used are: ACV, 6(a-aminoadipyl)-cysteinyl-

valine; kb, kilobase pairs; SDS, sodium dodecyl sulfate; PAGE, poly- acrylamide gel electrophoresis; ORF, open reading frame; bp, base pairs.

of four hydrogen atoms to form the @-lactam-thiazolidine nucleus of isopenicillin N. From here the pathway diverges to hydrophobic penicillins in filamentous fungi and to cephalo- sporins and cephamycins in various molds and actinomycetes (1,3).

Several factors affecting the ACV synthesis in vivo have been characterized in low and high penicillin-producing cul- tures (4). ACV formation is stimulated when protein synthesis is blocked with cycloheximide or anisomicin indicating that it is synthesized by a non-ribosomal mechanism (4). ACV synthesis might be the rate-limiting step in biosynthesis of penicillins and cephalosporins and is known to be regulated by glucose in Penicillium chrysogenum and Nocardia lactam- durans (5, 6), by phosphate in Streptomyces clavuligerus (7) and by ammonium in Cephalosporium acremonium (8, 9).

Two genes encoding enzymes of the penicillin biosynthetic pathway, pcbC (isopenicillin N synthase) (10, 11) and penDE (acyl-CoA:6-APA acyltransferase) (12), have been cloned. Lit- tle information is available on enzymes involved in ACV tripeptide biosynthesis. Cell-free systems catalyzing ACV for- mation have been described for C. acremonium (13-15) and in S. clauuligerus (16). A multifunctional peptide synthetase that catalyzes the formation of ACV has been recently purified from Aspergillus nidulans (17). Since the molecular weight of the ACV synthetase of A. nidulans is 220 kDa, at least 7 kb of DNA would be required to encode this multifunctional peptide synthetase. The finding of the linkage between the genes pcbC and penDE in a 5.1-kb Sal1 DNA fragment (18) suggested that the gene(s) encoding ACV synthetase @cbA and pcbB, or pcbAB if the two proposed enzyme activities are encoded by a single gene) (19) might be located in the same DNA region. Since we had available cosmid and phage (EMBL3) libraries of P. chrysogenum DNA (11, 12, 18), it was of great interest to clone the gene of P. chrysogenum encoding the ACV synthetase. This was done using two dif- ferent strategies, 1) complementation of mutants of P. chry- sogenum blocked in penicillin biosynthesis and 2) transcrip- tional mapping of the regions around thepcbc-penDE cluster.

We report in this article the isolation, sequentiation, and characterization of a 17.6-kb stretch of contiguous DNA en- coding the ACV synthetase which is linked to the pcbC and penDE genes that encode the two other enzymes involved in penicillin biosynthesis. The gene encoding ACV synthetase @cbAB) is transcribed into a main transcript of about 11.5 kb.

EXPERIMENTAL PROCEDURES

Microorganisms and Vectors Used-P. chrysogenum AS-P-78, a strain provided by Antibibticos, S. A. (Lebn, Spain) (5, 20) was used as a source of DNA. Mutants of P. chrysogenum Wis 54-1255 npe5

16358

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The a-Aminoadipyl-cysteinyl- Valine Synthetase Gene 16359

6-(La-Aminoadipyl)-L-cysteinyl-o-valine

FIG. 1. The first step of the penicillin biosynthetic pathway. Condensation of amino acids and isomerization reactions carried out by the ACV synthetase. Note the change in configuration from the L-valine to the D-configuration.

and npel0 deficient in ACV synthesis were isolated by Cantoral and Guti&rrez* among a number of mutants impaired in penicillin biosyn- thesis (ripe) (21).

Escherichia coli DH5a (22) was used as the recipient strain for high frequency plasmid transformation (lo’-IO8 transformants/pg of DNA) and E. coli LE392 as a host for XEMBLS phage derivatives (23). E. coli MV1193 (24) was used as the host strain for obtaining single-stranded DNA from the pBluescript plasmids.

XEMBLB (25) was used as a vector for cloning large DNA fragments (17-20 kb) of P. chrysogenum (11, 12). pBluescript plasmids KS(+) and KS(-) (Stratagene, La Jolla, CA) were used to subclone DNA fragments for sequencing. Phage M13K07 (26) was used as a “helper” in the infection of strain MV1193 with the pBluescript plasmids.

Southern Blotting and Sequencing-Southern blotting of DNA fragments was carried out according to Maniatis et al. (23). DNA fragments were sequenced by the dideoxynucleotide method (27) using the Sequenase system 2.0 (U. S. Biochemicals, Cleveland, OH). Double sequencing reactions with dGTP + dITP were used in some clones to avoid errors (28).

mRNA Isolation and Northern Blotting-Total RNA was obtained by the phenol/SDS method as described by Ausubel et al. (29). RNA (5 pg) was run in a 0.7% agarose-formaldehyde gel using RNA molecular weight markers (E. coli 16 S and 23 S rRNAs and Sets I and III, Boehringer Mannheim). The gel was blotted onto a nitrocel- lulose filter by standard methods, baked in a vacuum oven at 80 “C for 2 h, prehybridized at 42 “C overnight in 50% formamide, 5 X Denhardt, 5 x SSC, 0.1% SDS, and 500 pg/ml denatured salmon sperm DNA and hybridized in the same buffer containing 100 fig/ml of denatured salmon sperm DNA at 42 “C for 24 h. Afterward, the filter was washed twice in 2 x SSC (standard sodium citrate), 0.1% SDS at room temperature for 15 min, twice again in 0.1 X SSC, 0.1% SDS at room temperature for 15 min and once more in 0.1 x SSC, 0.1% SDS at 55 “C, and autoradiographed using Amersham Corp. x- ray film.

Transformation of Penicillium and Gene Expression-Transfor- mation of protoplasts of P. chrysogenum was carried out as described previously (30, 31). Expression of the cloned gene in the transform- ants was studied by measuring ACV synthetase activity.

ACV Synthetase Assay-ACV synthetase was quantified by the ATP-dependent [‘4C]valine (290 mCi/mM) incorporation into ACV assay as described by Van Liempt et al. (17). In SDS-PAGE (7.5%), cell-free extracts of transformed or untransformed strains were pre- cipitated with trichloroacetic acid at a final concentration of 10% and applied to the gel.

RESULTS

Cloning of Large DNA Fragments from P. chrysogenum- Initial characterization of mutants blocked in penicillin biosynthesis3 indicated that the ACV synthetase of P. chry-

‘J. M. Cantoral and S. Gutibrrez, unpublished results. ‘J. M. Cantoral, J. M. GutiBrrez, and J. F. Martin, unpublished

results.

sogenum in SDS-PAGE gels had an approximate molecular mass of 250 kDa (see below), slightly higher than the 220 kDa reported for the ACV synthetase of A. nidulans (17). Since a gene encoding this enzyme was expected to occupy about 9 kb, two genomic libraries of P. chtysogenum AS-P-78 DNA fragments were constructed in X-derived replacement vector EMBL3 and also in pPSO7, a pJB8-derived cosmid vector.

A 19.5-kb DNA fragment was cloned in phage 1A. Frag- ments of similar size were cloned in phages 1,6,6A, 12A, and 16A (12). All of them were initially selected by hybridization with probes corresponding to the amino-terminal ends of the isopenicillin synthase or the acyl-CoA:B-APA acyltransferase (11, 12). A large DNA fragment (22 kb) that contains 7.5 additional kb relative to the insert in phage 6A (upstream of the previously cloned DNA fragments in phages 1A or 6A that carried the p&C and penDE genes) was cloned in the vector pPSO7 yielding cosmid HM193. A simplified map of the DNA region, which contains the penicillin gene cluster, cloned in phage lA, 6A, and cosmid HM193 is shown in Fig. 2.

Complementation of ACV Synthetase-deficient Mutants- Mutants npe5 and npel0 of P. chrysogenum, which are defi- cient in ACV synthetase were isolated previously. Mutant npe5 contains a point mutation whereas npel0 is a deletion mutant. In order to test if the cloned fragments carried the ACV synthetase gene, mutants npe5 and npel0 were trans- formed with different recombinant phages. Since the phages or cosmids do not contain a selective marker, co-transforma- tion was made with plasmid pULJL43 that carries the phleo- mycin resistance marker.4 Alternatively, cotransformations of npe5 and npel0 were also made using cosmid HM193 and plasmid pGJO2 which contains both the phleomycin-resist- ante marker (expressed from the phosphoglycerate kinase promoter) (32) and the 5.1-kb Sal1 fragment (that carries the p&C and penDE genes) (Fig. 3). Transformants selected for resistance to phleomycin were tested for complementation of the ACV synthetase deficiency and for restoration of the penicillin biosynthetic pathway which is blocked in mutants npe5 and npel0.

Cotransformation with cosmid HM193 and pGJ02 comple- mented the ACV synthetase deficiency and restored penicillin production in both npe5 and npel0 (Table I), whereas trans- formation with HM193 and pULJL43 complemented ACV synthetase deficiency in both mutants but restored penicillin production only to mutant npe5.

ACV Synthetase Activity in npe5 Transformants-ACV synthetase activity was assayed in untransformed npe5 and also in npe5 transformed with pGJ02 (which carries the pcbC+penDE genes) or pGJ02 and HM193 (Table II). Control assays were run in parallel with extracts obtained from P. chrysogenum Wis 54-1255 (the parental strain from which the npe mutants were derived).

Since formation of the ACV tripeptide from the three amino acids in the L-configuration takes place directly in the in vitro reaction (without significative release of the dipeptide AC), the ACV synthetase carries out the entire synthesis of the tripeptide including 1) activation of the component amino acids, 2) condensation, and 3) epimerization of valine from the L to the D-configuration, as proposed previously (1, 17).

Formation of a Large Protein (250 kDa) in npe5 Transform- ants-Cell-free extracts of strains npe5 and npel0 mutants lack a large protein of about 250 kDa in SDS-PAGE gels which is present in the parental strain Wis 54-1255, that has been identified as the denatured form of the ACV synthetase.”

’ J. L. Barredo, unpublished results.

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16360 The (

FIG. 2. Restriction map of the P. chrysogenum DNA region which contains the gene cluster for peni- cillin biosynthesis indicating the po- sition and orientation of the pcbAB, pcbC, and penDE genes (thick ar- rows) (see text for details). The DNA fragments cloned in pHM193, EMBL3 6A, and EMBL3 1A are indicated by thin lanes in the bottom of the figure. Thin

pHM193 PcbAB pcb C pe”DE

EMElLS 6.4

arrows on the top correspond to the Sal1 fragments shown in detail in Fig. 6. The Sal1 indicated by an asterisk corresponds to the phage linker.

EMBW IA

FIG. 3. Plasmid pGJO2 used in P. chrysogenum cotransfor- mation experiments. pGJO2 carries thepcbC andpenDE genes and the phleomycin and ampicillin resistance markers (arrows). The phleomycin resistance gene is expressed from the P. chrysogenum phosphoglycerate kinase promoter @PGK).

The M, of 250,000 should be considered as tentative since it is difficult to determine precisely the molecular mass of a protein of such big size by PAGE. SDS-PAGE electrophoresis (Fig. 4) of transformant T20 (HM193) showed the presence of the 250-kDa protein with the same electrophoretic mobility as that in Wis 54-1255, i.e. this protein is encoded in the DNA fragment cloned in cosmid HM193. The correlation between the formation of this protein band and the presence in the extracts of the transformants of ACV synthetase activity further supports our initial observation that the 250-kDa protein corresponds to the denatured form of ACV synthetase.

Transcriptional Mapping of the Region Upstream of the pcbC-penDE Cluster-Total RNA of the high penicillin pro- ducing strain P. chrysogenum AS-P-78 (in which high expres- sion of thepcbAB gene was expected under optimal penicillin- producing conditions) was obtained as described in “Experi- mental Procedures.” Northern hybridizations were carried out with several probes corresponding to the subcloned Sal1 frag- ments of 1.7, 2.6, 6.0, and 2.2 kb (Fig. 2) and also with the small probes A, B, C, D, E, F, G, and H indicated in Fig. 6 to define the ends of the transcript. In addition, the RNA was hybridized with a NcoI fragment of 1004 nucleotides that contains the pcbC gene and with a XmnI-XbaI fragment that carries the penDE gene.

Results (Fig. 5) indicated that whereas thepcbC (orpenDE) gene forms a small discrete transcript of about 1.15 kb (lane B), a long ORF located upstream of the pcbC-penDE cluster (ORFl in Fig. 2) originates a transcript of about 11.5 kb (lane A). Some smearing was obtained in all hybridizations carried out with different ACV synthetase probes from either the 5’ (probes E, F, G) or 3’ (probes C, D) regions using three distinct preparations of RNA, but no smearing was observed when the same preparations of RNA were hybridized with pcbC or penDE probes (lane B). This suggests that the smearing is due to physical breakdown of the long ACV synthetase mRNA during isolation. In all cases the largest size found was con- stant (about 11.5 kb) in the three preparations of RNA by hybridization with 14 different internal probes.

Orientation of the Gene-The direction of transcription of the ACV synthetase gene has been determined by RNA hy-

t I 5kb

bridization. Dot blots were made using P. chrysogenum RNA, isolated from producing mycelium. A short piece of the inter- nal Hind111 fragment (1.5 kb, Fig. 2) was sequenced and oligonucleotide probes were synthesized in both polarities. Oligonucleotides were labeled using polynucleotide kinase and were hybridized according to standard procedures. The probes having a polarity opposite to the IPNS (pcbC gene) mRNA did not hybridize to the RNA spots, while probes with a polarity identical to the IPNS mRNA did hybridize. We concluded that the pcbAB gene is transcribed in opposite direction to the pcbC gene.

This result has been confirmed by nucleotide sequence data (see below). Therefore, the intergenic region between the pcbAB andpcbC genes (size 1.0 kb) contains the promoters of the pcbC and pcbAB genes which initiate transcription in opposite orientations.

Transcript Initiation and Termination Regions-The 3’ end of the gene (distal with respect to the pcbC and penDE genes) was mapped by Northern hybridizations with small probes A (450-bp SalI-EcoRV), B (187-bp EcoRV-XbaI), C (578-bp XbaI-EcoRI), and D (442-bp EcoRI-SalI) (Fig. 6). Results indicated that the transcript hybridized clearly with probes C and D, whereas it did not hybridize with probes A and B, i.e. the transcription terminates in the region delimited by the XbaI and EcoRI sites in Fig. 6.

Similarly, the 5’ end of the transcript was shown to occur in the BamHI-EcoRI (496 bp) fragment internal to the 2.2- kb Sal1 fragment and more exactly between the two XhoI sites indicated by an asterisk in Fig. 6. This result was con- firmed by the nucleotide sequence determination that identi- fied the ATG translation initiation triplet in this region.

Subcloningand Sequencing of thepcbAB-The entire region encoding the pcbAB, pcbC, and penDE genes was mapped with several restriction enzymes (Fig. 6). Five Sal1 fragments of 1.7, 2.6, 6.0, 2.2, and 5.1 kb (from left to right in Fig. 2) were subcloned in pBluescript KS(*) originating pSAL1.7, pSAL2.6, pSAL6.0, pSAL2.2, andpSAL5.1, respectively. Each plasmid was obtained in the two opposite orientations. Simi- larly, EcoRI and BamHI fragments (overlapping with the Sal1 fragments) were cloned in pEC00.5, pEC08.0, pEC02.5, pBAM2.4, pBAM2.6, and pBAM4.6 where the numbers indi- cate the size in kb of the respective DNA fragments inserted in pBluescript KS(?). Other small fragments were subcloned from these plasmids and used for sequentiation.

The nucleotide sequence of a 12.36-kb region encoding the entire ACV synthetase gene is indicated in Fig. 7. A very long ORF of 11,376 nucleotides was found that matched the tran- script initiation and termination regions identified by North- ern hybridization. This ORF contained a G+C content of 54.38% and the codon usage was typical of that of filamentous fungi (33). The presence of a long ORF was confirmed by a

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The a-Aminoadipyl-Cysteinyl-Valine Synthetase Gene

TABLE I

16361

Complementation of mutants blocked in penicillin biosynthesis by cosmid HM193 carrying the ACV synthetase gene Transformation of P. chrvsosenum nrotonlasts was carried out as described nreviouslv (30, 31).

DNA used in transformation

Complemented strains

npe5”

None

No. of transformants

tested

31

No. of pen+

clones

0

pGJOZ(pcbC+penDE) HM193+pGJOZ

No. of No. of No. of No. of transformants pen+ transformants pen+

tested clones tested clones

72 1” 19 5 &elO* 29 0 62 0 8 2

’ Reversion frequency of npe5 is 4%. ’ Mutant npel0 is a deletion mutant without reversion.

TABLE II ACV synthetase actiuity in npe5 transformed with cosmid HM193

Assay of ACV synthetase was essentially as described by Van Liempt et al. (17). Extraction with buffer A (0.1 M Tris-HCl, pH 7.5, 0.1 MKCl, 10 mM dithiothreitol, 1 mM EDTA, 45% glycerol) was for 30 min. [“C]Valine (290 mCi/mM) 12.5 rCi/reaction mixture was added, and the reaction was stopped after 30 min. The reaction mixture was precipitated with trichloroacetic acid (final concentra- tion 10%) and applied to Porapak Q (Serva, Heidelberg) columns. The column was washed with 2 ml of equilibration buffer and eluted twice with 1 ml of methanol. The ACV formed was quantified by high performance liquid chromatography. Samples of 100 ~1 were injected on a RP18 Waters column and eluted with 10% methanol in 50 mM KH2P04, pH 6.0, containing 0.1 M dithiothreitol at room temperature. Flow rate was 1.0 ml/min and detection was with a Berthold LB503 scintillation detector employing a 200-~1 cell. The labeled peak was collected, and the amount of label was determined by counting in a liquid scintillation counter.

A B

itskbw

- 74

- 53

- 2.8

tekbw

- 1.9 -16

w- 1.0 - 0.6

Transformants

P. chrysogenum Wis 54-1255 P. chrysogenum npe5 P. chrysogenum npe5 TlO

(pcbC+penDE) P. chrysogenum npe5 T20

(HM193+ncbC+oenDE)

ACV synthetase activitv Penicillin

formation +ATP -ATP

/“C]ualine incorporated into bioassay

ACV, IO” dpm 490.8 ND +

3.3 ND - 2.5 ND -

261.3 1.1 +

’ No incorporation detected.

1 2 3 4 5

-- Q 0250 KDa

-

FIG. 4. SDS-PAGE (7.5%) of cell-free extracts of control npe5 mutant and complemented clones. Cell-free extracts were prepared as described in Van Liempt et al. (17). Protein solutions were precipitated with trichloroacetic acid at a final concentration of 10% and applied to the gel. Lane 1, npe5; lane 2, npe5 transformed with pGJO2 (pcbC, penDE) and pHM193; lane 3, npe5 transformed with pGJO2 (pcbC, penDE); lane 4, Wis 54-1255; lane 5, size marker: the upper band is 250 kDa (provided by H. Van Liempt, unpublished data). Note the 250-kDa protein in Wis 54-1255 and in npe5 trans- formed with pHM193 (arrow).

FIG. 5. Northern hybridization of total RNA of penicillin- producing cells (48 h of culture) of P. chrysogenum AS-P-78. RNA isolation and Northern blotting were carried out as described under “Experimental Procedures.” Lane A, hybridization with probe D. Lane B, hybridization with a probe carrying the pcbC gene (1004- bp NcoI fragment) RNA size markers (set I from Boehringer) are indicated by bars on the right. The arrows indicate transcripts of about 11.5 and 1.15 kb.

computer analysis of all possible reading frames and codon preference study using the GCG Co. computer program. The protein encoded consisted of 3791 amino acids with a deduced M, of 425,971.

Presence of Three Repeated Dominia in the ACV Synthetase Protein-The deduced amino acid sequence of the ACV syn- thetase protein is shown in Fig. 7. A computer analysis of possible repeated dominia in the ACV synthetase showed the presence in this long protein of three repeated dominia which share extensive amino acid homology (Fig. 8). The first dom- inium extends from amino acid l-1068 (the homology is greater in the region of residues 301-935), the second from amino acid 1392-2154 (with higher interdominia homology in the region 1392-2020), and the third one from the residues 2474-3295. A good computer alignment was obtained between the three dominia of the enzyme.

A very interesting finding was the observation that the three dominia of the ACV synthetase shared an extensive amino acid homology with the tyrocidine synthetase I (TYl) and gramicidin synthetase I (GSl) of Bacillus breuis (small subunits of these peptide synthetases) encoded by the genes tycA and grsA, respectively (34, 35) but they do not show signifkative homology with the r-glutamylcysteine or gluta-

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The a-Aminoadipyl-Cysteinyl-Valine Synthetase Gene Sal I

XhoI Act I (Mbo I) EcoRV xbal Pvu II (Mbo 1) EcoRI Ava I C/a I Hind1

-A--B-

I 250 x0

AGT w

Him11 Sac I1 Sal I

sai I Accl “;“I

Hind111

Hind1 Ava I Hind1 PSI I Hind111

Sac 11 EcoRV Hind

Apn I Bsf EIl HincII EcoRV

\l I

85m

(Rsa I) Sal I Act 1

Sac11 BnmHI AccI Ava I EcoRV Pvu II

/

Sal I AvaI Sac1 Pvu II Sac II Avo I

Ace I \ ISac1 I /

HincIIxho I* xho I* BamI;f1 EcoRV\/ AvaI Am1 EcoRI

Sal I

GTA /

Sol I Act I Hin cII

XbOI 6% EII Apa I Ava I Sac I PSfI Nco I Him11 Hind1 Pst 1 EcoRV BumHI

\I ~/ / \/ _.i j i ,, ,, ,, /

FIG. 6. Detailed map of the 12.5-kb region encoding the ACV synthetase gene. Probes A-H used to establish the transcript-initiation and termination regions are indicated by arrowa above the DNA. Consecutive Sal1 fragments of 1.7, 2.6, 6.0, and 2.2 kb are shown starting at the top. The direction of transcription of thepcbAB gene is indicated by the thick arrow. The subclones used for sequencing are indicated by thin arrows in the bottom. The transcription initiation was mapped by hybridization inside the two XhoI sites indicated by asterisks.

thione synthetase of E. coli (36,37). The entire penicillin pathway is, therefore, encoded by a Linkage of the Three Penicillin Biosynthetic Genes--Re- 17.6-kb stretch of contiguous DNA (12.5 + 5.1-kb SalI frag-

striction mapping of the DNA fragments cloned in phages lA, ments, the latter, containing the p&C andpenDE genes). The 6A, and cosmid HM193 proved unequivocally that the ACV 17.6-kb stretch of native DNA forms part of a 35-kb region synthetase gene (pcbAB) is linked to pcbC and penDE genes that is greatly amplified (9-14-fold) in two high penicillin which encode isopenicillin N synthase and isopenicillin N producing strains (AS-P-78 and Pz) as compared with one of acyltransferase, respectively. the early parental strains Wis 54-1255 (38, 39). Nothing is

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The u-Aminoadipyl-cysteinyl- Valine Synthetase Gene 16363

known about the existence of regulatory genes controlling expression of the structural penicillin biosynthetic genes. Such regulatory genes, if they exist, may lay in this amplified region.

DISCUSSION

Condensation of the three precursor amino acids L-o-amino adipic acid, L-cysteine, and D-valine to form L-a-aminoadipyl- L-cysteinyl-D-valine, takes place by the action of a single enzyme (ACV synthetase) which activates the three amino acids in the L form, racemizes L to D-valine and carries out the polymerization steps to form the tripeptide. Two loci p&A and p&B were allocated for the enzymatic steps that form the dipeptide and tripeptide (19). However evidence of Banko et al. (15) and Van Liempt (17) in A. chrysogenum and A. niduluns, respectively, indicate that a single enzyme carries out the activation, racemization, and polymerization steps to form directly ACV.

The formation of ACV is similar in many aspects to the synthesis of other peptides of microbial origin which are formed by the nonribosomal enzyme template mechanisms

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16364 The a-Aminoadipyl-Cysteinyl- Valine Synthetase Gene

--------

GSSFRQWA---EAIQN RN---- SVCPKKRILFKAGHNG ADLLSE IALPEKTDSFKDWSIE ELFLEE

350 1445 2528

55 67

449 1540 2623

147 159

547 1636 2719

240 253

646 1736 2816

335 347

740 1832 2914

423 435

837 1924 3012

516 528

935 2020 3107

608 619

1028 2114 3202

700 711

1068 2154 3295

798 809

FIG. 8. Comparison of the amino acid sequences of the three dominia (A-C) of ACV synthetase with tyrocidine synthetase I and gramicidin synthetase I of B. brevis. Gaps have been introduced to get maximal alignment.Conservedamino acid sequences are shadowed.

(40, 41). Peptide synthetases require a specific spatial orga- nization to direct the activation and sequential polymerization of the component amino acids. The largest peptide synthe- tases consisting of a single polypeptide chain catalyze up to six consecutive steps corresponding to 18 reactions. This requires a very high molecular weight multienzymatic poly- peptide. The transcriptional mapping studies described in this work indicate that a long transcript of about 11.5 kb is formed from a region upstream of the p&C gene. This region contains a long ORF (11.37 kb) that encodes a protein of M, 425,971. This gene encodes the ACV synthetase as shown by comple- mentation of mutants npe5 (strain carrying a point mutation) and npel0 (a deletion mutant that lacks the ACV synthetase region and also the p&C and penDE genes).

A large protein of about 250 kDa was observed in rzpe5 and npel0 transformants that were missing in the untransformed

mutants. Since there is a correlation between the formation of this protein band and the presence in the extracts of the transformants of ACV synthetase activity, it seems that this protein band corresponds to a component of the ACV synthe- tase. This 250-kDa protein observed in SDS-PAGE may arise by denaturalization and cleavage of the native protein encoded by the 11.37-kb ORF. The native protein (Mr 426,000) may be processed into two subunits (see below).

An interesting result is the presence in the deduced ACV synthetase protein of three dominia with conserved amino acid sequences. These dominia show regions of very high similarity in the amino acid sequence with the B. breuis tyrocidine synthetase I and gramicidin synthetase I. The conserved amino acid sequences may represent centers in- volved in ATP-mediated activation of amino acids, since both TYl and GSl are involved in activation and racemization of

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The a-Aminoadipyl-Cysteinyl- Valine Synthetase Gene 16365

the amino acid phenylalanine (34, 35). The presence of three amino acid-activating dominia in the ACV synthetase is con- sistent with a similar organization that exists in the heavy tyrocydine synthetases II and III (TY2, 230 kDa; TY3, 460 kDa) which activate, respectively, three and six amino acids during tyrocidine biosynthesis. Biochemical data showed that TY2 and TY3 can be splitted after prolonged incubation into three or six amino acid-activating fractions of 70 kDa, respec- tively (42, 43). The 70-kDa fragments retained full activity for activating the appropriate amino acids (as determined by the amino acid-dependent ATP-pyrophosphate exchange) but could not carry out the polymerization reactions. During the initial steps of purification of the ACV synthetase, high mo- lecular forms observed by exclusion on gel filtration are trans- formed into the 250-kDa protein which loses most of the enzyme activity which suggests that the ACV synthetase is converted into a form with lower molecular weight. High molecular weight multienzyme synthetases are usually stable only within their natural environments or cellular compart- ments. This known instability of the peptide antibiotic syn- thetases may explain the differences between the deduced M, of the ACV synthetase and the protein band observed in SDS- PAGE gels.

The significance of the cluster of penicillin biosynthetic genes is intriguing. We have found that the penicillin biosyn- thetic genes are also linked in A. nidulans (44). Other fila- mentous fungi lack P-lactam biosynthetic genes or have them in a different arrangement (e.g. A. chrysogenum) (45) which indicates that linkage of genes has occurred during evolution and has been selected positively probably due to the ecological advantage conferred to the penicillin-producing strains by the coordinated expression resulting from the gene linkage in the fight against competing bacteria in the soil. The linkage of antibiotic biosynthetic genes is a well known phenomenon in many antibiotic-producing organisms (46). When this work was completed, a report has been published indicating that the npeA locus of A. niduluns consist of three contiguous genes (47). Although the authors do not provide evidence on the sequence of the ACV synthetase gene, their results in A. nidulum are consistent with the structural organization of the penicillin biosynthetic genes that we have found in P. chry- sogenum.

The lack of significative similarity between the pcbAB gene and the genes encoding the r-glutamyl-cysteine synthetase and the glutathione synthetase of E. coli is of interest since it indicates a different evolution of the E. coli glutathione syn- thesizing system and the ACV synthetase despite the apparent similarity in the structure of both tripeptides. Nothing is known, however, about the glutathione synthesizing enzymes in P. chrysogenum.

Acknowledgments-We thank J. Vara, Autonoma University of Madrid, for help with the GCG Co. computer program, and M. P. Puertas, B. Martin, and M. I. Corrales for technical assistance and to all colleagues (Lebn and Delft) for valuable discussions.

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B Díez, S Gutiérrez, J L Barredo, P van Solingen, L H van der Voort and J F Martínlinkage to the pcbC and penDE genes.

the pcbAB gene encoding the alpha-aminoadipyl-cysteinyl-valine synthetase and The cluster of penicillin biosynthetic genes. Identification and characterization of

1990, 265:16358-16365.J. Biol. Chem. 

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