cloning, characterization of the acyl-coa : 6-amino penicillanic acid acyltransferase gene of...

Mol Gen Genet (1990) 221:322-330 MGG- © Springer-Verlag !990

Cloning, characterization of the acyl-CoA:6-amino penicillanic acid acyltransferase gene of Aspergillus nidulans and linkage to the isopenicillin N synthase gene Eduardo Montenegro, Jose L. Barredo, Santiago Guti~rrez, Bruno Diez, Emilio Alvarez, and Juan F. Martin Section of Microbiology, Department of Ecology, Genetics and Microbiology, University of Le6n, 24071 Le6n, Spain

Summary. The penDE gene encoding acyl-CoA:6-amino penicillanic acid acyltransferase (AAT), the last enzyme of the penicillin biosynthetic pathway, has been cloned from the DNA of Aspergillus nidulans. The gene contains three introns which are located in the 5" region of the open reading frame. It encodes a protein of 357 amino acids with a molecular weight of 39 240 Da. The penDE gene of A. nidulans shows 73% similarity at the nucleotide level with the penDE gene of Penicillium chrysogenum. The A. nidulans gene was expressed in P. chrysogenum and complemented the AAT deficiency of the non-producer mutants of P. chrysogenum, ripe6 and npeS. The penDE gene of A. nidulans is linked to the pcbC gene, which encodes the isopenicillin N synthase, as also occurs in P. chrysogenurn. Both genes show the same orientation and are separated by an inter- genie region of 822 nucleotides.

Key words: Acyl-CoA:6-amino penicillanic acid acyltransferase - p e n D E g e n e - Aspergillus nidulans - Penicillin biosynthesis - Linkage of genes

Introduction

Aspergillus nidulans is known to produce benzylpenicillin, although at a much lower rate than most strains of Penicil- lium chrysogenum, some of which are used for industrial production of penicillin (Holt and Macdonald 1968). Mu- tants ofA. nidulans impaired in penicillin biosynthesis (npe) have been obtained (Edwards and Holt 1974) and partially characterized by cosynthesis studies (Makins et al. 1980; 1981 ; 1983).

Penicillins are formed by condensation of three amino acids, L-~-aminoadipic acid, L-cysteine and L-valine that are linked together to form the tripeptide 6(L-~-aminoadipyl)-L-cysteinyl-o-valine (ACV) by the multifunctional enzyme ACV synthetase (Van Liempt et al. 1989) (Fig. 1). ACV is then cyclized to form isopenicillin N, an intermediate which has an L-~-aminoadipyl side chain attached to the /Mactam-thiazolidine ring nucleus of penicillin (re- viewed by Martin and Liras 1989a). This cyclization reaction is carried out by isopenicillin N synthase (IPNS; Pang et al. 1984; Ramos et al. 1985). The gene (pcbC) encoding IPNS has been cloned from two different strains of P. chrysogenum (Carr et al. 1986; Barredo et al. 1989a) and from Cephalosporium acrernonium (synonym Acremonium chryso-

Offprint requests to: J.F. Martin

genum) (Samson et al. 1985) and Aspergillus nidulans (Ra- m6n et al. 1987; Weigel et al. 1988). In the last step of penicillin biosynthesis the ~-aminoadipyl side-chain is ex- changed for phenylacetic acid. This reaction, which is cata- lyzed by the enzyme acyl-CoA:6-amino penicillanic acid

L-o~-AA A L - CYS L -VAL l ACV Synthetase

HOOC-CH-(CH2) 3 - CO-NH - CH~CH2/SH jCH3 I I fCN~CH 3 NH 2 ~,C --N H - - CH

d " I coo H

TRIPEPTIDE ACV

lsopeniciliin N synthase cp_s.~ c)

HOOC - CH - (CH2) 3 - CO- NH ---.~---~S"'~CH3

NH2 H

ISOPENICILLIN N /

H2 ) lsopenici[[in N arnido|yase

c~-AminoadipiCacld / i S - - . /C H 3

O//---N K.COOH \

PhenyLacetyl - CoA

Isopenicillin N ocyltransferase ( pen DE )

x " ~ CoA

6-APA acyltronsferase

-Aminoadipie 6 - A P A ~ acid

/ / -..

CoA

PENICILLIN G

Fig. 1. Biosynthetic pathway of penicillin G from the three precur- sor amino acids L-~-aminoadipic acid, L-cysteine and L-valine, indi- caring the enzymes involved (Martin and Liras 1989a) and the genes which encode them (Martin et al. 1990). ACV, 6(L-c~-aminoadipyl)-L-cysteinyl-D-valine; 6-APA, 6-amino penicillanic acid

(APA) acyltransferase (AAT), occurs only in penicillin producing strains. This enzyme does not exist in C. acremonium and other cephalosporin producers, but occurs in A. nidulans (E. Alvarez, unpublished).

The AAT of P. chrysogenum has been purified to homogeneity (Alvarez et al. 1987) showing a molecular weight (M,) of 29 000 Da. The enzyme forms penicillin G in vitro from CoA derivatives of phenylacetic acid and either 6-APA or isopenicillin N (Alvarez et al. 1987; Martin et al. 1987).

The gene penDE (previously named aat) encoding AAT has been cloned from the genome of P. chrysogenum AS-P- 78 (Barredo et al. 1989b). The penDE gene encodes a protein of 357 amino acids with a deduced M r of 39943 Da which appears to be processed post-translationally into two non-identical polypeptides. A similar gene is likely to exist in A. nidulans. Since this filamentous fungus has been widely used as a model microorganism for basic genetic studies (Arst and Scazzocchio 1985), it was of great interest to characterize the gene encoding AAT. In this article we re- port the cloning and characterization of the penDE gene of A. nidulans. Furthermore, we provide evidence that, as in P. chrysogenum (Diez et al. 1989), this gene is closely linked to the pcbC gene.

Materials and methods

Microorganisms and vectors used. A. nidulans ATCC 28901, a white conidial biotinless mutant with increased penicillin production (Holt et al. 1968) was used as a source of DNA. Escherichia coli LE392 was used as host for 2EMBL3 phage derivatives (Maniatis et al. 1982) and E. coli DH5~ (Gibco BRL, New York) as recipient strain for high-frequency transformation (10 7 I 0 8 transformantsAtg ofDNA). E. coli MVl193 (Miller 1972) was used to obtain single-stranded DNA from the Bluescript plasmids.

P. chrysogenum AS-P-78 and P. chrysogenum Wis 54-1255 were used for comparative expression experiments and the non-producing mutants Wis 54-1255 npe6 and npe8 were used for complementation studies (Martin et al. 1987). Plasmid pULJL43 is a fungal vector which carries the phleomycin resistance marker (J.L. Barredo and S. Gutirr- rez, unpublished).

2EMBL3 (Frischauf et al. 1984) was used for cloning large fragments (14-17 kb) of A. nidulans DNA. Bluescript plasmids K S ( + ) and K S ( - ) (Stratagene, La Jolla, Califor- nia) were used to subclone DNA fragments for sequencing, as described previously (Barredo etal. 1989a). Phage M13K07 was used as a "helper" in the preparation of single-stranded DNA from the Bluescript plasmids.

323

Construction and screening of an A. nidulans genomic library. DNA of A. nidulans ATCC 28901 obtained as described by Specht et al. (1982) was partially digested with Sau3AI and fragments of 14-17 kb were selected in sucrose gradients (10%-40%). The size-selected fragments were li- gated to purified 2EMBL3 arms and the ligation mixture was packaged in vitro, using the Amersham 2 "in vitro" packaging system.

The entire genomic library was transferred to nitrocellulose papers (BA85, 0.45 pm Schleicher and Schuell) at 4 ° C for 1 rain and the filters soaked in a denaturing solution and baked as described by Barredo etal. (1989a). The baked filters were hybridized using the following condi- tions: 37 ° C for 12 h and washed sucessively at: (1) room temperature in 2 x SSC containing 0.1% SDS for 15 min; (2) 37°C in 2× SSC containing 0.1% SDS for 15 rain; (3) twice at 37°C in 0.1 × SSC with 0.1% SDS. SSC contains 3 M NaC1, 0.3 M sodium citrate, pH 7.0.

Southern blotting and sequencing. Southern blotting of DNA fragments was carried out using standard methods (South- ern 1975). Both strands of DNA were sequenced by the dideoxynucleotide method (Sanger etal. 1977) using pBluescript KS(+) and KS(--) as vectors and the Sequen- ase system (US Biochemicals, Cleveland, Ohio) for the DNA polymerization reaction.

Probes. Three different probes (indicated as A, B and C in Fig. 2) corresponding to the complete penDE gene of P. chrysogenum and the amino- and carboxyl-terminal regions, respectively, were used for the identification of the penDE gene of A. nidulans. In addition, a fourth probe D carrying the whole pcbC gene of P. chrysogenum (Barredo et al. 1989a, b) was used in linkage studies. They were labelled by nick translation with 0~-[S2P]dCTP (Amersham International) using standard methods (Maniatis et al. 1982).

Probes I, II and III were 20mer synthetic oligonucleo- tides complementary to internal sequences of each of the three introns, whereas probe IV was a 20mer oligonucleotide complementary to a translated region at the carboxyl- terminal half of the gene.

Probe I: 5 ' -GGCGTTAAATATGGGCAAAT-3 ' Probe II: 5 ' -CCGTCAACGGGGTGATTCAG-3 ' Probe III: 5 ' -GGCGGTTAGCAAGGAAAATA-3 ' Probe IV: 5 ' -GACAGCGACACCAGCACTGT-3 '

Transformation of P. chrysogenum and gene expression. Transformation of protoplasts of P. chrysogenum was carried out as described previously (Cantoral et al. •987; Diez et al. 1987). Protoplasts were transformed with pULEM25

Hinel l H nc Hincl l Hincl l H n d l l H inc ~ Hincl l Sai l a ba l l • I Xb I I I Hincl l EcoRI X b a

[.~ I L,. N~°' I I I n o' I i t I I "-q: .......... ! J ! i 4 t / L /

I I I I 1 2 3 4 5

D A B C

Fig. 2. Restriction map of the 5.1 kb SalI fragment of Penieillium ehrysogenum DNA carrying the pebC and penDE genes showing the location of probes A, B, C and D that have been used for hybridization experiments. The black arrows indicate the pebC (left) and penDE (right) genes. The open boxes in the penDE gene correspond to the three introns

324

a plasmid vector carrying the phleomycin-resistance marker of Streptoalloteichus hindustanus (G. Tiraby, personal com- munication) and a 2.1 kb BamHI fragment containing the penDE gene of A. nidulans (J.L. Barredo, S. Guti~rrez, and E. Montenegro, unpublished). Expression of the penDE gene of A. nidulans in P. chrysogenum Wis 54-1255 npe6 and npe8, two mutants deficient in AAT activity (Martin et al. 1987), was studied by assaying the AAT activity in transformed and control npe6 and npe8 strains (transformed with pULJL43) as described previously (Alvarez et al. 1987). AAT activity in A. nidulans is given as picokatals (picomoles of penicillin G formed per second; pkat) per mg of protein.

RNA isolation and Northern blotting. Total RNA was obtained by the phenol/SDS method as described by Ausubel et al. (1987). Five micrograms of RNA was run in an agar- ose (1.2%) formaldehyde gel using RNA molecular weight markers (16 S, 23 S ribosomal RNA and set III, Boehringer Mannheim).

The gel was blotted onto a nitrocellulose filter, baked in a vacuum oven, prehybridized and hybridized with probes I, II, I II and IV, as described previously (Barredo et al. 1989b).

Computer analysis. An analysis of the nucleotide and deduced amino acid sequences was made using DNASTAR computer programs.

Results

AAT activity in A. nidulans

An initial study of AAT activity in A. nidulans was made to confirm the presence of a gene encoding this enzyme and to obtain an estimation of the level of expression for RNA hybridization studies. A. nidulans ATCC 28901 pro- duces trace amounts of penicillin when compared with the standard laboratory strains of P. chrysogenum Wis 54-1255 or AS-P-78. Cell-free extracts of A. nidulans showed an AAT activity of 0.2 pkat/mg of protein as compared to 1.5 and 12.0 pkat/mg of protein for the P. chrysogenum Wis 54-1255 and AS-P-78 strains, respectively. Accumula- tion of 6-aminopenicillanic acid (6-APA; an intermediate of the penicillin pathway) in the broth (72 h after inocula- tion) was also low in the A. nidulans strain: 1.4 Ixg/ml versus 371 and 1360 pg/ml for P. chrysogenum strains Wis 54-1255 and AS-P-78, respectively.

EcoRI EcoRI BamHI SallBa~ mill Sail

X Anaat 1 2

I~mHI 1 i i Barn / '

II BarnHI BamH Bal HI E RI S~ / / Hindlll indlll

; ' 8 1'0 ' 1'2 i iSacl l lXbal \ BamHI

ISmal IPstl

~mHI ~mHI BamHI ~mHI IEcoRI Hindlll I EcoRV EcoRI ]Hindlll

I I I ~ , , / A o o D D

I I I I I 4 6

Kpnl ~oal I ~mHI ~ol Hindlll

"-t 2 pULEM18

10.3 kb

Sacl I Pstl B Yh"'amHi BamH, Pstl Pstl Smal Hindlll ~ BimHI

Pstl[ I Accl I sial EcoR I I

EcoRV ~ [ ] 1 ._ iii!!ii~iiiiiiiiiiiii~izll

pcbC I pULEM21 1 ~ 7.1 kb

Kpnl EcoRI Smal (673} (697) (320)

Sacll ~al

BamH L ~ t::::::::i::i:://::i!i!:;!::iiii/iiiiii~ili

pULEM1 5.0 kb

~mHI Kpnl_ Smat ~-¢oRI /

I 2 penDE

Accl (875)

i 1

p

Pstl BamHI Hindlll

~ al Sma Kpn I T×~o,

I

~ K p n l . . . ISa]l Pstl xoal IHindlll

(1585) (1913) IEcoRI } Pst l / S m a l

I J B a m H I ~ (2101)

i 2

Fig. 3. Comparative restriction maps of the 12.5 kb Aspergillus nidulans DNA insert in phage 2Anaatl (top) and the fragments that have been subcloned in pULEMt8, pULEM21 and pULEMI (bottom). Numbers indicate the size in kb. The position of the pcbC and penDE genes in each of the constructions is indicated by arrows. The dotted fragment upstream of each gene corresponds to the promoter region. The open boxes inside the penDE gene indicate the three introns. The sequencing strategy is shown by thin arrows at the bottom

Identification of a sequence analogous to the penDE gene in the genome of A. nidulans

Clear hybridizing bands were observed when total DNA of A. nidulans digested with BamHI (2.1 kb) or EcoRI (two fragments of 7.1 kb and about 5.7 kb) was hybridized with probe A (results not shown) suggesting that a gene analogous to the penDE of P. chrysogenum is present in the A. nidulans genome.

Cloning of the penDE gene

A genomic library of A. nidulans DNA was constructed as described in Materials and methods. About 20 000 plaque forming units were obtained. Three recombinant phages were identified after screening the library with probe A. One of them, 2Anaatl, (A. nidulans AAT clone 1), gave a stronger hybridization signal than the other two and was studied in detail. DNA of this clone was purified and mapped with four enzymes (SalI, BamHI, EcoRI and Hin- dIII; Fig. 3).

The gene is located in a 7.5 kb SalI fragment (one of the SalI ends belongs to the phage linker) and also in a 4.2 kb HindIII and in a 2.1 kb BamHI fragment internal to the SalI fragment. With EeoRI, two hybridizing fragments were observed which confirmed the results obtained in Southern hybridizations of total DNA.

The gene was subcloned in pBluescript KS(+) after di- gestion of 2Anaatl with SalI, HindIII or BamHI, and intro- duced into E. coli DH5e originating plasmids pULEM18, pULEM21 and pULEM1, respectively (Fig. 3; pULEM22, pULEM17 and pULEM2 in the opposite orientation). All three plasmids were mapped in detail with several restriction endonucleases (Fig. 3).

u

m

!

u

Complementation of P. chrysogenum Wis 54-1255 npe6 and npe8 and expression of the cloned gene

Definitive proof that the cloned DNA contained the AAT gene was obtained by complementation of the AAT-deficient P. ehrysogenum mutants npe6 and npe8 which are very stable mutants without a significant reversion frequency. A vector pULEM25 was constructed by subcloning the 2.1 kb BamHI fragment carrying thepenDE gene ofA. nidulans (Fig. 3) into the previously constructed pULJL43 vector which carries the phleomycin resistance marker (J.L. Barredo, and S. Guti6rrez, unpublished). About 800 transformants of each mutant were obtained by transformation with pULEM25 (transformation efficiency was 320 transformants per ~tg of DNA). Ninety six of these transformants (48 of each mutant) were assayed for penicillin production using the agar plug assay (Ichikawa et al. 1971). Of the phleomycin-resistant transformants, 62% were able to com- plement the AAT-deficiency of mutants npe6 and npe8.

To verify that the complementation (i.e. penicillin production) was due to the introduction of an intact penDE gene, transformants were assayed for AAT activity. The level of AAT activity in two transformants of P. chrysogenum npe6 with pULEM25 was 11.8% and 26.8% of the level in the parental P. chrysogenum Wis 54-1255. Two transformants of strain npe8 showed, respectively, 13.5% and 4.1% of the AAT activity of the Wis 54-1255 strain. Control npe6 and npe8 transformed with pULJL43 showed no acyltransferase activity.

325

Orientation of the penDE gene

In order to determine the orientation of the penDE gene, plasmid pULEM2 was digested with four restriction enzymes, and the fragments obtained were blotted to two

I

II 1 2 3

l ib

4

I

Q

B

O l i b

Fig. 4. Hybridization of pULEM2 carrying the A. nidulans penDE gene with probes B and C (see Fig. 1) to establish the orientation of the penDE gene. Center: pULEM2 was digested with BamHI (lane I), XbaI (lane 2), PstI (lane 3) and Sinai (lane 4) and hybridized with probes B (left) and C (right). C, size markers (HindIII- digested 2DNA and HinfI + Bg/I-digested pBR328 DNA)

1 2 3 4 5 1 2 3 4 5

' - 9 . 4 - - 6 . 5

'~"-4. 3

4 -2 .3 " -2 .0

.,-1.2

iiiiiii!ii! iii~i~!i~i' i~ i!i i?i i~ii!i!ili!!~ !~!*-o.4

Fig. 5. Linkage of the pcbC and penDE genes of A. nidulans shown by hybridization with probes A and D (Fig. 2). DNA of plasmid pULEM22 was digested with HindlII (lane 1), KpnI (lane 2), PstI (lane 3), SmaI (lane 4) and XbaI (lane 5) and hybridized with probes A (left panel, penDE gene) and D (right panel, pcbC gene). Note that single 4.2 kb HindIII (lane 1) and 6.5 kb XbaI (lane 5) fragments hybridize with both probes: they thus carry both pebC and penDE genes. The pattern of hybridization of KpnI, PstI and Sinai fragments with both probes further supports the linkage of the genes. One of the Sinai (1.82 kb) generated fragments also hybridizes with both probes. Size markers (in kb) are indicated by arrows

326

identical nitrocellulose filter papers. One of them was hybridized to probe B carrying the 5' end fragment of the penDE gene of P. chrysogenum; the other was hybridized to probe C carrying the 3' region of the same gene. Results (Fig. 4) indicated that the 5' region of the gene is located in the KpnI-E¢oRI fragment and the 3' end is located in the AccI-PstI fragment (Fig. 3, pULEM1).

Linkage of penDE and pcbC genes in A. nidulans

The genes encoding AAT and IPNS have been shown to be linked on a 5.1 kb SalI fragment in P. ehrysogenum (Diez et al. 1989). To determine if a similar gene organization exists in A. nidulans the DNA of phage 2Anaatl or plasmid

pULEM22 was digested with several restriction enzymes and the fragments obtained were analyzed by Southern hybridization with probe A (carrying the penDE gene) and probe D (carrying the pcbC gene) of P. chrysogenum (Fig. 2). Results (Fig. 5) indicated that the 4.2 kb HindIII fragment carries both genes. Both penDE and pcbC genes are also linked on the 6.5 kb XbaI fragment released from pULEM22 (Fig. 5). Analysis of the nucleotide (nt) sequences showed that both genes are separated by an intergenic region of 822 nt, and have the same orientation (Fig. 3) as in P. chrysogenum. The intergenic region of A. nidulans is shorter than that of P. ehrysogenum (1497 nt). Both regions showed a similarity of 50.2% with a gap of 600 nt in the region near the 3' end of the pcbC gene, indi-

GGATccA•GGGTTcCGT•TcAGTAGcGGcTG•GTA•Ac•ATccTAA••TAAAAGAAccAcAGG•cAAGcCcA•ATG•ATAG•ATGTTGATTTc••TGG•• 100 CGGCGCCGGCCGAAC•TTGGTCTAGGGCTTTGTGCGCTCTGTCGATTCCGTGTCAAAACTGGACCAGGCAGCAGGAACATGTCTGTATTCTAGATCTCAT 200

CGGTACTTGcCGCCAA~TCACCCAAATC~CGATCTC~AATG~ACCTATCTAGCTAGCCACCCTGTTCAATATTTTCTACTGGACGCTCCGAAGAAAA~' 300 m

CTTCACGTAACTTGCCAAGGTA•CCCCTCCGAAGTAAGTCCATACCCGAATGTATATTTGCCCATATTTAACG•CTATACTTCCAGATCGGCTATCACCA 400 L h v t c q g t p s e i g y h h

TGGCTCTGCTGCCAAAGGCGAGATTGCGAAAGCCATTGACTTCGCAACTGGCCTCATTCATGGCAAAACAAAAAAGACACAGGCGGAGCTTGAACAGCTC 500 g s a a k g e i a k a i d f a t g l i h g k t k k t q a e l e q l

CTCAGGGAGTTGGAGCAGGTGATGAAACAGCGCTGGCCGAGATACTATGAGGAAATCTGCGGTATGTTTACCTACGTTCTTTACTTGCTGAATCACCC~ 600 l r e l e q v m k q r w p r y y e e i c g

TTGACGGAATTTTCAGGAATCGCAAAGGGTGCGGAACGCGAAGTATCGGAGATTGTCATGCTCAACACTCGTACGGAATTCGCGTACGGGCTCGTAGAAG 700 i a k g a e r e v s e i v m l n t r t e f a y g l v e a

CCCGGGACGGGTGCACCACTGTTTACTGCAAAACCCCCAATGGAGCGCTACAGGGCCAGAACTGGGACGTAGGTTATTAGATCCACGGTCTGCTATCTAT 800 r d g c t t v y c k t p n g a l q g q n w d

TTTCCTTGCTAACCGCCATTCCGGCAGTTCTTCACCGCAACCAAAGAAAACTTGATCCAGTTAACAATTTGTCAGCCGGGTCTACCCACTATCAAAATGA 900 f f t a t k e n l i q l t i c q p g l p t i k m i

TTACAGAAGCTGGTATCATTGGCAAAGTGGGTTTCAACAGTGCTGGTGTCGCTGTCAATTACAATGCACTACACCTACATGGCCTCCGTCCCACTGGCCT 1000 t e a g i i g k v g f n s a g v a v n y n a l h [ h g l r p t g l

CCCCTCGCATCTCGCGCTGCGCATGGCCCTCGAAAGTACATCTCCGTCTGAGGCGTATGAAAAAATCGTCTCGCAAGGGGGCATGGCGGCTAGCGCGTTC 1100 p s h l a l r m a l e s t s p s e a y e k i v s q g g m a a s a f

ATCATGGTGGGCAACGCACACGAGGCCTACGGGCTAGAGTTCTCGCCCATCAGCTTGTGCAAGCAAGTTGCTGACACCAATGGGCGGATAGTGCATACGA 1200 i m v g n a h e a y g l e f s p i s l c k q v a d t n g r i v h t n

ACCACTGCCTCCTCAACCATGGGCCATCGGCGCAAGAGCTTAATCCCCTGCCGGACTCGTGGAGCCGCCACGGGCGGATGGAACATCTCCTCTCTGGTTT 1300 h c l l n h g p s a q e l n p l p d s w s r h g r m e h l l s g f

TGACGGCACGAAGGAGGCATTTGCGAAGTTGTGGGAGGACGAAGACAACTACCCTCTCTCGATCTGCCGGGCATATAAGGAAGGGAAAAGTAGAGGCTCC 1400 d g t k e a f a k l w e d e d n y p l s i c r a y k e g k s r g s

ACTCTTTTCAACATCGTCTTCGATCATGTGGGCCGGAAGGCAACAGTGCGGCTGGGCCGGCCCAATAACCCTGATGAGACCTTTGTCATGACCTTTAGCA 1500 t l f n i v f d h v g r k a t v r l g r p n n p d e t f v m t f s n

ATCTGGATACCAAGTCCGCGATC•AAGCCAACATTTGACCAATATCTCTTTCCAACCGATGCCTTCTGCAGTCTTCTCCAGCCATAGCGTACTACACCGT 1600 I d t k s a i q a n i

GAACACTTCTATGTATCTTGACAACTAGATACAAGTCAAAAGG•GAATCAAATAAAGTCGCATGTTTGGGAGAATGCACGC•AGAGTGAAATGCTGAATC 1700 CTGTTGGCTTTCACAACCAGGTGCCTAAAGTATAGCAATGCAACGAAGGCATGCCTAGTCTAGCTTCTTTCCCTAAACATGCTTTAAATCTAATACGAGA 1800 ATTATTTAAAGCATTCCAGAACTACTACTAACTT•CTATTACCCTGGAGTTTCCTGTTTCGGAAGGGTTGACCTCTCAATCCTTCCTAACCTTGTGAGTC 1900 TAGAGTAAG•TCTGCAATCTTGTTTAACCTACCTAGGACTCA•GAAGACTCCCTcAGTATGAAGTTGATAAAGGTTCTTTGTGCTCCGAGCCCGCAAGGG 2000 AAATCAGTGCTGAGATATACTGGCTGGGCTCTATATGTTGTAGAGTCAAGTGTTGACAATTTGAATCCTCGCTAATGTCACGGCTCTGCAAGCCGGGATC 2100 CCGAGTTGGTAAAGCATG•AACAGCAAGCCC•ATTTTATCTACATATTAGCTACGATTATCCTAATCATTGCTTTTAGAATAATCTCTAAATTTGGAGCC 2200 AGCAGGTCACAGAGCTCACTCTAGGCACCCCGGGCACAGTGCTCGAAATAGCGTCGTGCCGTTATCCAGCTTCATACATTGGTGTAAAAGAAGAATCGAG 2300 CCATTTCTGGCCACCCATCTGATAAGTTAGGCCTAGCTCCGTGAAGACATTCCACAAAATC 2361

Fig. 6. Nucleotide and deduced amino acid sequences of a 2361 nucleotide f f a ~ e n t containing the A. nidu~ns penDE gene and upstream and downstream regions. The ATG initiation triplet is boxed and the intron/exon boundary sequences and splicing signals of the three introns are u n ~ r ~ e & The T G A termination codon and a TAG triplet internal to the ~ i r d intron are also u n ~ r ~ e d and labelled w i ~ a s t e r ~ . A putative polyadenylation signal downstream of the gene is overlmed

327

Table 1. Sequence comparison of the intron/exon splicing sites and the splicing signal (lariat formation) of the penDE genes of Aspergillus nidulans and Penicillium chrysogenum

5' Splice site Splicing signal 3' Splice site

Consensus

Intron 1 A. nidulans P. chrysogenum



A C TGCTAAC ACAG GTAYGTT AA G CT

5'-GTAAGTCCATACCCGAATGTATATTTGCCCAT 5'-GTAAGTG

ATTTAACGCCTATACTTCCAG-3' TTCTGAC CCAG-3'

5'-GTATGTTTACCTACGTTCTTTACTTGCTGAATCACCC CGTTGACGGAATTT TCAG-3' 5'-GTGAGTG TGCTGAC CCAG-3'

5'-GTAGGTTATTAGATCCACGGTCTGCTATCTATTTTCCTTGCTAACCGCCATTCCGGCAG-3' 5'-GTACGTT GACTAAT CAAG-T

cating that the intergenic region is less conserved than the open reading frames (ORFs) of the penDE genes (see be- low).

Analysk~ of the nucleotide and deduced amino acid sequence of the penDE gene

Thirteen fragments (Fig. 3, thin arrows) covering the entire penDE gene and par t of the upstream and downstream regions were subcloned into pBluescript plasmids.

A total of 2.36 kb of D N A including the 2.1 kb BamHI fragment was sequenced on both strands. An open reading

frame of 1071 nt, (separated by three introns), was found, encoding a protein of 357 amino acids having a total Mr of 39240 Da (Fig. 6), Three putative introns of 53, 55 and 59 nt, respectively, were identified by comparison with the consensus exon/intron splicing sequences (Ballance 1986; Table 1). All of them are located in the 5' region of the gene as in the penDE gene of P. chrysogenum. A translation termination triplet (TAG) was found in-frame within the third intron (Fig. 6). The presence of the three introns was confirmed by hybridization of R N A with probes I, II, I I I and IV (see Materials and methods). Probe IV gave hybridization whereas probes I, II and I I I internal to the three

10v 20v 30v 40v 50v 60v 70v 80v 90v 100v

MLHVTCQGTPSEIGYHHGSAAKGEIAKAIDFATGLIHGKTKKTQAELEQLLRELEQVMKQRWPRYYEEICGIAKGAEREVSEIVMLNTRTEFAYGLVEARD

MLH: CQGTP EIGY:HGSAAK: IA::IDFA.:LI:GKTKKT:.EL.Q:L.:L.:V:.:RWP:YYEEI GIAKGAER:VSEIVMLNTRTEFAYGL .ARD

MLHILCQGTPFEIGYEHGSAAKAVIAR•IDFAVDLIRGKTKKTDEELKQVLSQLGRVIEERWPKYYEEIRG•AKGAERDVSEIVMLNTRTEFAYGLKAARD

10 ̂ 20 ̂ 30 ̂ 40 ̂ 50 ̂ 60 ̂ 70 ̂ 80 ̂ 90 ̂ 100 ̂

110v 120v 130v 140v 150v 160v 170v 180v 190v 200v

GCTTVYCKTPNGALQGQNWDFFTATKENLIQLTICQPGLPTIKMITEAGIIGKVGFNSAGVAVNYNALHLHGLRPTGLPSHLALRMALESTSPSEAYEKIV

GCTT.YC: PNGALQGQNWDFF:ATKENLI:LTI Q:GLPTIK.ITEAGIIGKVGFNSAGVAVNYNALHL:GLRPTG:PSH:ALR:ALESTSPS:AY::IV

GCTTAYCQLPNGALQGQNWDFFSATKENLIRLTIRQAGLPTIKFITEAGIIGKVGFN•AGVAVNYNALHLQGLRPTGVPSHIALRIALE•TSPSQAYDRIV

110 ̂ 120 ̂ 130 ̂ 140 ̂ 150 ̂ 160 ̂ 170 ̂ 180 ̂ 190 ̂ 200 ̂

210v 220v 230v 240v 250v 260v 270v 280v 290v 300v

SQGGMAASAFIMVGNAHEAYGLEF•PI$L•KQVADTNGRIVHTNHCLLNHGPSAQELNPLPDSWSRHGRMEHLLSGFDGTKEAFAKLWEDEDNYPLSICRA

.QGGMAASAFIMVGN:HEA:GLEFSP.S: KQV D:NGR:VHTNHCLL:HG :.:EL:PLPDSW:RH RME LL.GFDGTK:AFA:LW.DEDNYP:SICRA

EQGGMAASAFIMVGNGHEAFGLEFS•TSIRKQVLDANGRMVHTNHCLLQHGKNEKELD•LPDSWNRHQRMEFLLDGFDGTKQAFAQLWADEDNYPFSICRA

210 ̂ 220 ̂ 230 ̂ 240 ̂ 250 ̂ 260 ̂ 270 ̂ 280 ̂ 290 ̂ 300 ̂

310v 320v 330v 340v 350v

YKEGKSRGSTLFNIVFDHVGRKATVRLGRPNNPDETFVMTFSNLDTKSAIQANIX Aspergi[tus nidu[ans ATCC 28901

Y.EGKSRG:TLFNI::DH. R.ATVRLGRP.NPDE FVM F.: D.:SA::A.:X

YEEGKSRGATLFNIIYDHARREATVRLGRPTNPDEMFVMRFDEEDERSALNARLX Penicilt ium chrysogenum AS-P-78

310 ̂ 320" 330 ̂ 340" 350"

Fig. 7. Comparison of the amino acid sequences of the acyl-CoA:6-amino penicillanic acid transferases of A. nidulans (upper line) and P. chrysogenum (lower) showing the identical or functionally conserved (: , .) amino acids (center line). The conserved 11 amino acid sequence surrounding the processing sites (vertical arrow) of the P. chrysogenum enzyme is underlined

328

10v 20v 30v 40v 50v 60v 70v 80v 90v 100v

CATCCATGCT•AATCCAGCTCCTCGGCAAGACTAGGCGGATG•AGCAGGGATACTCGAGGTGCCCCAGTTGATGTCCCATCAGTGTCATGCTATGGTCCCA

CAT CAT T A TCC GGC G C G C G C GTGC C TGTC TC GTGTCA TGG CCA

CATGCATAGCATGTTGATTTCCCTGGCCCGGCGCCGGCCGAACCTTGGTCTAGGGCTTTGTGCGCTC ..... TGTCGATTCCGTGTCAAAAC-TGGA-CCA

10 ̂ 20 ̂ 30 ̂ 40 ̂ 50 ̂ 60 ̂ 70 ̂ 80 ̂ 90 ̂

110v 120v 130v 140v 150v 160v 170v 180v 190v

GATTGGTGGCTACGGCCAATATAA•-•ATCTCAGCATGCAGTTCCGCCTGCATGATCATCC•CAGGACGCGTTTGTCATCTCCGTCAGCCAGGTCTCA••

G G GG G C TAT ATCTCA C T CCGCC C TCA CCCA CGCG ATCTC C TCT

GGCAGCAGGAACATGTCTGTATTCTAGATCTCATCGGTACTTGCCGCCAAC .... TCA--CCCAAATCGCG ...... ATCTCGAATGCACCTATCTAGCT

100 ̂ 110 ̂ 120 ̂ 130 ̂ 140 ̂ 150 ̂ 160 ̂ 170 ̂ 180 ̂

200v 210v 220v 230v .

. . . . . . . . . GTTGTTTACCCATCTTCCGACCCGCAGCAGAA--ATG P, chrysogenurn

GTT TA GAC C C G AGAA

AGCCACCCTGTTCAATATTTTCTACTGGACGCTCCGAAGAAAAATG A. ni du tans

190 ̂ 200 ^ 210 ^ 220 ^

Fig. 8. Analysis of the nucleotide sequences of the upstream regions of the penDE genes of A. nidulans and P. chrysogenum which carry the promoters of the genes. Regions of significant homology are underlined. Overlined sequences correspond to the conserved motif PuTCTCPu

introns failed to give hybridization, providing confirmation that the latter are spliced and are not present in the mature transcript. There is a strong conservation of the nucleotide sequences of the exon/intron splicing sites and to a lesser extent of the lariat sequences of the three introns in A. nidulans and P. ehrysogenum (Table 1).

The reading frame shows 73.0% similarity with the penDE gene of P. chrysogenum at the nucleotide level. The G + C content in the ORF is 54.1% and the upstream region (300 nt) which includes the promoter region has a similar (53%) G + C content.

By comparing the deduced amino acid sequence of the AAT of A. nidulans with that of P. chrysogenum (Fig. 7) in which a post-translational cleavage has been proposed (Barredo et al. 1989b), it may be concluded that the AAT of A. nidulans is also synthesized as a 40 amino acid precur- sor protein that might be processed later to give the mature protein. An 11 amino acid sequence around the Cys-Thr- Thr protein cleavage site of the P. chrysogenum protein is also present in A. nidulans (underlined in Fig. 7),

Analysis of the upstream and downstream sequences of the penDE genes ofA. nidulans and P. chrysogenum

A region of about 300 nt upstream of the penDE ORF of A. nidulans carries the promoter, since it confers tran- scription-initiating activity as shown by complementation of npe6 and npe8 mutants. Comparison of the 245 bp upstream of the penDE genes of A. nidulans and P. chrysogenum (Fig. 8) revealed considerable similarity (J02 match- ing bases out of 245), although the nucleotide similarity was lower than within the ORFs of both genes. Some of the conserved features are likely to be important in promoter structure. A conserved AGAAA sequence precedes the ATG triplet in both promoters. A sequence PuTCTCPu is repeated three times. Other conserved motifs are underlined in Fig. 8, but the role of all of these conserved sequences in the penDE promoter structure is still unclear.

A consensus polyadenylation sequence AATAAA was found in the A. nidulans penDE gene downstream of the

ORF (Fig. 6). The same sequence is also present in a similar position in the penDE gene of P. chrysogenum.

Discussion

This paper describes the cloning of a gene encoding the AAT enzyme of A. niduIans, as established by complementation of two npe mutants of P. chrysogenum lacking AAT activity (J.M. Cantoral, and S. Guti~rrez, unpublished; Martin et al. 1987). Further support for the identification of the penDE gene was provided by the relatively high similarity of the gene with the previously cloned penDE gene of P. chrysogenum (Barredo et al. 1989b).

Three introns are present in the penDE gene, at similar positions to those found in the P. chrysogenum gene (Ta- ble 1). They were identified by comparison with the fungal consensus intron/exon splicing sequences (Ballance 1986) and confirmed by m R N A hybridization experiments using three oligonucleotide probes internal to the three introns. In addition, an in-frame translation termination triplet was found within the third intron which, if the intron were not spliced, would cause termination of translation resulting in an abnormally small polypeptide. No further potential intron/exon splicing sites could be identified in the 3' region of the ORF, although we cannot exclude the existence of other introns.

The size of the translated AAT polypeptide (Mr 39240 Da) agrees well with the size of the pre-AAT previously characterized in P. chrysogenum (Mr 39943 Da; Barredo et al. 1989b). The similarity of the penDE genes of P. chrysogenum and A. nidulans is very high. The proteins encoded by both genes contain the same number of amino acids, and when the two amino acid sequences were compared, no gaps were required to achieve maximal alignment (Fig. 7). Due to the high percentage (76.5%) of identical or functionally conserved amino acids it is not possible to determine by amino acid comparison which amino acids are likely to be involved in the active center of the enzyme. The similarity between the pcbC genes of A. nidulans (Ra- m6n et al. 1987) and P. chrysogenum (Carr et al. 1986; Bar-

329

redo et al. 1989a) is also very high (75.7% homology at the nucleotide level and 81% at the amino acid level). Phy- logenetic da ta o f the Ascomycetae based on 5 S r R N A led to the hypothesis that A. nidulans and P. chrysogenum der- ive from a common ancestor (Chen et al. 1984). Our results suppor t this hypothesis.

Based on biochemical and genetical evidence we have proposed (Barredo et al. 1989b) that the P. chrysogenum A A T is processed post- t ransla t ional ly giving rise to two polypept ides of 102 and 255 amino acids (29 kDa). The N-terminal end of the 29 k D a protein purified to homogeneity from extracts of P. chrysogenum starts with amino acids Cys-Thr-Thr-Ala-Tyr . . . (Fig. 7) internal to the A A T protein. A structural ly conserved 11 amino acid sequence is present in the same posi t ion in A. nidulans AAT, suggesting that it might be processed in a similar form. The active form of the A. nidulans A A T is currently being purified to determine whether processing of the 40 kDa protein takes place, as occurs in P. chrysogenum.

The penDE gene of A. nidulans is l inked to the pcbC gene on a 4.2 kb HindIII fragment in the D N A of A. nidulans and both are expressed in the same direction. We have previously found a similar organizat ion of the pcbC-penDE cluster in P. chrysogenum (Diez et al. 1989). Clusters of antibiotic biosynthet ic genes are very frequently found in Streptomyces producing antibiotics (Mart in and Liras 1989b). I t is likely that the same clustered ar rangement of the biosynthetic genes for secondary metaboli tes occurs in f i lamentous fungi.

Since an extensive body of knowledge on classical genetics in A. nidulans is available (see review by Arst and Scaz- zocchio 1985), the availabil i ty of a D N A fragment carrying two penicillin biosynthetic genes will contr ibute to advanc- ing our studies of the control of gene expression during penicillin biosynthesis in fungi (Mart in et al. 1990).

Acknowledgements. This work was supported by a grant of Gist- Brocades (Delft, The Netherlands). J.L.B., E.A. and E.M. were granted fellowships of the Ministry of Education and Science of Spain; B.D. was supported by a fellowship of the Diputaci6n de Le6n (Spain). We acknowledge the excellent technical assistance of M.I. Corrales, M.P. Puertas, B. Martin, R. Barrientos and S. Llamas.

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Communica ted by C.A. van den Hondel

Received November 16, 1989

cloning, characterization of the acyl-coa : 6-amino penicillanic acid acyltransferase gene of...

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