Journal of Biotechnology, 17 (1991) 81-90 81 Elsevier

BIOTEC 00571

Strain improvement of Penicillium chrysogenum by recombinant DNA techniques

A.E. Veenstra, P. van Solingen, R.A.L. Bovenberg and L.H.M. van der Voort

Gist-brocades Research and Development, Delft, The Netherlands

(Received 27 February 1990; revision accepted 12 June 1990)

Summaff

The penDE gene from Penicillium chrysogenum has been isolated; the gene is located in close vicinity of the pcbC gene. Amplification of the pcbC-penDE gene cluster in Penicillium chrysogenum Wis54-1255 leads to a significant increase in penicillin production. In selected transformants an increase of up to 40% is observed.

Penicillin; fl-Lactam; Gene-amplification; pcbC, penDE

Introduction

Penicillin production by the filamentous fungus Penicillium chrysogenum has been improved enormously during the past 40 years, using the conventional strain improvement techniques of mutation and subsequent selection of better producing strains (e.g. Van der B e e k e t al., 1984). Several years ago the application of recombinant D N A techniques has been extended to the filamentous fungi, e . g . P . chrysogenum; hence strain improvement can now also be tried using recombinant D N A technology.

A prerequisite for the ability to transform any organism is the availability of a good selection system, that can discriminate between transformed and non-trans- formed cells. Several transformation systems have now been described for P.

Correspondence to." A.E. Veenstra, Gist-brocades Research and Development, PO Box 1, 2600 MA Delft, The Netherlands.

0168-1656/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)

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chrysogenum. These systems use for selection either the complementation of an auxotrophic mutation (Diez et al., 1987; Picknett et al., 1987; Sanchez et al., 1987) or a dominant selectable marker like resistance to oligomycin (Bull et al., 1988) or phleomycin (Kolar et al., 1988) or growth on acetamide using the amdS gene from Aspergillus nidulans (Beri and Turner, 1987; Kolar et al., 1988). For application in industrial strains, the dominant systems are favoured, since they can be applied to any strain that has to be transformed. Use of auxotrophic markers requires the isolation of the corresponding mutants, a procedure which can be rather laborious and time-consuming. A more serious disadvantage is that auxotrophic mutations have been reported to influence the production of penicillin (MacDonald et al., 1963). We have applied resistance to phleomycin for transformation of P. chry- sog, enum, using the gene isolated from Streptoalloteichus hindustanus under control of the P. chrysogenum pgk promoter.

The first fl-lactam biosynthetic gene that has been isolated is the pcbC gene encoding isopenicillin N synthase (IPNS). The gene has first been isolated from Acremonium chrysogenum (Samson et al., 1985), but the isolation of this gene has now been described for various organisms (Carr et al., 1986; Leskiw et al., 1988; Ramon et al., 1987; Shiffman et al., 1988; Weigel et al., 1988). Also the isolation of the gene encoding the expandase/hydroxylase activities (cefEF) from A. chry- sogenum has been described (Samson et al., 1987). The availability of these genes allows for their application in strain improvement. The pcbC gene has been reintroduced into an industrial production strain of P. chrysogenum and the penicillin production of several transformants has been studied (Skatrud et al., 1987). No increase in penicillin production could be demonstrated in the transfor- mants analysed. Similar results have been obtained after introduction of the pcbC gene of A. chrysogenum back into this fungus (strain ATCC 11550; Chapman et al., 1987).

pcbC [

L-~L--arninoadipic acid L--cyste[ne L-valine

&-(L--~-aminoodipyl)-L-cysteinyI-D-voline

isopeniciliin N (IPN)

6-eminopenicillonlc acid (6-APA)

phenylocetic ocicl

penicillin-G Fig. 1. Schematic representation of the biosynthetic pathway for penicillin G.

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Reintroduction of the cefEF gene in A. chrysogenum strain 394-4, on the other hand, has resulted in transformants that do show an increase in the production of cephalosporin, due to an increased activity of the expandase/hydroxylase enzyme (Skatrud et al., 1989).

We present data that also in P. chrysogenum amplification of penicillin biosyn- thetic genes can increase the production of the antibiotic in selected transformants. To this end we have isolated and characterized the penDE gene (compare Fig. 1) and its product, the acylCoA : 6APA acyltransferase or AT. This gene was shown to be clustered with the pcbC gene; this gene cluster has been reintroduced into P. chrysogenum.

Materials and Methods

Strains and plasmids

P. chrysogenum: strain Wisconsin 54-1255 was used throughout this study. E. coli: propagation of recombinant DNA was performed using strain HB101.

P~smids

pPS47 contains the phleomycin resistance gene from Streptoalloteichus hindustanus, isolated from pUT702 (Cayla, Toulouse Cedex, France), under the control of the P. chrysogenum pgk promoter. Insertion of the 5.1 kb SalI fragment containing the pcbC and penDE genes into pPS47 yielded pGJ02.

Purification of acylCoA : 6APA acyltransferase (AT)

Purification of AT was done essentially as described by Alvarez et al. (1987) with minor modifications. Mycelium was disrupted by freezing in liquid nitrogen, fol- lowed by mortaring and the 0-45% ammoniumsulphate fraction (cell-free extract) was used for further purification.

Sodiumdodecylsulphate-polyacrylamide gel electrophoresis (SDS-PA GE)

SDS-PAGE was done as described by Laemmli (1970).

Immunoblotting

Polypeptides were separated by SDS-PAGE in 13.3% APt (w/v) separating gels. The polypeptides were electrophoretically transferred from the slab gel onto nitro- cellulose sheets using a Multiphor II Nova blot apparatus (LKB) according to the procedure given by the manufacturers. Immunostaining was done with the Proto- Blot Western Blot AP system (Promega) according to the procedure given by the manufacturers.

84

Size exclusion chromatography

The purified enzyme preparation was lyophilized, the pellet was dissolved in 50 mM KzHPO4/KH2PO4, pH 7.0, and was applied to a gelfiltration column (TSK G 2000 SW, LKB). Fractions were freeze-dried and analysed by SDS-PAGE.

P. chrysogenum transformation

The P. chrysogenum strains were transformed using the procedure described by Cantoral et al. (1987). Transformants were selected for their resistance to phleomy- cin (30 ~g m1-1) (Cayla, Toulouse, France).

Penicillin production

The penicillin production of the strains has been analysed qualitatively using a biological assay. The strains were grown on plugs of complex agar medium for 3 d at 25°C. The plugs were transferred to agar plates, containing spores of Bacillus subtilis (ATCC 6633) in complex medium. After 16 h at 37°C clear inhibition zones around the plugs of the penicillin producing strains could be observed. Penicillin production data were obtained by growing selected strains in shake flasks in production medium (e.g. Luengo et al., 1979) containing 0.5% (w/v) of pheno- xyacetic acid. The amount of penicillin V in the culture medium was determined by HPLC, after centrifugation at 15 000 x g for 10 min.

Biochemical analyses

Assays for the determination of the activity of the IPNS and AT enzymes were performed according to Ramos et al. (1985) and Alvarez et al. (1987), respectively. High performance liquid chromatography was used to detect isopenicillin N and penicillin G.

Results

Cloning of the gene encoding the acylCoA:6APA acyltransferase (penDE) from Penicillium chrysogenum and analysis of the gene product

The isolation of the gene encoding the acylCoA-6APA acyltransferase (AT) enzyme (penDE) has been described elsewhere (Veenstra et al., 1989; Barredo et al., 1989). Briefly, a mixed oligonucleotide probe has been designed based on the N-terminal aminoacid sequence of the purified enzyme. Using this probe, a genomic library of P. chrysogenum in the vector lambda EMBL3 has been screened and one positive clone, B21, has been further isolated and characterized. The presence of the penDE gene on this clone was verified by complementation of a nonproducing mutant, known to lack AT enzyme activity (Veenstra et al., 1989). Enzyme activity was shown to be restored in only those transformants having regained the ability to

85

8 8

t

s I /

f

! S

s S s

s "

8s! I I I

1'.o

B E 8 B H E 8 8 B EE H H 8 : ; j " ,* , . . . . . . : , ;

I i i 2 .0 3 .0 4 .0 5 .0 kb

Fig. 2. Genetic organization of the pcbC-penDE gene cluster present in the genome of P. chrysogenum. The upper drawing shows the insert of phage lambda B21; the 5.1 kb SalI fragment containing the pcbC-penDE gene cluster is enlarged in the lower drawing. The introns in the penDE gene are indicated

by arrowheads or shading.

produce penicillin. Hybridization studies have shown that this clone also contained the gene encoding the isopenicillin N synthase (pcbC); hence both genes are closely linked in the genome of P. chrysogenum (Fig. 2; Veenstra et al., 1989; Diez et al., 1989). The DNA nucleotide sequence of the penDE gene has been determined; this sequence has been published elsewhere (Barredo et al., 1989). The penDE gene has been shown to contain three introns making this gene the first fl-lactam biosynthetic gene to contain introns.

Purified enzyme preparations contained two major polypeptides with apparent molecular masses of 29 and 10 kDa. The N-terminal amino acid sequences of both polypeptides were found to be encoded by the penDE gene, the 10 kDa polypeptide preceding the 29 kDa polypeptide. By Northern analysis it was shown that the penDE gene is transcribed into a mRNA of about 1.5 kb long, a size that is very similar to that of the pcbC mRNA. The isopenicillin N synthase has a molecular weight of 39 kDa.

These data suggest that AT is synthesized as a 40 kDa precursor polypeptide, which is processed post-translationally into a 10 kDa and a 29 kDa polypeptide. A protein of this size has been demonstrated once in a purified enzyme preparation (Veenstra et al., 1989); this protein has not been identified, however. In more recent enzyme purifications, detection of the 40 kDa protein could not be reproduced. Moreover, in immunoblotting experiments, using a polyclonal antiserum raised against the 29 kDa polypeptide band and with cell-free extracts as a substrate the 29 kDa band was detected, but no staining was observed in the 39 kDa region (Fig. 3).

The presence of two polypeptides in the active enzyme was also suggested by the demonstration of one single protein band after electrophoresis of the purified enzyme preparation in non-denaturing conditions. After excision and SDS-PAGE both the 10 and the 29 kDa polypeptides were found to be present in this band, while determination of the N-terminal amino acid sequence of the excised band revealed the N-terminal amino acid sequences of both the 10 and the 29 kDa polypeptides, in a 1 :1 ratio (data not shown). However, the two polypeptides in purified enzyme preparation could not be separated by size exclusion chromatogra- phy Both polypeptides were found in one fraction, whereas ct-lactalbumin (14.4

86

- - 9 2 . 5

- - 6 7

- - 4 6

m 3 0

- - 2 1 , 5

~ 1 4 . 3

1 2 3

Fig. 3. Immunoblot of a cell-free extract prepared from Wisconsin 54-1255 stained with antiserum against the 29 kDa polypeptide. Lane 1: polypeptides stained with NHS-biotin. Lane 2: reaction with the

a-29 kDa serum. Lane 3: molecular weight markers.

kDa) and carbonic anhydrase (30 kDa) were separated. These data suggest a linkage of both proteins in an enzyme complex. Finally, SDS-PAGE of the purified AT enzyme preparation gave similar patterns in reducing and non-reducing conditions. This indicates that the 10 and the 29 kDa polypeptides are not covalently bound by sulfur-sulfur bonds.

Strain improvement using the pcbC and penDE gene cluster

The availability of genes from the penicillin biosynthetic pathway opens the way to strain improvement by amplification of the cloned genes. The results that have been described so far using the isolated pcbC gene (Chapman et al., 1987; Skatrud et al., 1987) show that the enzyme encoded by this gene is not rate limiting in the biosynthesis of either cephalosporin or penicillin, at least not in the strains that have

87

8 n t z ~ ' ~ pPS47

pGJ02 6

5

4

3

2 -

1

10~00 ' t2'oo ' 14b0 ' te'o0 t a o 0 2 0 o 0 --,-units of penicillin

Fig. 4. Strain improvement of P. chrysogenum Wis54-1255 using the pcbC-penDE gene cluster. The number of transformants of either pPS47(///) or pGJ02 ( \ \ \ ) is plotted versus the penicillin V

production in shake flasks.

been used in these experiments. We have studied the effect of introduction of the gene cluster, containing both the pcbC and penDE genes. To this end the gene cluster, contained in a 5.1 kb SalI fragment (Fig. 2), was cloned into a vector containing the phleomycin-resistance gene as a selectable marker (pPS47). The resulting construct, pGJ02, as well as the vector pPS47 have been transformed to P. chrysogenum Wisconsin 54-1255, and phleomycin-resistant transformants have been isolated and were preliminarily tested for penicillin V production in a bioassay. Transformants that produced a halo with a diameter significantly larger than that of the control transformants (pPS47 only) were analysed further in shake flasks. The results are represented graphically in Fig. 4.

The average penicillin V production of 26 transformants containing the gene cluster is significantly larger (18%) than that of control transformants. The 2 transformants having the largest penicillin V production produce about 40% more penicillin V than the average value of the control transformants. Inspection of these transformants at the DNA level learned that the phleomycin gene has been integrated into the genome (results not shown).

D i s c u s s i o n

The cloning of the penDE gene from P. chrysogenum is described. The identity of the pen DE gene has been unequivocally proven by complementation of nonpro- ducing mutants, restoration of enzyme activity in these transformants and by comparison of protein and nucleotide sequence data. The penDE gene was shown to contain introns, which has never been demonstrated before in fl-lactam-biosynthetic

88

genes. Since introns are not found in prokaryotic genes, this finding suggests a eukaryotic origin of the penDE gene, in contrast to the proposed prokaryotic origin of other fl-lactam biosynthetic genes (Weigel et al., 1988).

The presence of a 1.5 kb transcript of the penDE gene, combined with the comparison of protein- and DNA sequence data, suggests that AT is synthesized as a 39 kDa precursor polypeptide; however, a polypeptide with this molecular mass could not be detected in cell free extracts using immunoblotting. In non-denaturing conditions the 10 and the 29 kDa polypeptides could not be separated. Moreover, N-termini of both polypeptides were demonstrated in the native protein. These results indicate that the two polypeptides remain non-covalently bound after post- translational processing of the 39 kDa precursor. The failure to detect the precursor protein suggests a rapid processing of this precursor into the 29 and 10 kDa polypeptides. At present it is unclear whether acyltransferase activity is associated with the complex of the 10 and the 29 kDa polypeptides or with the 29 kDa polypeptide alone.

The close vicinity of the pcbC and penDE genes on the genome suggests the possibility of clustering of the genes encoding the entire penicillin biosynthetic pathway. This assumption has been proven to be correct. In a stretch of chro- mosomal DNA extending approximately 20 kb upstream of the pcbC gene, another gene, encoding an mRNA longer than 10 kb, has been detected. This gene has been identified as the pebAB gene by its ability to complement non-producing mutants of Wis54-1255. Restoration of penicillin biosynthesis in the transformants is accompa- nied with restored ACVS activity and the detection of a large protein. These results will be published separately (Diez et al., submitted for publication).

With the detection of the pcbAB gene, all three key genes involved in the biosynthesis of penicillin are now shown to be clustered in the genome of Penicil- lium chrysogenum. A similar situation is thought to occur in streptomycetes (Chen et al., 1988; Kovacevic et al., 1989). In A. chrysogenum, on the other hand, two genes involved in cephalosporin biosynthesis, the pcbC and cefEF genes, are present on different chromosomes (Skatrud and Queener, 1989).

Reintroduction of the pcbC and penDE genes on a 5.1 kb SalI fragment into the Penicillium strain Wis54-1255 results in an increased production of penicillin V in shake flasks. Although the results of individual transformants are rather variable, the mean of the penicillin V concentration is significantly larger for pGJ02 transfor- mants than for transformants containing the vector only. Differences in penicillin V concentrations between individual transformants may be explained by integration of the plasmid at different chromosomal locations or by a difference in copy number. From these experiments it may be concluded that the final step in the biosynthesis of penicillin, exchange of the hydrophilic by a hydrophobic side chain by the AT enzyme, may be the rate limiting step in the biosynthetic pathway, at least in the strain tested herein. Alternatively, a balanced increased expression of both the pcbC and penDE genes may be required for an increase in the production of penicillin. Further experiments will be required to elucidate this problem.

The experiments described herein confirm that cloned penicillin biosynthetic genes can be sucessfully applied in strain improvement of P. chrysogenum. The

89

availability of a third penicillin biosynthetic gene allows for a variety of new experiments in strain improvement of P. chrysogenum. This is the first report of strain improvement of P. chrysogenum using molecular biological techniques.

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