Analysis of promoter activity by transformation of Acremonium chrysogenum

Gene. 114 (1992) 211-216 O 1992 Elsevier Science Publishers B.V. All rights reserved. 0378-1 ! 19/92/$05.00

211

GENE 06459

Short Communications Analysis o f promoter activity by transformation of

Acremonium chrysogenum

(Recombinant DNA; Cephalosporium acremonium; isopenicillin N synthetase; integration; hygromycin B)

Andrew W. Smith a*, Martin Ramsden b and John F. Peberdy a "Microbial Biochemisto' and Genetics Group. Department of Life Sciences, Universityof Nottingham. Nottingham. NG7 2RD (UK); and Molecular Biology Laboratory. Glaxochem Limited, Ulverston, Cumbria LA 12 9DR (UK) Tel. (44)22952261; Fax (44)22956180 Received by J.R. Kinghorn: 24 August 1991 Revised/Accepted: 8 December/9 December 1991 Received at publishers: 25 February 1992

SUMMARY Promoter ac~',;~vity was examined in the/1-1actam-producing fungus, Acremonium chrysogenum, by assessment of the properties of transformant isolates. Transformation was achieved using plasmid constructs specifying hygromycin B resistance (Hy R) linked to the promoter elements of gpdA (the glucose-6-phosphate dehydrogenase-encoding gene of A spergillus nidulans), and pcbC [the gene encoding the isopeniciUin N synthetase (IPN S) enzyme of A. chrysogenum]. Transformation frequency, Hy R levels, and Hy phosphotransferase (HPT) levels suggested that the transformants of constructs using the gpdA promoter showed a higher level of expression of the Hy R gene than in transformants obtained using the pcbC promoter. The patterns of integration of the transforming DNA also differed in that pcbC promoter construct transformants appeared to have tandem repeats. All integrations of plasmid DNA occurred on a single chromosome which was different in four out of five transformants studied. Multiple copy transformants of constructs using the pcbC promoter did not show the regulated pattern of expression of HPT activity observed with IPNS in untransformed strains.

INTRODUCTION The filamentous fungus A. chrysogenum ( Cephalosporium acremonium) is the major producer of the commercially

Correspondenceto: Dr. J.F. Peberdy. Department of Life Sciences, University of Nottingham, Nottingham, NG7 2RD (UK) Tel. (44)602484848, ext. 3650; Fax (44)602424270. * Present address: SmithKlineBeecham Research and Technical Centre, St. George's Ave., Weybridge, Surrey KTI3 ODE (UK) Tel. (44)932822!29; Fax (44)932822!00. Abbreviations: A., Acremonium; aa, amino acid(s); As., Aspergillus; bp, base pair(s); DTT, dithiothreitoi; gpdA, glueose-6-phosphate dehydrogenase-encoding gene; Hy, hygromycinB; hph, gene encoding HPT; HPT, Hy phosphotransferase; IPNS, isopenieillin N synthetase; kb, kilobase(s) or 1000bp; Mb, megabase(s) or 106bp; nt, nueleotide(s); pcbC, gene encoding IPNS; PFGE, pulsed-field gel eleetrophoresis; n resistance/ resistant; rpm, revolutions/rain; PMSF, phenylmethylsulfonylfluoride; Tf, transformation frequency; TSA, tryptone soya agar (Oxoid Co.); (), denotes integrated plasmid.

important /~-lactam antibiotic cephalosporin C. Recent technical advances in molecular biology have resulted in the cloning of DNA fragments with gene promoter activity from this microorganism (Samson et ai., 1985; 1987). When these promoter fragments are incorporated into a vector, whereby they control the expression of a dominant selectable marker, they facilitate the integrative transformation of A. chrysogenum (Skatrud et al., 1987) and A. niger (Ktlck et ai., 1989). Thus the successful transformation of these fungi has been achieved with vectors comprising promoter fragments from the native pcbC gene fused to the bacterial gene encoding hph (Skatrud et al., 1987; Ktlck et al., 1989). The pcbC gene encodes IPNS which catalyses an early step in/l-lactam biosynthesis (Queener, 1990). Transformation of A. chrysogenum using pcbC promoter-hph constructs occurs at a low frequency of 0.05 to 25 transformants per #g of DNA, by integration of vector DNA into the host genome (Skatrud et al., 1987). There could be a number of explanations for the low Tf. One

212 A

80.

l"

[] plH2 (91 tested) O plH3 (73 tested) [] pINS4 (95 tested)

7O

b

so

|

,o

LI-i T:I

so 20 10 ..

o

• ...]---]

200

400 800 1600 Hy resistance level (pg/ml)

80

'il

>1600

• pANT-1 (135 tested)

I

40 J

~

30

TABLE I Transformation of Acremonium chrysogenum with pcbC and gpdA promoter-containing vectors

lo

J

0 200

400 800 1600 Hy resistance level (pg/ml)

Plasmld hob flene

I

~' I

KDnl

II Ncal

I

NcoINcol

Kanl

II Ncm

I(,~l I

O,5kb

Insert size (kb)

plHO

3,9

plH2

4.4

plH3

';,8

piNS4

1,5

uf,qt I

~i~l

I

No_9.~.Nc.__m

Ncol N~I

I

ecbC 5'-region

Plasmid"

Number of experimentsb

Mean Tf ~ Standard deviation

Mean abortive Tf d

plH0 pill2 pill3 pINS4 pAN7- !

2 4 5 6 4

<0.05 6.00 6.78 6.73 12.00

-<0.05 < 0.05 <0.05 970.00

>1600

rr~l

Ncol Ncol

Hindlll

possibility could relate to the natural expression pattern of pcbC being highly regulated (Smith et al., 1990) allowing the expression of structural genes efficiently only in the later phase of growth and not in the stage of protoplast regeneration. Hence the linking of the pcbC promoter to other genes may result in low-level expression in the protoplast regeneration and selection procedures. We have investigated the possibility of utilising expression of hph as a marker system for examining pcbC promoter activity. Results are compared to hph expression using the strong constitutive promoter of the As. nidulans gpdA-encoding gene.

-cbC 3'.region

Fig. I. Resistance levels (Hy I~) in transformant populations and linear maps of transforming plasmids. (A) Hy-resistance levels of stable integrative transformants obtained using three plasmids containing pcbC promoter-hph constructs. (B)Hy-resistance levels of stable integrative transformants obtained using pAN7-1. (C) Linear schematic maps of the pcbC-hph regions of the plasmids used to transform A. cho,sogenumin this study. Plasmid pAN7-1 is characterised elsewhere (Punt et al., 1987). Methods. The Hy R phenotype was determined in transformants by preparing TSA plates (Oxoid) containing concentrations of Hy (BCL) at 0-1600 llg/ml (Waterworth, 1978). A portion of a sub-cultured transformant colony to be tested was removed from a TSA regeneration plate with a sterile toothpick and touched to the surface of the lowest concentration Hy-containing plate, This inoculation was continued through the increasing concentration of Hy-containing plates, without leaving any visible trace of the colony on the agar surface. The plates were incubated for five days at 25:C and the lowest concentration of Hy that inhibited growth was defined as the Hy-resistance level. An untransformed M8650 strain did not grow on plates containing 200/zg Hy/ml but growth was observed on control plates containing no antibiotic.

-i.88 2.46 !.72 4.40

Standard deviation

--

926.38

L, Plasmids pill2 and pill3 (Smith, 1990) contain the hph gene fused to 850 bp and 3 kb, respectively, of the 5' pcbC DNA and about !.5 kb of 3' pcbCDNA. Plasmid pill0 is an Ncol deletion ofpIH3 removing 1.8 kb of proximal promoter DNA, but leaving the hph gene intact. Plasmid piNS4 includes only 476 bp ofthe 5' pcbCDNA with no terminator fused to the hph gene as a 1.5-kb insert in pUCI9 (Fig. 2C). Plasmid pAN7-1 contains the hph gene fused to the As. nidula,s gpdA promoter and trpC terminator regions as described previously (Punt et al., 1987). The pcbC terminator fragment used in constructs pIH0, plH2 and pill3 was not in the same reading frame as hph and included 8-nt of linker sequence 3' to hph followed by 45 nt of pcbC encoding the C-terminal 15 aa of IPNS. b The number of experiments performed with each transforming plasmid. Methods. Protoplasts of A. cho'soge,um strain M8650 (ATCC1455.al were prepared and transformed according to the method of Chapman et al. (1987) modified by Smith (1990). Transformation experiments always contained 4 x 10" protoplasts/ml and 20,ug of plasmid DNA. The results are the average of between four and six experiments and Tf was the number of transformants per/~g of DNA that were viable when sub-cultured to selective media. All stable transformants erA. chrysogenum strain M8650 produced in this study were assumed to be integrative transformants. ,i Abortive Tf was determined by dilutin~ the transformation mixture 1:5 and I: 10 in stabilisation buffer and plating out aliquots in duplicate onto regeneration medium. The large (viable) and the tiny transformants were counted after five days of incubation. From each transforming plasmid 100 tiny transformants were, sub-cultured to selective media. The nonviable transformants were counted after a further five days of incubation and the number used to express the total abortive transformants from a % of those sub-cultured.

213

A

EXPERIMENTAL AND DISCUSSION

m

I

I ~ I

~.I~

kb

U 9 ebC

I

I O

I

4 23.1

,9.4 , e.e

LL

44.4 ~,d I =

.

O 41 0.6

B u~.D

Mb m

6.3

~ 1 m m

3.5 3.4 3.0 2.4 2.3

Fig. 2. Southern analysis of transformant-digested genomic and chromosomal DNA. (Panel A)Southern analysis of piNS4 and pAN7-1 integrative transformants of strain M8650; lanes are numbered with either the transformant isolate number, Mr markers, or M8650, the untransformed recipient strain. Either the untransformed recipient strain (M8650), or transformant mycelium were freeze-dried and powdered in a mortar and pestle with the addition of a small amount of acid-washed sand. Genomic

(a) T r a n s f o r m a t i o n of Acremonium chrysogenum Table I shows the T f results for various constructs. A schematic linear representation o f the plasmids containing the pcbC p r o m o t e r is sl'3wn in Fig. IC. The plasmid p I H 0 was constructed from pIH3 by deletion of 1.8 kb o f A. chrysogenum pcbC promoter D N A leaving the hphencoding gene intact. This construct did not yield any transformants suggesting that a specific promoter activity is required for transformation. There was no statistical difference between the T f o f the three plasmids containing the pcbC promoter. This shows that the 476-bp pcbC promoter fragment is sufficient to express hph. A construct similar to p I N S 4 has been shown to result in high-frequency transformation using the pcbC-hph selection marker in As. niger (K0ck et al., 1989; Smith, 1990). T h e pcbCterminator fragment present in constructs p I H 2 and p I H 3 does not affect Tf; this is in line with previous findings (Skatrud et al., 1987). The T f for pAN7-1 was about twice that of constructs containing the pcbC promoter. T h e presence of large numbers of small colonies, less than 1 ~,~ o f which failed to grow on further incubation or sub-culture (abortive transformants), was a characteristic o f transformations with this vector. T h e n u m b e r of abortive :ransfor,nants produced

DNA was extracted by the method of Raeder and Broda (1985) with the addition of repeated phenol extraction and an ammonium acetate precipitation (Smith, 1990). Either 100 ng of 2 DNA, 5 ng of plasmid DNA or 5/,g of genomic DNA were digested with restriction enzymes (either Hindlll for ). DNA, pINS4 or pINS4 transformants, or BamH! for pAN7-I or pAN7-I transformants)and separated in 40 m M Tris. acetate/ 2 mM EDTA/0.8°,o agarose gels, as described in Maniatis et al. (1982). Agarose gels were alkali-blotted onto Hybond-N (Amersham) nylon membrane, hybridised and washed according to the manufacturer's instructions. Hybridised and washed membranes were exposed to Fuji RX X-ray film with intensifying screens for 18 h. Probe DNA was linearised (50 ng plasmid and 5 ng ). DNA), labelled by random priming using an oligodeoxyribonucleotidekit (Pharmacia) and [',J2P]dCTP (Amersham) according to the manufacturer's instructions to a specific activity of between 0.7 to ! x 109dpm//,g of DNA. The lanes labelled 2Hindlll are Mr markers in kb from a HindIll digest of ). DNA. Transforming plasmid DNA was used as the probe in each case. (Panel B)Transformant protoplasts were prepared as in Table I, footnote c. The preparation and PFGE analysis of chromosomal DNA by CHEF-DR T M (Bio-Rad) were as previously described (Smith et al., 1991). After separation, gels were alkali-blotted, hybridised and washed as in the legend to panel A. The probe was pAN7-I-prepared as in the legend to panel A. Transformants 4/8 and 4/2 are stable integrativetransformants of the pcbCpromoter-hph construct piNS4. The transformants 71/5, 6 and 4 are stable integrative transformants of pAN7-1. The loading position of the protoplast plugs and faint hybridisation bands are arrowed. The designation of the hybridising chromosome in each transformant is noted as previously determined in the untransformed strain M8650 (Smith et al., 1991). in the schematic representation of the strain M8650 karyotype (on the right margin), chromosome ! (0.3 Mb) would lie far below.

214 transformations using pAN7-1 ranged between 205 and 2000 per #g. One possible explanation for this large number of abortive transformants may be high-level expression from the gpdA promoter prior to and interfering with integration.

(b) Colony Hy-resistance levels About 100 transformants obtained using each plasmid were screened for their Hy a level and the patterns over the range 0-1600 #g/ml are shown for pIH2, pIH3 and pINS4 in Fig. 1A. In each case the modal Hy a value was 800 #g/ ml and probably represents the most common level of Hy R in a population of transformants derived from pcbC promoter-hph constructs. The distribution of Hy R transformants of pAN7-1 is shown in Fig. lB. Of the transformants of pAN7-1, 89% were Hy R to at least 1600 #g/ml, a level of resistance reached by only 25-45% (pIH3 and pIH2, respectively) of constructs containing pcbC promoter-hph combination. (c) Southern analysis of transformant DNA Genomic DNA from five representative transformants obtained using pINS4, pAN7-1 and the untransformed recipient strain (M8650) were digested with restriction enzymes cutting at one site (HindIII for pINS4 and BamHI for pAN7-1) within the transforming plasmid (Fig. 2A). In the two plasmids containing the pcbC promoter (4/8 and 4/2) a hybridization band was observed which corresponded to the same electrophoretic mobility as Hi~idllldigested pINS4 (far righthand lane in Fig. 2A). This relatively intense band in transformant DNA could be compared to the faint band for the single copy of the pcbC promoter in M8650 (Fig. 2A) and indicated that more than one intact integration of the plasmid had occurred probably as a tandem repeat at a nonhomologous sites(Fincham, 1989). Hybridisation bands that indicated fragments either larger or smaller than the digested plasmids possibly represent sequences flanking tandemly repeated integrations, rearrangements of probe-related sequences or other ectopic integrations on the same chromosome (see section d). The BarnHI hybridisation pattern of the pAN7- l transformants was different in each transformant with the exception of a single band having the same electrophoretic mobility as BamHI-digested pAN7-1. Transformant 71/5 showed many integration events probably involving deletion and rearrangement of the transforming DNA.

(d) Pulsed-field gel electrophoresis (PFGE) of transformant protoplasts The separation by PFGE and hybridisation of chromospinal DNA from protoplast lysates of the same six representative strains in Fig. 2A, is shown in Fig. 2B. In each case a single hybridisation band is observed (faint bands

are arrowed). This suggested that a single chromosome was involved in integrations in each transformant. Even in the extreme case of transformant 5 of pAN7-1 (Fig. 2A) which showed many integrations probably with rearrangements or deletions on the genomic digests, all the integrations occur on a single chromosome. It is not possible without exhaustive analysis to determine whether a single site on the chromosome is involved or whether there are multiple integrations along the same chromosome. If the model of a primary event forming a 'hot-spot' for further events is true (Fincham, 1989), then the former would apply.

(e) HPT activity in transformants The HPT activities of two transformants and an untransformed recipient strain were assessed during the time course of cephalosporin C fermentation (Fig. 3). The levels of enzyme in the transformant of pAN7-1 (71/5) are significantly higher than in either the transformant of piN $4 (4/8) or the untransformed control over the time tested. The high HPT activity and Hy R phenotype in pAN7-1 transformant 71/5 can be directly correlated. In contrast, transfor;aaats of pINS4 produce low levels of HPT activity throughout the fermentation and have a low mean Hy R phenotype. The continuously low H PT expression in trans-

800 "1 700"~ 4

~

A

7511=Me650(pANT-1)

:

418=M8650(plNS4) M8650

tX

'°°t\

f-,, i

Z[

,°°

100 I 1

.

2

.

.

3

.

.

4

.

5 Time

.

6

.

7

8

9

(dsys)

Fig. 3. HPT assays for piNS4, pAN7-1 transformants and untransformed M8650. Mycclium was recovered from a batch fermentation under the conditions previously described (Smith ct al., 1990), with growth in seed medium (Smith ct al., 1990) until day 2; then a 20% inoculum from this into production medium (Smith et al., 1990) for all subsequent days. Mycdium was freeze-dried, powdered as in Fig. 2A, suspended in 500 pl of enzyme extraction buffer (10 mM Tris pH 7.5/1 mM MgCI2/0.15 mM EDTA/I mM DTT/I mM PMSF/2 pg pepstatin A per ml/0.5 #g leupeptin per ml) and stored on ice for 30 rain with occasional vortex mixing. The supernatant was removed after centrifugation at 13 500 rpm for 30 rain at 4~C and stored at -70°C as the cell-free extract. The protein levels in cell-free extracts were quantified by the method of Bradford (1976) (Bio-Rad microassay) and the extracts were assayed for HPT activity by the method of Mohr (1989), modified for scintillation counting (Smith, 1990). Transformant 75/1 was a stable integrative chromosomal transformant of strain M8650 obtained using pAN7-1 that is M8650(pAN7I). Transformant 4/8 was a stable integrative chromosomal transformant of strain M8650 obtained using pINS4 that is M8650(pINS4).

215 formant 4/8 contrasts with the dramatic increases seen in endogenous pcbC expression under similar conditions (Ramsden et al., 1989; Smith et al., 1990). This suggests that the pcbC promoter fragments used in the current studies are not subject to normal regulatory control when linked to hph. These results differ from the regulated expression obtained using Peniciilium chrysogenum or As. nidulans pcbC promoter constructs to express lacZ as a reporter gene in As. nidulans transformant, s (G6mez-Pardo and Peflalva, 1990; Kolar et al., 1991). The reasons for the lack of normal regulation of the pcbC-hph constructs remain unclear. One possibility is that the position and state of the integrated construct copies, in particular on different chromosomes, could affect expression (Miller et al., 1987). A further possibility is that multiple copies of the pcbC promoter element present in these transformants may cause titration of a putative positive trans-acting effector (Hynes et al., 1988) of pcbC expression and thus transformants have multicopy deregulated hph e~pression by virtue of dominant marker selection. The origin of such an effector is unclear but there is some evidence of a role for the activity of IPNS enzyme or its product in the regulation ofpcbC (Ramsden et al., 1989). (f) Conclusions (i) The gpdA promoter causes efficient expression ofhph as assessed by Tf, Hy R phenotype and HPT activity. By the same criteria, the pcbC promoter is less effective at expressing hph. (2) Transformants carrying gpdA promoter constructs show multiple copies of transforming DNA which are likely to be rearranged. Conversely, the pcbC promoter construct transformants appear to carry a higher proportion of integrations in tandem repeats. These contrasting events on integration may have been due to the level of expression of the hph gene, perhaps before integration. (3) All transformants so far studied, regardless of the nature of the transforming DNA, had integration events which occurred at a single chromosome and this target chromosome was different in four out of five transformants analysed. (4) The transformed pcbC promoter-hph constructs were not subject to the normal regulatory control acting on pcbC. Further investigation of the regulation of endogenous pcbC gene expression in transformants, possibly at the level of transcription, may result in the identification of tranr-acting effectors of pcbC expression. (5) Expression of hph can only be utilised as a simple system for assay of the presence of gene promoter activity. The system is not suitable as a means of investigating the regulation of pcbC-encoding gene expression especially as alternative reporter genes are now available (Roberts et al., 1989).

ACKNOWLEDGEMENTS We would like to thank Dr. C.A.M.J.J. van den Hondel (TNO, The Netherlands) for the plasmid pAN7-1, Mr. B.V. Case (Nottingham) and Miss C.S. Pink (SmithKline Beecham) for photographic assistance. A.W.S. wishes to thank the Science and Engineering Research Council for a CASE studentship awarded in collaboration with Glaxochem Ltd.

REFERENCES Bradford, M.M.: A rapid and sensitive method for the quantitation ofpg quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72 (I 976) 248-254. Chapman, J.L., Skatrud, P.L., lngolia, T.D., Samson, S.M., Kaster, K.R. and Queener, S.W.: Recombinant DNA studies in Cephalosporium acremonium. In: Pierce, G. (Ed.), Developments in Industrial Microbiology, Vol. 27. Society for Industrial Microbiology, Arlington, Virginia, 1987, pp. 165-174. Fincham, J.R.S.: Transformation in fungi. Microbiol. Rev. 53 (1989) 148-170. G6mez-Pardo, E. and Peflalva, M.A.: The upstream region of the IPNS gene determines expression during secondary metabolism in Aspergillusnidulam. Gene 89 (1990) 109-115. Hynes, M.J., Corrick, C.M., Kelly, J.M. and Littlejohn, T.G.: Identification of the sites of action for regulatorygenes controlling the amdS gene of Aspergillusnidulans. Mol. Cell. Biol. 8 (1988) 2589-2596. Kolar, M., Holzmann, K., Weber, G., Leitner, E. and Schwab, H.: Molecular characterization and functional analysis in Aspergillusnidulans of the 5'-region of the Penicilliumcho'sogenumisopemcillin N synthetase gene. J. Bioteeh. 17 (1991) 67-80. K0ck, U., Walz, M., Mohr, G. and Mracek, M.: The 5'-sequence of the isopenicillin N-synthctase gene (pcbC) from Cephalosporium acremonium directs the expression of the prokaryotic hygromycin B phosphotransferase gene (hph) in Aspergillusuiger. Appl. Microbiol. Biotechnol. 31 (1989)358-365. Maniatis, T., Fritsch, E.F. and Sambrook, J.: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982. Miller, B.L., Miller, K.Y., Roberti, K.A. and Timberlake, W.E.: Positiondependent and -independent mechanisms regulatecell-specificexpression of spoCl gene cluster ofAspergillus nidulans. Mol. Cell. Biol. 7 (1987) 427-434. Mohr, G.: Rapid detection of bacterial hygromycin B phosphatase in Aspergillusnigertransformants. Appl. Microbiol. Biotechnol. 30 ( i 989) 371-374. Punt, P.J., Oliver, R.P., Dingemanse, M.A., Pouwels, P.H. and Van den Hondel, C.A.M.J.J.: Transformation of Aspergillusbased on the h~gromycin B resistance marker from Escherichiacoli. Gene 56 (1987) 117-124. Queener, S.W.: Molecular biology of penicillin and cephalosporin biosynthesis. Antimicrob. Agents Chemother. 34 (1990) 943-948. Raeder, U. and Broda, P.: Rapid preparation of DNA from filamentous fungi. Lett. Appl. Microbiol. I (1985) 17-20. Ramsden, M., McQuade, B.A., Saunders, K., Turner, M.K. and Harford, S.: Characterization of a loss-of-function mutation in the isopenicillin N synthetase gene of Acremoniumcho'~ogenum.Gene 85 (1989) 267273. Roberts, I.N., Oliver, R.P., Punt, P.J. and Van den Hondel, C.A.M.J.J.:

216 Expression ofthe Escherichiacoil beta-glucuronidase gene in industrial and phytopathogenic filamentous fungi. Curt. Genet. 15 (1989) 177180. Samson, S.M., Belagaje, R., Blankenship, D.T., Chapman, J.L., Perry, D., Skatrud, P.L., Van Frank, R.M., Abraham, E.P., Baldwin, J.E., Queener, S.W. and Ingolia, T.D.: Isolation, sequence determination and expression in Escherichia coil of the isopenicdlin N synthetase gene from Cephalosporium acremonium. Nature 318 (1985) 191-194. Samson, S.M., Dotzlaf, J.E., Slisz, M.L., Becker, G.W., Van Frank, R.M., Veal, L.E., Yeh, W.-K., Miller, J.R., Queener, S.W. and Ingolia, T.D.: Cloning and expression of the fungal expandase/hydroxylase gene involved in cephalosporin biosynthesis. Bic/Technology 5 (1987) 1207-1214. Skatrud, P.L., Queener, S.W., Cart, L.G. and Fisher, D.L.: Efficient integrative transformation of Cephalosporium acremouium. Curr. Genet. 12 (1987) 337-348. Skatrud, P.L., Tietz, A.J., ingolia, T.D., Cantwell, C.A., Fisher, D.L.,

Chapman, J.L. and Queener, S.W.: Use of recombinant DNA to improve production of cephalosporin C by Cephalosporium acremonium. Bio/Teehnology 7 (1989) 477-485. Smith, A.W.: The Isopenicillin N Synthetase (IPNS) Gene (pcbC) Promoter of Acremonium chrysogenum. Ph.D. Thesis, University of Nottingham, Nottingham, UK, 1990. Smith, A.W., Ramsden, M., Dobson, M.J., Hafford, S. and Peberdy, J.F.: Regulation of isopenicillin N synthetase (iPNS)gene expression in Acremonium chrysogenum. Bio/Technology 8 (1990) 237-240. Smith, A.W., Collis, K., Ramsden, M., Fox, H.M. and Peberdy, J.F.: Chromosome rearrangements in improved cephalosporin C producing strains ofAcremonium chrysogem,n. Curt. Genet. 19 (1991) 235237. Waterworth, P.: Quantitative methods for bacterial sensitivity testing. In: Reeves, D., Phillips, I., Williams, J.D. and Wise, R. (Eds.), Laboratory Methods in Antimicrobial Chemotherapy. Churchill Livingstone, London, 1978, pp. 31-40.