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