Skip to main content
Log in

Expression of the penDE gene of Penicillium chrysogenum encoding isopenicillin N acyltransferase in Cephalosporium acremonium: production of benzylpenicillin by the transformants

  • Published:
Molecular and General Genetics MGG Aims and scope Submit manuscript

Summary

No DNA sequence homologous to the penDE gene of Penicillium chrysogenum was found in the genome of three different strains of Cephalosporium acremonium. The pcbC-penDE gene cluster of P. chrysogenum complemented the isopenicillin N synthase deficiency of C. acremonium mutant N2 and resulted in the production of penicillin, in addition to cephalosporin, in cultures supplemented with phenylacetic acid. The penicillin formed was identified as benzylpenicillin by HPLC and NMR studies. The penDE gene of P. chrysogenum is expressed in C. acremonium forming a transcript of 1.15 kb. The transcript is processed and translated in C. acremonium resulting in the formation of acyl CoA isopenicillin N acyl transferase. When the penDE gene was introduced into a cephalosporin producing strain, the total titre of β-lactam antibiotics comprised distinct proportions of penicillin and cephalosporin in different transformants. Analysis of the hybridization patterns of the DNA of C. acremonium transformed with the pcbC or penDE genes indicated that integration occurs by non-homologous recombination.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Alvarez E, Cantoral JM, Barredo JL, Diez B, Martin JF (1987) Purification to homogeneity and characterization of acylcoenzyme A:6-aminopenicillanic acid acyltransferase of Penicillium chrysogenum. Antimicrob Agents Chemother 31:1675–1682

    Google Scholar 

  • Ausubel FM, Brent R, Kingston RE, Moore D, Smith JA, Seidman JG, Struhl K (1987) Current protocols in molecular biology. Wiley, New York

    Google Scholar 

  • Ballance DJ (1986) Sequences important for gene expression in filamentous fungi. Yeast 2:229–236

    Google Scholar 

  • Barredo JL, Cantoral JM, Alvarez E, Diez E, Martin JF (1989a) Cloning, sequence analysis and transcriptional study of the isopenicillin N synthase of Penicillium chrysogenum AS-P-78. Mol Gen Genet 216:91–98

    Google Scholar 

  • Barredo JL, van Solingen P, Diez B, Alvarez E, Cantoral JM, Kattevilder A, Smaal EB, Groenen MAM, Veenstra AE, Martin JF (1989b) Cloning and characterization of the acylcoenzyme A:6-aminopenicillanic acid acyltransferase gene of Penicillium chrysogenum. Gene 83:291–300

    Google Scholar 

  • Barredo JL, Diez B, Alvarez E, Martin JF (1989c) Large amplification of a 35-kb DNA fragment carrying two penicillin biosynthetic genes in high penicillin producing strains of Penicillium chrysogenum. Curr Genet 16:453–459

    Google Scholar 

  • Caltrider PG, Niss HF (1966) Role of methionine in cephalosporin synthesis. Appl Microbiol 14:746–753

    Google Scholar 

  • Cantoral JM, Diez B, Barredo JL, Alvarez E, Martin JF (1987) High frequency transformation of Penicillium chrysogenum. Bio/Technology 5:494–497

    Google Scholar 

  • Carr LG, Skatrud PL, Scheetz III ME, Queener SW, Ingolia TD (1986) Cloning and expression of the isopenicillin N synthetase gene from Penicillium chrysogenum. Gene 48:257–266

    Google Scholar 

  • Díez B, Alvarez E, Cantoral JM, Barredo JL, Martín JF (1987) Selection and characterization of pyrG mutants of Penicillium chrysogenum lacking orotidine-5′-phosphate decarboxylase and complementation by the pyr4 gene of Neurospora crassa. Curr Genet 12:277–282

    Google Scholar 

  • Díez B, Barredo JL, Alvarez E, Cantoral JM, van Solingen P, Groenen MAM, Veenstra AE, Martin JF (1989) Two genes involved in penicillin biosynthesis are linked in a 5.1 kb Sall fragment in the genome of Penicillium chrysogenum. Mol Gen Genet 218:572–576

    Google Scholar 

  • Díez B, Gutierrez S, Barredo JL, van Solingen P, van der Voort LHM, Martín JF (1990) The cluster of penicillin biosynthetic genes. Identification and characterization of the pcbAB gene encoding the α-aminoadipyl-cysteinyl-valine synthetase and linkage to the pcbC and penDE genes. J Biol Chem 265:16358–16365

    Google Scholar 

  • Elander RP (1983) Strain improvement and preservation of β-lactam-producing fungi. In: Demain AL, Solomon NA (eds) Antibiotics containing the beta-lactam structure. Springer-Verlag, Berlin, pp 97–146

    Google Scholar 

  • Fincham JRS (1989) Transformation in fungi. Microbiol Rev 53:148–170

    Google Scholar 

  • Hopwood DA, Malpartida F, Kieser HM, Ikeda H, Duncan J, Fujii I, Rudd BAM, Floss HG, Omura S (1985) Production of “hybrid” antibiotics by genetic engineering. Nature 314:642–644

    Google Scholar 

  • Kolar M, Punt PJ, van den Hondel CAMJJ, Schwab H (1988) Transformation of Penicillium chrysogenum using dominant selection markers and expression of an Escherichia coli lacZ fusion gene. Gene 62:127–134

    Google Scholar 

  • Láiz L, Liras P, Castro JM, Martin JF (1990) Purification and characterization of the isopenicillin N epimerase from Nocardia lactamdurans. J Gen Microbiol 136:663–671

    Google Scholar 

  • MacCabe AP, Riach MBR, Unkless SE, Kinghorn JF (1990) The Aspergillus npeA consists of three contigous genes required for penicillin biosynthesis. EMBO J 9:279–287

    Google Scholar 

  • Martin JF, Liras P (1989) Enzymes involved in penicillin, cephalosporin and cephamycin biosynthesis. In: Fiechter A (ed) Advances in biochemical engineering and biotechnology. Springer-Verlag, Berlin, pp 153–187

    Google Scholar 

  • Martín JF, Ingolia T, Queener S (1990) Molecular genetics of penicillin and cephalosporin antibiotic biosynthesis. In: Leong SA, Berka R (eds) Molecular industrial mycology: Systems and applications. Marcel Dekker, New York, pp 149–195

    Google Scholar 

  • Montenegro E, Barredo JL, Gutierrez S, Díez B, Alvarez E, Martín JF (1990) Cloning, characterization of the acyl-CoA:6-aminopenicillanic acid acyltransferase gene of Aspergillus nidulans and linkage to the isopenicillin N synthase gene. Mol Gen Genet 221:322–330

    Google Scholar 

  • Onions AHS, Brady BL (1987) Taxonomy of Penicillium and Acremonium. In: Peberdy JF (ed) Penicillium and Aeremonium. Plenum, New York, pp 1–36

    Google Scholar 

  • Pérez-Martínez G, Peberdy JF (1985) Production of cephalosporin C, and its intermediates, by raised-titre strains of Acremonium chrysogenum. Enzyme Microb Technol 7:389–394

    Google Scholar 

  • Queener SW, Ingolia TD, Skatrud PL, Chapman JL, Kaster KR (1985) A system for genetic transformation of Cephalosporium acremonium. In: Leive L (ed) Microbiology 1985. ASM Publications, Washington, DC, pp 468–472

    Google Scholar 

  • Ramos FR, López-Nieto MJ, Martín JF (1986) Co-ordinate increase of isopenicillin N synthetase, isopenicillin N epimerase and deacetoxycephalosporin C synthetase in a high cephalosporin-producing mutant of Acremonium chrysogenum and simultaneous loss of the three enzymes in a non-producing mutant. FEMS Microbiol Lett 35:123–127

    Google Scholar 

  • Ramsden M, McQuade BA, Saunders K, Turner MK, Harford S (1989) Characterization of a loss-of-function mutation in the isopenicillin N synthetase gene of Acremonium chrysogenum. Gene 85:267–273

    Google Scholar 

  • Samson SM, Belagaje R, Blankenship DT, Chapman JL, Perry D, Skatrud PL, Van Frank RM, Abraham EP, Baldwin JE, Queener SW, Ingolia TD (1985) Isolation, sequence determination and expression in Escherichia coli of the isopenicillin N synthetase gene from Cephalosporium acremonium. Nature 318:191–194

    Google Scholar 

  • Samson SM, Dotzlaf JE, Slisz ML, Becker GW, Van Frank RM, Veal LE, Miller JR, Yeh W-K, Queener SW, Ingolia TD (1987) Cloning and expression of the fungal expandase/hydroxylase gene involved in cephalosporin biosynthesis. Bio/Technology 5:1207–1214

    Google Scholar 

  • Shirafuji H, Fujisawa Y, Kida M, Kanzaki T, Yoneda M (1979) Accumulation of tripeptide derivatives by mutants of Cephalosporium acremonium. Agric Biol Chem 43:155–160

    Google Scholar 

  • Smith DJ, Brunham MKR, Edwards J, Earl AJ, Turner G (1990) Cloning and heterologous expression of the penicillin biosynthetic gene cluster from Penicillium chrysogenum. Bio/Technology 8:39–41

    Google Scholar 

  • Usui S, Yu C-A (1989) Purification and properties of isopenicillin N epimerase from Streptomyces clavuligerus. Biochim Biophys Acta 9:76–85

    Google Scholar 

  • Yelton MM, Hamer JE, Timberlake WE (1984) Transformation of Aspergillus nidulans by using a trpC plasmid. Proc Natl Acad Sci USA 81:1470–1474

    Google Scholar 

  • Zanca DM, Martin JF (1983) Carbon catabolite regulation of the conversion of penicillin N into cephalosporin C. J Antibiot 36:700–708

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Additional information

Communicated by C. van den Hondel

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gutiérrez, S., Díez, B., Alvarez, E. et al. Expression of the penDE gene of Penicillium chrysogenum encoding isopenicillin N acyltransferase in Cephalosporium acremonium: production of benzylpenicillin by the transformants. Molec. Gen. Genet. 225, 56–64 (1991). https://doi.org/10.1007/BF00282642

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00282642

Key words

Navigation