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. 1992 Jun;174(11):3789–3799. doi: 10.1128/jb.174.11.3789-3799.1992

Regulation of Aspergillus nidulans penicillin biosynthesis and penicillin biosynthesis genes acvA and ipnA by glucose.

A A Brakhage 1, P Browne 1, G Turner 1
PMCID: PMC206070  PMID: 1592830

Abstract

Expression of the Aspergillus nidulans penicillin biosynthesis genes acvA and ipnA, encoding delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase and isopenicillin N synthetase, respectively, was analyzed. The intergenic region carrying the divergently oriented promoters was fused in frame in both orientations to Escherichia coli lacZ and E. coli uidA reporter genes. Each construct permits simultaneous expression studies of both genes. Transformants of A. nidulans carrying a single copy of either plasmid integrated at the chromosomal argB locus were selected for further investigations. Expression of both genes was directed by the 872-bp intergenic region. ipnA- and acvA-derived gene fusions were expressed from this region at different levels. ipnA had significantly higher expression than did acvA. Glucose specifically reduced the production of penicillin and significantly repressed the expression of ipnA but not of acvA gene fusions. The specific activities of isopenicillin N synthetase, the gene product of ipnA, and acyl coenzyme A:6-aminopenicillanic acid acyltransferase were also reduced in glucose-grown cultures.

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Selected References

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  1. Alvarez E., Cantoral J. M., Barredo J. L., Díez B., Martín J. F. Purification to homogeneity and characterization of acyl coenzyme A:6-aminopenicillanic acid acyltransferase of Penicillium chrysogenum. Antimicrob Agents Chemother. 1987 Nov;31(11):1675–1682. doi: 10.1128/aac.31.11.1675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bailey C., Arst H. N., Jr Carbon catabolite repression in Aspergillos nidulans. Eur J Biochem. 1975 Feb 21;51(2):573–577. doi: 10.1111/j.1432-1033.1975.tb03958.x. [DOI] [PubMed] [Google Scholar]
  3. Ballance D. J., Turner G. Development of a high-frequency transforming vector for Aspergillus nidulans. Gene. 1985;36(3):321–331. doi: 10.1016/0378-1119(85)90187-8. [DOI] [PubMed] [Google Scholar]
  4. Barredo J. L., Cantoral J. M., Alvarez E., Díez B., Martín J. F. Cloning, sequence analysis and transcriptional study of the isopenicillin N synthase of Penicillium chrysogenum AS-P-78. Mol Gen Genet. 1989 Mar;216(1):91–98. doi: 10.1007/BF00332235. [DOI] [PubMed] [Google Scholar]
  5. Barredo J. L., van Solingen P., Díez B., Alvarez E., Cantoral J. M., Kattevilder A., Smaal E. B., Groenen M. A., Veenstra A. E., Martín J. F. Cloning and characterization of the acyl-coenzyme A: 6-aminopenicillanic-acid-acyltransferase gene of Penicillium chrysogenum. Gene. 1989 Nov 30;83(2):291–300. doi: 10.1016/0378-1119(89)90115-7. [DOI] [PubMed] [Google Scholar]
  6. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  7. Brakhage A. A., Putzer H., Shazand K., Röschenthaler R. J., Grunberg-Manago M. Bacillus subtilis phenylalanyl-tRNA synthetase genes: cloning and expression in Escherichia coli and B. subtilis. J Bacteriol. 1989 Feb;171(2):1228–1232. doi: 10.1128/jb.171.2.1228-1232.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Buxton F. P., Radford A. Cloning of the structural gene for orotidine 5'-phosphate carboxylase of Neurospora crassa by expression in Escherichia coli. Mol Gen Genet. 1983;190(3):403–405. doi: 10.1007/BF00331067. [DOI] [PubMed] [Google Scholar]
  9. Carr L. G., Skatrud P. L., Scheetz M. E., 2nd, Queener S. W., Ingolia T. D. Cloning and expression of the isopenicillin N synthetase gene from Penicillium chrysogenum. Gene. 1986;48(2-3):257–266. doi: 10.1016/0378-1119(86)90084-3. [DOI] [PubMed] [Google Scholar]
  10. Davis M. A., Hynes M. J. Regulatory genes in Aspergillus nidulans. Trends Genet. 1989 Jan;5(1):14–19. doi: 10.1016/0168-9525(89)90006-1. [DOI] [PubMed] [Google Scholar]
  11. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  12. Fincham J. R. Transformation in fungi. Microbiol Rev. 1989 Mar;53(1):148–170. doi: 10.1128/mr.53.1.148-170.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Foster J. W., Karow E. O. Microbiological Aspects of Penicillin: VIII. Penicillin from Different Fungi. J Bacteriol. 1945 Jan;49(1):19–29. doi: 10.1128/jb.49.1.19-29.1945. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Guthrie E. P., Chater K. F. The level of a transcript required for production of a Streptomyces coelicolor antibiotic is conditionally dependent on a tRNA gene. J Bacteriol. 1990 Nov;172(11):6189–6193. doi: 10.1128/jb.172.11.6189-6193.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gómez-Pardo E., Peñalva M. A. The upstream region of the IPNS gene determines expression during secondary metabolism in Aspergillus nidulans. Gene. 1990 Apr 30;89(1):109–115. doi: 10.1016/0378-1119(90)90212-a. [DOI] [PubMed] [Google Scholar]
  16. Hynes M. J., Kelly J. M. Pleiotropic mutants of Aspergillus nidulans altered in carbon metabolism. Mol Gen Genet. 1977 Jan 18;150(2):193–204. doi: 10.1007/BF00695399. [DOI] [PubMed] [Google Scholar]
  17. Johnstone I. L., Hughes S. G., Clutterbuck A. J. Cloning an Aspergillus nidulans developmental gene by transformation. EMBO J. 1985 May;4(5):1307–1311. doi: 10.1002/j.1460-2075.1985.tb03777.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lennard A. C., Fried M. The bidirectional promoter of the divergently transcribed mouse Surf-1 and Surf-2 genes. Mol Cell Biol. 1991 Mar;11(3):1281–1294. doi: 10.1128/mcb.11.3.1281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. MacCabe A. P., Riach M. B., Unkles S. E., Kinghorn J. R. The Aspergillus nidulans npeA locus consists of three contiguous genes required for penicillin biosynthesis. EMBO J. 1990 Jan;9(1):279–287. doi: 10.1002/j.1460-2075.1990.tb08106.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. MacCabe A. P., van Liempt H., Palissa H., Unkles S. E., Riach M. B., Pfeifer E., von Döhren H., Kinghorn J. R. Delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Aspergillus nidulans. Molecular characterization of the acvA gene encoding the first enzyme of the penicillin biosynthetic pathway. J Biol Chem. 1991 Jul 5;266(19):12646–12654. [PubMed] [Google Scholar]
  21. Marahiel M. A., Zuber P., Czekay G., Losick R. Identification of the promoter for a peptide antibiotic biosynthesis gene from Bacillus brevis and its regulation in Bacillus subtilis. J Bacteriol. 1987 May;169(5):2215–2222. doi: 10.1128/jb.169.5.2215-2222.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Martin J. F., Demain A. L. Control of antibiotic biosynthesis. Microbiol Rev. 1980 Jun;44(2):230–251. doi: 10.1128/mr.44.2.230-251.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Miller J. R., Ingolia T. D. Cloning and characterization of beta-lactam biosynthetic genes. Mol Microbiol. 1989 May;3(5):689–695. doi: 10.1111/j.1365-2958.1989.tb00217.x. [DOI] [PubMed] [Google Scholar]
  24. Montenegro E., Barredo J. L., Gutiérrez S., Díez B., Alvarez E., Martín J. F. Cloning, characterization of the acyl-CoA:6-amino penicillanic acid acyltransferase gene of Aspergillus nidulans and linkage to the isopenicillin N synthase gene. Mol Gen Genet. 1990 May;221(3):322–330. doi: 10.1007/BF00259395. [DOI] [PubMed] [Google Scholar]
  25. Nüesch J., Heim J., Treichler H. J. The biosynthesis of sulfur-containing beta-lactam antibiotics. Annu Rev Microbiol. 1987;41:51–75. doi: 10.1146/annurev.mi.41.100187.000411. [DOI] [PubMed] [Google Scholar]
  26. Pobjecky N., Rosenberg G. H., Dinter-Gottlieb G., Käufer N. F. Expression of the beta-glucuronidase gene under the control of the CaMV 35s promoter in Schizosaccharomyces pombe. Mol Gen Genet. 1990 Jan;220(2):314–316. doi: 10.1007/BF00260500. [DOI] [PubMed] [Google Scholar]
  27. Punt P. J., Dingemanse M. A., Kuyvenhoven A., Soede R. D., Pouwels P. H., van den Hondel C. A. Functional elements in the promoter region of the Aspergillus nidulans gpdA gene encoding glyceraldehyde-3-phosphate dehydrogenase. Gene. 1990 Sep 1;93(1):101–109. doi: 10.1016/0378-1119(90)90142-e. [DOI] [PubMed] [Google Scholar]
  28. Punt P. J., Greaves P. A., Kuyvenhoven A., van Deutekom J. C., Kinghorn J. R., Pouwels P. H., van den Hondel C. A. A twin-reporter vector for simultaneous analysis of expression signals of divergently transcribed, contiguous genes in filamentous fungi. Gene. 1991 Jul 31;104(1):119–122. doi: 10.1016/0378-1119(91)90476-r. [DOI] [PubMed] [Google Scholar]
  29. Queener S. W. Molecular biology of penicillin and cephalosporin biosynthesis. Antimicrob Agents Chemother. 1990 Jun;34(6):943–948. doi: 10.1128/aac.34.6.943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ramos F. R., López-Nieto M. J., Martín J. F. Isopenicillin N synthetase of Penicillium chrysogenum, an enzyme that converts delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to isopenicillin N. Antimicrob Agents Chemother. 1985 Mar;27(3):380–387. doi: 10.1128/aac.27.3.380. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ramón D., Carramolino L., Patiño C., Sánchez F., Peñalva M. A. Cloning and characterization of the isopenicillin N synthetase gene mediating the formation of the beta-lactam ring in Aspergillus nidulans. Gene. 1987;57(2-3):171–181. doi: 10.1016/0378-1119(87)90120-x. [DOI] [PubMed] [Google Scholar]
  32. Revilla G., López-Nieto M. J., Luengo J. M., Martín J. F. Carbon catabolite repression of penicillin biosynthesis by Penicillium chrysogenum. J Antibiot (Tokyo) 1984 Jul;37(7):781–789. doi: 10.7164/antibiotics.37.781. [DOI] [PubMed] [Google Scholar]
  33. Revilla G., Ramos F. R., López-Nieto M. J., Alvarez E., Martín J. F. Glucose represses formation of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine and isopenicillin N synthase but not penicillin acyltransferase in Penicillium chrysogenum. J Bacteriol. 1986 Nov;168(2):947–952. doi: 10.1128/jb.168.2.947-952.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. SOLTERO F. V., JOHNSON M. J. The effect of the carbohydrate nutrition on penicillin production by Penicillium chrysogenum Q-176. Appl Microbiol. 1953 Jan;1(1):52–57. doi: 10.1128/am.1.1.52-57.1953. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Shah A. J., Tilburn J., Adlard M. W., Arst H. N., Jr pH regulation of penicillin production in Aspergillus nidulans. FEMS Microbiol Lett. 1991 Jan 15;61(2-3):209–212. doi: 10.1016/0378-1097(91)90553-m. [DOI] [PubMed] [Google Scholar]
  37. Smith D. J., Bull J. H., Edwards J., Turner G. Amplification of the isopenicillin N synthetase gene in a strain of Penicillium chrysogenum producing high levels of penicillin. Mol Gen Genet. 1989 Apr;216(2-3):492–497. doi: 10.1007/BF00334395. [DOI] [PubMed] [Google Scholar]
  38. Smith D. J., Burnham M. K., Bull J. H., Hodgson J. E., Ward J. M., Browne P., Brown J., Barton B., Earl A. J., Turner G. Beta-lactam antibiotic biosynthetic genes have been conserved in clusters in prokaryotes and eukaryotes. EMBO J. 1990 Mar;9(3):741–747. doi: 10.1002/j.1460-2075.1990.tb08168.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Smith D. J., Earl A. J., Turner G. The multifunctional peptide synthetase performing the first step of penicillin biosynthesis in Penicillium chrysogenum is a 421,073 dalton protein similar to Bacillus brevis peptide antibiotic synthetases. EMBO J. 1990 Sep;9(9):2743–2750. doi: 10.1002/j.1460-2075.1990.tb07461.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Timberlake W. E., Marshall M. A. Genetic regulation of development in Aspergillus nidulans. Trends Genet. 1988 Jun;4(6):162–169. doi: 10.1016/0168-9525(88)90022-4. [DOI] [PubMed] [Google Scholar]
  41. Unkles S. E., Hawker K. L., Grieve C., Campbell E. I., Montague P., Kinghorn J. R. crnA encodes a nitrate transporter in Aspergillus nidulans. Proc Natl Acad Sci U S A. 1991 Jan 1;88(1):204–208. doi: 10.1073/pnas.88.1.204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Ward M., Wilkinson B., Turner G. Transformation of Aspergillus nidulans with a cloned, oligomycin-resistant ATP synthase subunit 9 gene. Mol Gen Genet. 1986 Feb;202(2):265–270. doi: 10.1007/BF00331648. [DOI] [PubMed] [Google Scholar]
  43. Whiteman P. A., Abraham E. P., Baldwin J. E., Fleming M. D., Schofield C. J., Sutherland J. D., Willis A. C. Acyl coenzyme A: 6-aminopenicillanic acid acyltransferase from Penicillium chrysogenum and Aspergillus nidulans. FEBS Lett. 1990 Mar 26;262(2):342–344. doi: 10.1016/0014-5793(90)80224-7. [DOI] [PubMed] [Google Scholar]
  44. van Gorcom R. F., Pouwels P. H., Goosen T., Visser J., van den Broek H. W., Hamer J. E., Timberlake W. E., van den Hondel C. A. Expression of an Escherichia coli beta-galactosidase fusion gene in Aspergillus nidulans. Gene. 1985;40(1):99–106. doi: 10.1016/0378-1119(85)90028-9. [DOI] [PubMed] [Google Scholar]
  45. van Gorcom R. F., Punt P. J., Pouwels P. H., van den Hondel C. A. A system for the analysis of expression signals in Aspergillus. Gene. 1986;48(2-3):211–217. doi: 10.1016/0378-1119(86)90079-x. [DOI] [PubMed] [Google Scholar]
  46. van Liempt H., von Döhren H., Kleinkauf H. delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Aspergillus nidulans. The first enzyme in penicillin biosynthesis is a multifunctional peptide synthetase. J Biol Chem. 1989 Mar 5;264(7):3680–3684. [PubMed] [Google Scholar]

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