Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Environmentally safe production of 7-aminodeacetoxycephalosporanic acid (7-ADCA) using recombinant strains of Acremonium chrysogenum

Nature Biotechnology volume 18, pages 857–861 (2000)Cite this article

Abstract

Medically useful semisynthetic cephalosporins are made from 7-aminodeacetoxycephalosporanic acid (7-ADCA) or 7-aminocephalosporanic acid (7-ACA). Here we describe a new industrially amenable bioprocess for the production of the important intermediate 7-ADCA that can replace the expensive and environmentally unfriendly chemical method classically used. The method is based on the disruption and one-step replacement of the cefEF gene, encoding the bifunctional expandase/hydroxylase activity, of an actual industrial cephalosporin C production strain of Acremonium chrysogenum. Subsequent cloning and expression of the cefE gene from Streptomyces clavuligerus in A. chrysogenum yield recombinant strains producing high titers of deacetoxycephalosporin C (DAOC). Production level of DAOC is nearly equivalent (75–80%) to the total β-lactams biosynthesized by the parental overproducing strain. DAOC deacylation is carried out by two final enzymatic bioconversions catalyzed by D-amino acid oxidase (DAO) and glutaryl acylase (GLA) yielding 7-ADCA. In contrast to the data reported for recombinant strains of Penicillium chrysogenum expressing ring expansion activity, no detectable contamination with other cephalosporin intermediates occurred.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Four different strategies for 7-ADCA production.
Figure 2: Disruption of the cefEF gene.
Figure 3: HPLC and mass spectrum analysis of fermentation broth samples from A. chrysogenum transformants.
Figure 4: β-Lactam antibiotic production by the parental strain A. chrysogenum AC7, the transformant ΔcefEF-T1 lacking the enzymatic activities DAOCS/DACS, and the transformant ΔcefEF-T1/cefE-T1 lacking the enzymatic activities DAOCS/DACS but expressing the DAOCS (cefE) from S. clavuligerus.
Figure 5: Genetic map of the plasmid pALC88 used to introduce ring expansion activity (DAOCS) into A. chrysogenum ΔcefEF.
Figure 6: Southern analysis of genomic DNA from selected A. chrysogenum transformants using as probe a 0.4 kb NcoI-SmaI fragment corresponding to the cefE gene of S. clavuligerus28.

Similar content being viewed by others

References

  1. Hersbach, G.J.M., Van der Beek, C.P. & Van Dijck, P.W.M. The penicillins: Properties, biosynthesis and fermentation. In Biotechnology of industrial antibiotics. (ed. Vandamme, E.J.) 45–140 (Marcel Dekker, Inc., New York; 1984).

    Google Scholar 

  2. Samson, S.M. et al. Cloning and expression of the fungal expandase\hydroxylase gene involved in cephalosporin biosynthesis. Bio/Technology 5, 1207–1214 (1987).

    CAS  Google Scholar 

  3. Cantwell, C.A., Beckmann, R., Whiteman, P., Queener, S.W. & Abraham, E.P. Isolation of deacetoxycephalosporin C from fermentation broths of Penicillium chrysogenum transformants: construction of a new fungal biosynthetic pathway. Proc. R. Soc. London B 248, 283–289 (1992).

    Article  CAS  Google Scholar 

  4. Beckman, R., Cantwell, C.A., Whiteman, P., Queener, S.W. & Abraham, E.P. Production of deacetoxycephalosporin C by transformants of Penicillium chrysogenum: Antibiotic biosynthetic pathway engineering. In Industrial microorganisms: basic and applied molecular genetics. (eds Baltz, R.H., Hegeman, G.D. & Skatrud, P.L.) 177–182 (American Society for Microbiology, Washington, DC; 1993).

    Google Scholar 

  5. Cantwell, C.A. et al. Cloning and expression of a hybrid Streptomyces clavuligerus cefE gene in Penicillium chrysogenum. Curr. Genet. 17, 213–221 (1990).

    Article  CAS  Google Scholar 

  6. Queener, S.W. et al. Improved expression of a hybrid Streptomyces clavuligerus cefE gene in Penicillium chrysogenum. Ann. N.Y. Acad. Sci. 721, 178–193 (1994)

    Article  CAS  Google Scholar 

  7. Crawford, L. et al. Production of cephalosporin intermediates by feeding adipic acid to recombinant Penicillium chrysogenum strains expressing ring expansion activity. Bio/Technology 13, 58–62 (1995).

    CAS  PubMed  Google Scholar 

  8. Ballio, A. et al. Incorporation of α,ω-dicarboxylic acids as side chains into the penicillin molecule. Nature 185, 97–99 (1960).

    Article  CAS  Google Scholar 

  9. Kupka, A., Shen, Y.-Q., Wolfe, S. & Demain, A.L. Partial purification and properties of the α-ketoglutarate-linked ring-expansion enzyme of β-lactam biosynthesis of Cephalosporium acremonium. FEMS Microbiol. Lett. 16, 1–6 (1983).

    CAS  Google Scholar 

  10. Yeh, W.-K., Dotzlaf, J.E. & Huffman, G.W. Biochemical characterization and evolutionary implication of β-lactam expandase/hydroxylase, expandase and hydroxylase. In 50 Years of penicillin application: history and trends. (eds Kleinkauf, H. & von Doehren, H.) 208–223 (Public Ltd., Prague; 1994).

    Google Scholar 

  11. Maeda, K. et al. The substrate specificity of deacetoxycephalosporin C synthase (“expandase”) of Streptomyces clavuligerus is extremely narrow. Enz. Microb. Technol. 17, 231–234 (1995).

    Article  CAS  Google Scholar 

  12. Baldwin, J.E. et al. The enzymatic ring expansion of penicillins to cephalosporins: side chain specificity. Tetrahedron 43, 3009–3014 (1987).

    Article  CAS  Google Scholar 

  13. Demain, A.L. Enzymatic 7-ADCA: I said it couldn't be done. Bio/Technology 13, 23–24 (1995).

    CAS  PubMed  Google Scholar 

  14. Cho, H. et al. Elucidation of conditions allowing conversion of penicillin G and other penicillins to deacetoxycephalosporins by resting cells and extracts of Streptomyces clavuligerus NP1. Proc. Natl. Acad. Sci. USA 95, 11544–11548 (1998).

    Article  CAS  Google Scholar 

  15. Fernández, M.J. et al. Stimulatory effect of growth in the presence of alcohols on biotransformation of penicillin G into cephalosporin-type antibiotics by resting cells of Streptomyces clavuligerus NP1. Appl. Microbiol. Biotechnol. 52, 484–488 (1999).

    Article  Google Scholar 

  16. Isogai, T. et al. Construction of a 7-aminocephalosporanic acid (7ACA) biosynthetic operon and direct production of 7ACA in Acremonium chrysogenum. Bio/Technology 9, 188–191 (1991).

    CAS  PubMed  Google Scholar 

  17. Isogai, T. New processes for production of 7-aminocephalosporanic acid from Cephalosporium. In Biotechnology of antibiotics. (ed. Strohl, W.R.) 733–751 (Marcel Dekker, Inc. New York; 1997).

    Google Scholar 

  18. Alonso, J. et al. D-amino-acid oxidase gene from Rhodotorula gracilis (Rhodosporidium toruloides) ATCC 26217. Microbiology 144, 1095–1101 (1998).

    Article  CAS  Google Scholar 

  19. Alonso, J. et al. Engineering the D-amino-acid oxidase from Trigonopsis variabilis to facilitate its overproduction in Escherichia coli and its downstream processing by tailor-made metal chelate supports. Enz. Microb. Tech. 25, 88–95 (1999).

    Article  CAS  Google Scholar 

  20. Matsuda, A. & Komatsu, K.I. Molecular cloning and structure of the gene for 7β-(4-carboxybutanamido) cephalosporanic acid acylase from a Pseudomonas strain. J. Bacteriol. 163, 1222–1228 (1985).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Croux, C., Costa, J., Barredo, J.L. & Salto, F. Process for the enzymatic preparation of 7-aminocephalosporanic acid. US 05354667 (1994).

  22. Cambiaghi, S., Tomaselli, S. & Verga, R. Enzymatic process for preparing 7-aminocephalosporanic acid and derivatives. US 5424196 (1995).

  23. Rothstein, R.J. One-step gene disruption in yeast. Methods Enzymol. 101, 202–211 (1983).

    Article  CAS  Google Scholar 

  24. Kolar, M., Holzmann, K., Weber, G., Leitner, E. & Schwab, H. Molecular characterization and functional analysis in Aspergillus nidulans of the 5′-region of the Penicillium chrysogenum isopenicillin N synthetase gene. J. Biotechnol. 17, 67–80 (1991).

    Article  CAS  Google Scholar 

  25. Feng, B., Friedlin, E. & Marzluf, G.A. A reporter gene analysis of penicillin biosynthesis gene expression in Penicillium chrysogenum and its regulation by nitrogen and glucose catabolite repression. Appl. Environ. Microbiol. 60, 4432–4439 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Gutiérrez, S. et al. Expression of the cefG gene is limiting for cephalosporin biosynthesis in Acremonium chrysogenum. Appl. Microbiol. Biotechnol. 48, 606–614 (1997).

    Article  Google Scholar 

  27. Díez, B., Mellado, E., Rodríguez, M., Bernasconi, E. & Barredo, J.L. The NADP-dependent glutamate dehydrogenase gene from Penicillium chrysogenum and the construction of expression vectors for filamentous fungi. Appl. Microbiol. Biotechnol. 52, 196–207 (1999).

    Article  Google Scholar 

  28. Kovacevic, S., Tobin, M.B. & Miller, J.R. The β-lactam biosynthesis genes for isopenicillin N epimerase and deacetoxycephalosporin C synthetase are expressed from a single transcript in Streptomyces clavuligerus. J. Bacteriol. 172, 3952–3958 (1990).

    Article  CAS  Google Scholar 

  29. LePage, G.A. & Campbell, E. Preparation of streptomycin. J. Biol. Chem. 162, 163–171 (1946).

    CAS  Google Scholar 

  30. Queener, S.W., Ingolia, T.D., Skatrud, P.L., Chapman, J.L. & Kaster, K.R. A system for genetic transformation of Cephalosporium acremonium. In Microbiology-1985. (ed. Lieve, L.) 468–472 (American Society of Microbiology, Washington D.C.; 1985).

    Google Scholar 

  31. Shen, Y.Q., Wolfe, S. & Demain, A.L. Levels of isopenicillin N synthetase and deacetoxycephalosporin C synthetase in Cephalosporium acremonium producing high and low levels of cephalosporin C. Bio/Technology 4, 61–64 (1986).

    CAS  Google Scholar 

  32. Gutiérrez, S., Velasco, J., Fernández, F.J. & Martín, J.F. The cefG gene of Cephalosporium acremonium is linked to the cefEF and encodes a deacetylcephalosporin C acetyltransferase closely related to homoserine O-acetyltransferase. J. Bacteriol. 174, 3056–3064 (1992).

    Article  Google Scholar 

  33. Punt, P.J., Oliver, R.P., Dingemanse, M.A., Pouwels, P.H. & van den Hondel, C.A.M.J.J. Transformation of Aspergillus based on the hygromycin B resistance marker from Escherichia coli. Gene 56, 117–124 (1987).

    Article  CAS  Google Scholar 

  34. Maniatis, T., Sambrook, J. & Fritsch, E.F. Molecular Cloning: a laboratory manual. Edn. 2 (Cold Spring Harbor Laboratory Press, New York; 1989).

    Google Scholar 

  35. Dotzlaf, J.E. & Yeh, W.K. Copurification and characterization of deacetoxycephalosporin C synthase/hydroxylase from Cephalosporium acremonium. J. Bacteriol. 169, 1611–1618 (1987).

    Article  CAS  Google Scholar 

  36. Specht, C.A., DiRusso, C.C., Novotny, C.P. & Ullrich, R.C. A method for extracting high-molecular-weight deoxyribonucleic acid from fungi. Anal. Biochem. 119, 158–163 (1982).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors thank P. Merino, J.A. González, M. Sandoval, M.T. García, J. Morán, and C. Alonso for their excellent technical assistance, M.A. Cortés (Waters Cromatografía, S.A. Madrid) for mass spectrometry analysis, and E. Bernasconi, M. Esteban, M. Rodríguez, and A.T. Marcos for helpful comments.

Author information

Authors and Affiliations

  1. Laboratorios de Biotecnología and Bioquímica. Antibióticos S.A., Avenida de Antibióticos 59-61, León, 24009, Spain

    Javier Velasco, José Luis Adrio, Miguel Ángel Moreno, Bruno Díez, Gloria Soler & José Luis Barredo

Authors
  1. Javier Velasco
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. José Luis Adrio
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Miguel Ángel Moreno
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Bruno Díez
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Gloria Soler
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. José Luis Barredo
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to José Luis Barredo.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Velasco, J., Luis Adrio, J., Ángel Moreno, M. et al. Environmentally safe production of 7-aminodeacetoxycephalosporanic acid (7-ADCA) using recombinant strains of Acremonium chrysogenum. Nat Biotechnol 18, 857–861 (2000). https://doi.org/10.1038/78467

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/78467

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing