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Expression of the Acremonium chrysogenum cefT gene in Penicillum chrysogenum indicates that it encodes an hydrophilic β-lactam transporter

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Abstract

The Acremonium chryrsogenum cefT gene encoding a membrane protein of the major facilitator superfamily implicated in the cephalosporin biosynthesis in A. chrysogenum was introduced into Penicillium chrysogenum Wisconsin 54-1255 (a benzylpenicillin producer), P. chrysogenum npe6 pyrG − (a derivative of Wisconsin 54-1255 lacking a functional penDE gene) and P. chrysogenum TA98 (a deacetylcephalosporin producer containing the cefD1, cefD2, cefEF and cefG genes from A. chrysogenum). RT-PCR analysis revealed that the cefT gene was expressed in P. chrysogenum strains. HPLC analysis of the culture broths of the TA98 transformants showed an increase in the secretion of deacetylcephalosporin C and hydrophilic penicillins (isopenicillin N and penicillin N). P. chrysogenum Wisconsin 54-1255 strain transformed with cefT showed increased secretion of the isopenicillin N intermediate and a drastic decrease in the benzylpenicillin production. Southern and northern blot analysis indicated that the untransformed P. chrysogenum strains contain an endogenous gene similar to cefT that may be involved in the well-known secretion of the isopenicillin N intermediate. In summary, the cefT transporter is a hydrophilic β-lactam transporter that is involved in the secretion of hydrophilic β-lactams containing α-aminoadipic acid side chain (isopenicillin N, penicillin N and deacetylcephalosporin C).

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Acknowledgments

This work was supported by grants of the European Union (Eurofung QLRT-1999-00729I) and DSM (Delft, Holland). F. Teijeira received a MEC-FPU predoctoral fellowship from the Spanish Government. We thank Barredo JL (Antibioticos S.A.) for providing the pASG418 plasmid and the excellent technical assistance of A. Sánchez, B. Martín, J. Merino, A. Casenave and B. Aguado (Instituto de Biotecnología, INBIOTEC).

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Authors and Affiliations

  1. Instituto de Biotecnología (INBIOTEC) de León, Avda. Real Nº1, 24006, León, Spain

    Ricardo V. Ullán & Juan F. Martín

  2. Área de Microbiología, Facultad de Ciencias Biológicas y Ambientales, Universidad de León, 24071, León, Spain

    Fernando Teijeira & Juan F. Martín

Authors
  1. Ricardo V. Ullán
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  2. Fernando Teijeira
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  3. Juan F. Martín
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Correspondence to Juan F. Martín.

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Communicated by U. Kueck.

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294_2008_207_MOESM1_ESM.tif

Figure S.1. (A) Southern blot hybridization of EcoRI-digested genomic DNAs of P. chrysogenum npe6 pyrG -; transformed with pORF7 using as probe a 1400 bp HindIII-XhoI fragment of the cefT gene: lane M, size markers (Lambda DNA / HindIII digested); lane 1, positive control A. chrysogenum C10; lane 2, negative control P. chrysogenum npe6 pyrG -; lane 3, P. chrysogenum npe6-T24 and lane 4, P. chrysogenum npe6-T45 (see description of the bands in Fig. 2). Volumetric (B) and specific IPN production in the culture broths of P. chrysogenum npe6 pyrG -, P. chrysogenum npe6-T24 and P. chrysogenum npe6-T45 strains. All cultures were grown in CP medium for 96 hours. The error bars indicate the standard deviations of three independent cultures. (DOC 357 kb)

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Ullán, R.V., Teijeira, F. & Martín, J.F. Expression of the Acremonium chrysogenum cefT gene in Penicillum chrysogenum indicates that it encodes an hydrophilic β-lactam transporter. Curr Genet 54, 153–161 (2008). https://doi.org/10.1007/s00294-008-0207-9

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