Biochemistry and molecular characterization of the isopenicillin N epimerization during cephalosporin biosynthesis in Acremonium chrysogenum
Ullán
R.V., Casqueiro J., Bañuelos O., Gutiérrez S., Naranjo
L., Campoy S.,
Vaca I., and Martín J.F.
Área
de Microbiología, Facultad de Biología, Universidad de León,
24071 León,
Instituto de biotecnología (INBIOTEC), Avda. del Real Nº1,
24006 León.
The epimerization step that converts isopenicillin N into penicillin N during cephalosporin biosynthesis has remained uncharacterised in spite of its industrial relevance due to the impossibility of purifying the isopenicillin N epimerase that is extremely unstable. A transcriptional analysis of a 9 kb region located downstream of the pcbC gene revealed the presence of two transcripts that correspond to the genes named cefD1 and cefD2 encoding proteins with high similarity to acyl-CoA synthetases and acyl-CoA racemases from Mus musculus, Homo sapiens and Ratus norvegicus. Both genes are expressed in opposite orientations from a bidirectional promoter region.
Targeted inactivation of cefD1 and cefD2 was achieved by the two marker gene replacement procedure. Disrupted strains lacked isopenicillin N epimerase activity, were blocked in cephalosporin C production and accumulated isopenicillin N. Complementation in trans of the disrupted non-producer mutant with both genes restored epimerase activity and cephalosporin biosynthesis. However, when cefD1 or cefD2 were introduced separately into the double-disrupted mutant, no epimerase activity was detected indicating that the concerted action of both proteins encoded by cefD1 and cefD2 is required for epimerisation of isopenicillin N into penicillin N in the cephalosporin biosynthesis pathway. This epimerisation system occurs in eukaryotic cells and is entirely different from the known epimerisation systems involved in the biosynthesis of bacterial b-lactam antibiotics.
