A Novel Epimerization System in Fungal Secondary Metabolism Involved in the Conversion of Isopenicillin N into Penicillin N inAcremonium chrysogenum

The epimerization step that converts isopenicillin N into penicillin N during cephalosporin biosynthesis has remained uncharacterized despite its industrial relevance. A transcriptional analysis of a 9-kb region located downstream of the pcb C gene revealed the presence of two transcripts that correspond to the genes named cef D1 and cef D2 encoding proteins with high similarity to long chain acyl-CoA synthetases and acyl-CoA racemases from Mus musculus , Homo sapiens , and Rattus norvegicus . Both genes are expressed in opposite orientations from a bidirectional promoter region. Targeted inactivation of cef D1 and cef D2 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 nonproducer mutant with both genes restored epimerase activity and cephalosporin biosynthesis. However, when cef D1 or cef D2 were introduced separately into the double-disrupted mutant, no epimerase activity was detected, indicating that the concerted action of both proteins encoded by cef D1 and cef D2 is required for epimerization of isopenicillin N into penicillin N. This epimerization system occurs in eukaryotic cells and is entirely different from the known epimerization systems involved in the biosynthesis of bacterial β-lactam antibiotics.