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

Abstract

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 pcbC gene revealed the presence of two transcripts that correspond to the genes named cefD1 and cefD2 encoding proteins with high similarity to long chain acyl-CoA synthetases and acylCoA racemases from Mus musculus, Homo sapiens, and Rattus 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 nonproducer 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 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 ␤-lactam antibiotics.The molecular genetics of cephalosporin biosynthesis is an excellent model for secondary metabolism, since considerable information on the enzymology (reviewed in Refs. 1 and 2), molecular genetics, and gene expression mechanisms (3-5) has accumulated in the last few years. The biosynthesis of cephalosporins by Acremonium chrysogenum and cephamycins by Amycolatopsis (Nocardia) lactamdurans and Streptomyces clavuligerus (reviewed in Refs. 6 and 7) begins with the formation1 by the ACV synthetase (8), followed by cyclicization of ACV to isopenicillin N (IPN). IPN is later converted into penicillin N by an epimerase activity that has remained uncharacterized so far in A. chrysogenum. After the epimerization step, penicillin N is transformed by a deacetoxycephalosporin C synthase (expandase) into deacetoxycephalosporin C, and finally, deacetoxycephalosporin C is converted into deacetylcephalosporin C (DAC) and cephalosporin C by DAC synthase (hydroxylase) and DAC acetyltransferase, respectively (Fig. 1).The genes pcbAB (9) and pcbC (10) encoding ACV synthetase and IPN synthase are linked together in chromosome VII in the so-called "early cephalosporin gene cluster" (11). The genes cefEF, encoding the bifunctional expandase-hydroxylase (12), and cefG, encoding the DAC acetyltransferase (13,14), are linked together in the so-called "late cephalosporin cluster" in chromosome I (11).The isopenicillin N epimerization step still remains unclear. Demain and co-workers (15, 16) reported that isopenicillin N was converted into penicillin N by extracts of A. chrysogenum, although the epimerizing enzyme was extremely labile (17, 18), preventing ...

Mutational Analysis and Characterization of Nocardicin C-9′ Epimerase

“…These bacterial isopenicillin N epimerases are PLP-dependent enzymes, belonging to the aspartate aminotransferase structural family (56). Surprisingly, this same epimerization in the fungus Acremonium chrysogenum, which produces cephalosporin C, was discovered to be due to the action of two separate enzymes: an acyl-CoA synthetase and an acyl-CoA racemase (15). Homologs for these genes have also been identified in the marine fungus Kallichroma tethys, suggesting this may be a universal solution to fungal epimerization of isopenicillin N (21).…”

“…It has been recently determined, however, that in fungal cephalosporin producers two proteins are required, an acyl-CoA synthetase and an acyl-CoA epimerase. This is the first example of such a two-protein epimerization system in secondary metabolism (15).…”

Matching the proteome to the genome: the microbody of penicillin-producing Penicillium chrysogenum cells

“…CefD1 and CefD2 catalyze the first step that commits the pathway in A. chrysogenum to the production of cephalosporins, the isomerization of the l-α-aminoadipate side chain of IPN to the D-enantiomer to give penicillin N. The subsequent activities of the CefEF and CefG proteins will result in ring expansion of the β-lactam ring and production of cephalosporin C (Brakhage 1998). CefD1 is highly similar to very-long-chain-fattyacid-CoA synthetases, while CefD2 is highly similar to human racemases (Ullan et al 2002). Members of both families have been found in mammalian microbodies (Wanders 2004).…”

“…This is remarkable since P. chrysogenum does not synthesize this β-lactam antibiotic. Pc20g13500 and Pc22g13680 are orthologs of Acremonium chrysogenum CefD1 and CefD2, respectively (Ullan et al 2002), while Pc22g24630 represents a small (truncated) protein that also shows weak similarity to CefD2. In the filamentous fungus A. chrysogenum, CefD1 and CefD2 are involved in cephalosporin biosynthesis at the stage of isopenicillin N epimerization, a prerequisite for subsequent expansion of the β-lactam ring (for review, see Brakhage 1998).…”

Genome sequencing and analysis of the filamentous fungus Penicillium chrysogenum

“…These may include identified orthologs of 4-coumarateCoA ligase. Surprisingly, several orthologs of bacterial 25 and fungal 26 isopenicillinN epimerase were identified. The predicted protein sequence of Pc12g11540 shares 40% homology with S. clavuligerus isopenicillinN epimerase, although it probably functions as an aminotransferase.…”