The transporter CefM involved in translocation of biosynthetic intermediates is essential for cephalosporin production

Abstract

The cluster of early cephalosporin biosynthesis genes (pcbAB, pcbC, cefD1, cefD2 and cefT of Acremonium chrysogenum) contains all of the genes required for the biosynthesis of the cephalosporin biosynthetic pathway intermediate penicillin N. Downstream of the cefD1 gene, there is an unassigned open reading frame named cefM encoding a protein of the MFS (major facilitator superfamily) with 12 transmembrane domains, different from the previously reported cefT. Targeted inactivation of cefM by gene replacement showed that it is essential for cephalosporin biosynthesis. The disrupted mutant accumulates a significant amount of penicillin N, is unable to synthesize deacetoxy-, deacetyl-cephalosporin C and cephalosporin C and shows impaired differentiation into arthrospores. Complementation of the disrupted mutant with the cefM gene restored the intracellular penicillin N concentration to normal levels and allowed synthesis and secretion of the cephalosporin intermediates and cephalosporin C. A fused cefM-gfp gene complemented the cefM-disrupted mutant, and the CefM–GFP (green fluorescent protein) fusion was targeted to intracellular microbodies that were abundant after 72 h of culture in the differentiating hyphae and in the arthrospore chains, coinciding with the phase of intense cephalosporin biosynthesis. Since the dual-component enzyme system CefD1–CefD2 that converts isopenicillin N into penicillin N contains peroxisomal targeting sequences, it is probable that the epimerization step takes place in the peroxisome matrix. The CefM protein seems to be involved in the translocation of penicillin N from the peroxisome (or peroxisome-like microbodies) lumen to the cytosol, where it is converted into cephalosporin C.