Molecular characterization of the Acremonium chrysogenum cefG gene product: the native deacetylcephalosporin C acetyltransferase is not processed into subunits

Abstract

Constructions starting at each of the three in-frame ATG codons of the Acremonium chrysogenum cefG gene (Met1, Met46 and Met60) were expressed in Escherichia coli, obtaining proteins of 49, 44 and 43 kDa, respectively. All three proteins showed deacetylcephalosporin C (DAC) acetyltransferase activity. The native A. chrysogenum DAC acetyltransferase was purified to electrophoretic homogeneity by immunoaffinity chromatography. It showed a molecular mass of 50 kDa by filtration in calibrated Sephadex G-75 SF or Superose 12 (FPLC) columns. The N-terminal end of the pure DAC acetyltransferase was Met-Leu-Pro-Ser-Ala-Gln-Val-Ala-Arg-Leu, which matched perfectly the deduced amino acid sequence starting at Met1. The putative α- and β-subunits of DAC acetyltransferase were also obtained in E. coli but showed no enzymic activity either separately or in combination. Immunoblotting (Western) analysis revealed that the 50 kDa DAC acetyltransferase showed high protein levels in A. chrysogenum cultures at 72 and 96 h and decreased sharply thereafter, but in all cases no detectable processing of the enzyme into subunits was found. Three different A. chrysogenum strains (including the wild-type Brotzu strain and two high-cephalosporin-producing mutants) showed the same unprocessed 50 kDa DAC acetyltransferase. The non-producer mutant ATCC 20371 showed no DAC acetyltransferase protein band but formed a normal transcript of 1.4 kb.

Role of peroxisomes in the biosynthesis and secretion of β-lactams and other secondary metabolites

“…Both activities are located in a single polypeptide that is encoded by the cefEF gene [112]. The last step of the cephalosporin biosynthesis pathway involves the conversion of DAC to cephalosporin C (CPC) by the DAC-acetyltransferase that is encoded by the cefG gene [42,140] (Fig. 3a).…”

Expression of cefD2 and the conversion of isopenicillin N into penicillin N by the two-component epimerase system are rate-limiting steps in cephalosporin biosynthesis

“…The cephalosporin biosynthetic genes in A. chrysogenum are organized in two clusters: pcbAB, pcbC, cefD1 and cefD2 are located in the so-called ''early'' cluster (encoding the enzymes that catalyze the early steps in the pathway), while cefEF and cefG genes are located in the so-called ''late'' cluster (Gutie´rrez et al 1992Velasco et al 1999). In P. chrysogenum a tandem amplification of the penicillin biosynthetic gene cluster has been found in high penicillin-producing strains Fierro et al 1995;Newbert et al 1997).…”

The cefT gene of Acremonium chrysogenum C10 encodes a putative multidrug efflux pump protein that significantly increases cephalosporin C production

“…The IPN is then isomerized to the D-configuration, giving rise to penicillin N, which is later converted to deacetoxycephalosporin and deacetylcephalosporin C by a bifunctional enzyme named deacetoxycephalosporin C synthase-hydroxylase (Scheidegger et al 1984;Dotzlaf and Yeh 1987;Samsom et al 1987). The last step in the cephalosporin biosynthesis pathway involves the conversion of DAC to cephalosporin C by the DAC acetyltransferase that is encoded by the cefG gene (Gutie´rrez et al 1992;Matsuyama et al 1992;Velasco et al 1999).…”