The cefG gene of Cephalosporium acremonium is linked to the cefEF gene and encodes a deacetylcephalosporin C acetyltransferase closely related to homoserine O-acetyltransferase

Abstract

The gene (cefG) encoding the acetyl coenzyme A.deacetylcephalosporin C acetyltransferase of Cephalosporium acremonium (synonym Acremonium chrysogenum) C10 has been cloned. It contains two introns and encodes a protein of 444 amino acids with an Mr of 49,269 that correlates well with the Mr deduced by gel filtration. The cefG gene is linked to the ceJEF gene (encoding the bifunctional deacetoxycephalosporin C synthase/hydroxylase), but it is expressed in an orientation opposite that of the ceJEF gene. Two transcripts of 1.2 and 1.4 kb were found in C. acremonium that correspond to the cefEF and cefG genes, respectively; the degree of expression of the ceiG gene was clearly lower than that of the ceJEF gene in 48-h cultures. The cloned cefG complemented the deficiency of deacetylcephalosporin acetyltransferase in the nonproducer mutant C.acremonium ATCC 20371 and restored cephalosporin biosynthesis in this strain. Heterologous expression of the cefG genes took place in Penicilium chrysogenum. The deacetylcephalosporin acetyltransferase showed a much higher degree of homology with the O-acetylhomoserine acetyltransferases of Saccharomyces cerevisiae and Ascobolus immersus than with other O-acetyltransferases. The cefEF-ceIG cluster of genes encodes the enzymes that carry out the three late steps of the cephalosporin biosynthetic pathway and is not linked to the pcbAB-pcbC gene cluster that encodes the first two steps of the pathway.Cephalosporin is a P-lactam antibiotic formed in Cephalosporium acremonium (synonym Acremonium chrysogenum) and other filamentous fungi by condensation of the three precursor amino acids L-a-aminoadipic acid, L-cysteine, and D-valine to form the tripeptide b-L-a-aminoadipyl-L-cysteinyl-

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

“…The cultures were incubated at 25°C in a rotary shaker at 250 rpm. The levels of cephalosporin antibiotics were determined by bioassay using E. coli ESS2231 as the test strain in plates with penicillinase from Bacillus cereus UL1 as described previously (13).…”

“…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).…”

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

“…Four strains of A. chrysogenum were used in this study, including the wild-type (Brotzu) isolate ATCC 11550, the improved cephalosporin producers CW19 (ATCC 36225) [13] and C10 (ATCC 48272), a high-cephalosporin-producing strain released by PanLabs [13,14] and the non-producer mutant ATCC 20371 that is deficient in DAC-AT [9,15]. Strains were grown in LPE medium [16] for 7 days at 28 mC for sporulation.…”

Molecular characterization of three loss-of-function mutations in the isopenicillin N-acyltransferase gene (penDE) of Penicillium chrysogenum

“…Northern blotting and hybridization with probes D, C, E, and G (see Fig. 1), corresponding to the pcbAB,pcbC, penDE, and pyrG genes, were carried out as described previously (11,14).…”

“…Escherichia coli DH5cx (15) was used as the recipient strain for high-frequency plasmid transformation (106 to 108 transformants per ,ug of DNA); single-stranded DNA from the pBluescript plasmids was obtained from E. coli WK6 (14). Phage M13K07 was used as helper in the infection of strain WK6 with the pBluescript plasmids (20).…”