Deacetylcephalosporin C production in Penicillium chrysogenum by expression of the isopenicillin N epimerization, ring expansion, and acetylation genes.
Penicillium chrysogenum npe6 lacking isopenicillin N acyltransferase activity is an excellent host for production of different beta-lactam antibiotics. We have constructed P. chrysogenum strains expressing cefD1, cefD2, cefEF, and cefG genes cloned from Acremonium chrysogenum. Northern analysis revealed that the four genes were expressed in P. chrysogenum. The recombinant strains TA64, TA71, and TA98 secreted significant amounts of deacetylcephalosporin C, but cephalosporin C was not detected in the culture broths. DAC-acetyltransferase activity was found in all transformants containing the cefG gene. HPLC analysis of cell extracts showed that transformant TA64, TA71, and TA98 accumulate intracellularly deacetylcephalosporin C and, in the last strain (TA98), also cephalosporin C. Mass spectra analysis confirmed that transformant TA98 synthesize true deacetylcephalosporin C and cephalosporin C. Even when accumulated intracellularly, cephalosporin C was not found in the culture broth.
Ullán
Ricardo V
RV
Instituto de Biotecnología (INBIOTEC) de León, Avda. Real No 1, 24006 León, Spain.
Campoy
Sonia
S
Casqueiro
Javier
J
Fernández
Francisco J
FJ
Martín
Juan F
JF
D000107:Acetylation; D000164:Acremonium / Q000235:genetics; D002511:Cephalosporins / Q000096:biosynthesis* / Q000737:chemistry; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D005656:Fungal Proteins / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D007535:Isomerases / Q000235:genetics; D010406:Penicillins / Q000096:biosynthesis*; D010408:Penicillium chrysogenum / Q000235:genetics* / Q000378:metabolism
D002511:Cephalosporins; D004271:DNA, Fungal; D005656:Fungal Proteins; D010406:Penicillins; C025071:deacetylcephalosporin C; D007535:Isomerases