Expression of the cefG gene is limiting for cephalosporin biosynthesis in Acremonium chrysogenum.
The conversion of deacetylcephalosporin C to cephalosporin C is inefficient in most Acremonium chrysogenum strains. The cefG gene, which encodes deacetylcephalosporin C acetyltransferase, is expressed very poorly in A. chrysogenum as compared to other genes of the cephalosporin pathway. Introduction of additional copies of the cefG gene with its native promoter (in two different constructions with upstream regions of 1056 bp and 538 bp respectively) did not produce a significant increase of the steady-state level of the cefG transcript. Expression of the cefG gene from the promoters of (i) the glyceraldehyde-3-phosphate dehydrogenase (gpd) gene of Aspergillus nidulans, (ii) the glucoamylase (gla) gene of Aspergillus niger, (iii) the glutamate dehydrogenase (gdh) and (iv) the isopenicillin N synthase (pcbC) genes of Penicillium chrysogenum, led to very high steady-state levels of cefG transcript and to increased deacetylcephalosporin-C acetyltransferase protein concentration (as shown by immunoblotting) and enzyme activity in the transformants. Southern analysis showed that integration of the new constructions occurred at sites different from that of the endogenous cefG gene. Cephalosporin production was increased two- to threefold in A. chrysogenum C10 transformed with constructions in which the cefG gene was expressed from the gdh or gpd promoters as a result of a more efficient acetylation of deacetylcephalosporin C.
Gutiérrez
S
S
Department of Ecology Genetics and Microbiology, Faculty of Biology, University of León, Spain.
Velasco
J
J
Marcos
A T
AT
Fernández
F J
FJ
Fierro
F
F
Barredo
J L
JL
Díez
B
B
Martín
J F
JF
D000123:Acetyltransferases / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D000164:Acremonium / Q000235:genetics* / Q000378:metabolism*; D002511:Cephalosporins / Q000096:biosynthesis*; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000032:analysis / Q000235:genetics; D005784:Gene Amplification; D018628:Gene Dosage; D015966:Gene Expression Regulation, Fungal; D005087:Glucan 1,4-alpha-Glucosidase / Q000235:genetics; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006836:Hydro-Lyases / Q000235:genetics; D015151:Immunoblotting; D010088:Oxidoreductases / Q000235:genetics; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D014158:Transcription, Genetic
D002511:Cephalosporins; D004271:DNA, Fungal; D010088:Oxidoreductases; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; C038370:isopenicillin N synthetase; D000123:Acetyltransferases; C018707:acetyl CoA-deacetylcephalosporin C acetyltransferase; D005087:Glucan 1,4-alpha-Glucosidase; D006836:Hydro-Lyases; C022771:dTDPglucose 4,6-dehydratase