Cloning and disruption of the cefG gene encoding acetyl coenzyme A: Deacetylcephalosporin C O-acetyltransferase from Acremonium chrysogenum

Abstract

Acetyl CoA: deacetylcephalosporin C o-acetyltransferase(DCPC-ATF) catalyses the final step in the biosynthesis of cephalosporin C (CPC) in Acremonium chrysogenum. The gene encoding DCPC-ATF, cefG, has been isolated from an A. chrysogenum genomic library using a DCPC-ATF cDNA probe. Nucleotide sequence analysis revealed that cefG contains two short introns of 79bp and 65bp. The gene was found to be closely linked to the cefEF gene encoding deacetoxycephalosporin C synthetase/deacetylcephalosporin C synthetase, which catalyses the preceding two steps in the pathway. The two genes are separated by a 1114 bp segment from which they are divergently transcribed. Introduction of the cloned cefG gene to A.chrysogenum resulted in an increased level of DCPC-ATF activity. A plasmid carrying a cefG gene interrupted in the coding region by a selectable marker for resistance to hygromycin B was constructed and used to disrupt the cefG locus in A.chrysogenum. The cefG-disrupted strains were found to lack the ability to produce CPC, and accumulated its precursor, deacetylcephalosporin C in the culture broth. Southern hybridization analysis confirmed that the disruption resulted from a gene replacement event at the cefG locus.