Characterization of the Cephalosporium acremonium pcbAB gene encoding alpha-aminoadipyl-cysteinyl-valine synthetase, a large multidomain peptide synthetase: linkage to the pcbC gene as a cluster of early cephalosporin biosynthetic genes and evidence of multiple functional domains.
A 24-kb region of Cephalosporium acremonium C10 DNA was cloned by hybridization with the pcbAB and pcbC genes of Penicillium chrysogenum. A 3.2-kb BamHI fragment of this region complemented the mutation in the structural pcbC gene of the C. acremonium N2 mutant, resulting in cephalosporin production. A functional alpha-aminoadipyl-cysteinyl-valine (ACV) synthetase was encoded by a 15.6-kb EcoRI-BamHI DNA fragment, as shown by complementation of an ACV synthetase-deficient mutant of P. chrysogenum. Two transcripts of 1.15 and 11.4 kb were found by Northern (RNA blot) hybridization with probes internal to the pcbC and pcbAB genes, respectively. An open reading frame of 11,136 bp was located upstream of the pcbC gene that matched the 11.4-kb transcript initiation and termination regions. It encoded a protein of 3,712 amino acids with a deduced Mr of 414,791. The nucleotide sequence of the gene showed 62.9% similarity to the pcbAB gene encoding the ACV synthetase of P. chrysogenum; 54.9% of the amino acids were identical in both ACV synthetases. Three highly repetitive regions occur in the deduced amino acid sequence of C. acremonium ACV synthetase. Each is similar to the three repetitive domains in the deduced sequence of P. chrysogenum ACV synthetase and also to the amino acid sequence of gramicidin synthetase I and tyrocidine synthetase I of Bacillus brevis. These regions probably correspond to amino acid activating domains in the ACV synthetase protein. In addition, a thioesterase domain was present in the ACV synthetases of both fungi. A similarity has been found between the domains existing in multienzyme nonribosomal peptide synthetases and polyketide and fatty acid synthetases. The pcbAB gene is linked to the pcbC gene, forming a cluster of early cephalosporin-biosynthetic genes.
Gutiérrez
S
S
Department of Ecology, Genetics and Microbiology, University of León, Spain.
Díez
B
B
Montenegro
E
E
Martín
J F
JF
D000164:Acremonium / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D002511:Cephalosporins / Q000096:biosynthesis*; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D008040:Genetic Linkage; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics*; D010204:Pantetheine / Q000031:analogs & derivatives / Q000378:metabolism; D010453:Peptide Synthases / Q000235:genetics*; D012329:RNA, Bacterial / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D013869:Thiolester Hydrolases / Q000235:genetics; D014158:Transcription, Genetic
D002511:Cephalosporins; D009097:Multienzyme Complexes; D012329:RNA, Bacterial; D012333:RNA, Messenger; D010204:Pantetheine; D013869:Thiolester Hydrolases; D010453:Peptide Synthases; C095368:alpha-aminoadipyl-cysteinyl-valine synthetase; C003129:4'-phosphopantetheine