The cluster of penicillin biosynthetic genes. Identification and characterization of the pcbAB gene encoding the alpha-aminoadipyl-cysteinyl-valine synthetase and linkage to the pcbC and penDE genes.

B Díez,S Gutiérrez,J L Barredo,P Van Solingen,L H Van Der Voort,J F Martín

doi:10.1016/s0021-9258(17)46231-4

Abstract

Penicillium chrysogenum DNA fragments cloned in EMBL3 or cosmid vectors from the upstream region of the pcbC-penDE cluster carry a gene (pcbAB) that complemented the deficiency of alpha-aminoadipyl-cysteinyl-valine synthetase of mutants npe5 and npe10, and restored penicillin production to mutant npe5. A protein of about 250 kDa was observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels of cell-free extracts of complemented strains that was absent in the npe5 and npe10 mutants but exists in the parental strain from which the mutants were obtained. Transcriptional mapping studies showed the presence of one long transcript of about 11.5 kilobases that hybridized with several probes internal to the pcbAB gene, and two small transcripts of 1.15 kilobases that hybridized with the pcbC or the penDE gene, respectively. The transcription initiation and termination regions of the pcbAB gene were mapped by hybridization with several small probes. The region has been completely sequenced. It includes an open reading frame of 11,376 nucleotides that encodes a protein with a deduced Mr of 425,971. Three repeated dominia were found in the alpha-aminoadipyl-cysteinyl-valine synthetase which have high homology with the gramicidin synthetase I and tyrocidine synthetase I. The pcbAB is linked to the pcbC and penDE genes and is transcribed in the opposite orientation to them.

Highlights

From the $Section of Microbiology, Department of Ecology, Genetics and Microbiology, University and the IIGist Brocades, Wateringseweg 1, P. 0
We report in this article the isolation, sequentiation, and characterization of a 17.6-kb stretch of contiguous DNA encoding the ACV synthetase which is linked to the pcbC and penDE genes that encode the two other enzymes involved in penicillin biosynthesis
Cloning of Large DNA Fragments from P. chrysogenumInitial characterization of mutants blocked in penicillin biosynthesis3 indicated that the ACV synthetase of P. chry

Summary

PROCEDURES

Escherichia coli DH5a [22] was used as the recipient strain for high frequency plasmid transformation (lo’-IO8 transformants/pg of DNA) and E. coli LE392 as a host for XEMBLS phage derivatives [23]. E. coli MV1193 [24] was used as the host strain for obtaining single-stranded DNA from the pBluescript plasmids. XEMBLB [25] was used as a vector for cloning large DNA fragments (17-20 kb) of P. chrysogenum [11, 12]. Southern Blotting and Sequencing-Southern blotting of DNA fragments was carried out according to Maniatis et al [23]. DNA fragments were sequenced by the dideoxynucleotide method [27]. Transformation of Penicillium and Gene Expression-Transformation of protoplasts of P. chrysogenum was carried out as described previously [30, 31]. In SDS-PAGE (7.5%), cell-free extracts of transformed or untransformed strains were precipitated with trichloroacetic acid at a final concentration of 10% and applied to the gel

RESULTS

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DISCUSSION