Transcriptional and bioinformatic analysis of the 56.8 kb DNA region amplified in tandem repeats containing the penicillin gene cluster in Penicillium chrysogenum.
High penicillin-producing strains of Penicillium chrysogenum contain 6-14 copies of the three clustered structural biosynthetic genes, pcbAB, pcbC, and penDE [Barredo, J.L., Díez, B., Alvarez, E., Martín, J.F., 1989. Large amplification of a 35-kb DNA fragment carrying two penicillin biosynthetic genes in high penicillin producing strains of Penicillium chrysogenum. Curr. Genet. 16, 453-459; Smith, D.J., Bull, J.H., Edwards, J., Turner, G., 1989. Amplification of the isopenicillin N synthetase gene in a strain of Penicillium chrysogenum producing high levels of penicillin. Mol. Gen. Genet. 216, 492-497.] . The cluster is located in a 56.8 kb DNA region bounded by a conserved TGTAAA/T hexanucleotide that undergoes amplification in tandem repeats [Fierro, F., Barredo, J.L., Díez, B., Gutiérrez, S., Fernández, F.J., Martín, J.F., 1995. The penicillin gene cluster is amplified in tandem repeats linked by conserved hexanucleotide sequences. Proc. Natl. Acad. Sci. USA 92, 6200-6204; Newbert, R.W., Barton, B., Greaves, P., Harper, J., Turner, G., 1997. Analysis of a commercially improved Penicillium chrysogenum strain series: involvement of recombinogenic regions in amplification and deletion of the penicillin biosynthesis gene cluster. J. Ind. Microbiol. Biotechnol. 19, 18-27]. Transcriptional analysis of this amplified region (AR) revealed the presence of at least eight transcripts expressed in penicillin producing conditions. Three of them correspond to the known penicillin biosynthetic genes, pcbAB, pcbC, and penDE. To locate genes related to penicillin precursor formation, or penicillin transport and regulation we have sequenced and analyzed the 56.8 kb amplified region of P. chrysogenum AS-P-78, finding a total of 16 open reading frames. Two of these ORFs have orthologues of known function in the databases. Other ORFs showed similarities to specific domains occurring in different proteins and superfamilies which allowed to infer their probable function. ORF11 encodes a D-amino acid oxidase that might be responsible for the conversion of D-amino acids in the tripeptide L-alpha-aminoadipyl-L-cysteinyl-D-valine or other beta-lactam intermediates to deaminated by-products. ORF12 encodes a predicted protein with similarity to saccharopine dehydrogenases that seems to be related to biosynthesis of the penicillin precursor alpha-aminoadipic acid. A deletion mutant, P. chrysogenum npe10 lacking the entire AR including ORF12, shows a partial requirement of L-lysine for growth. ORF13 encodes a putative protein containing a Zn(II)2-Cys6 fungal-type DNA-binding domain, probably a transcriptional regulator. Although some of the ORFs in the AR may play roles in increasing penicillin production, none of the 13 ORFs other than pcbAB, pcbC, and penDE seem to be strictly indispensable for penicillin biosynthesis. The genes located in the P. chrysogenum AR have been compared with those found in the Aspergillus nidulans 50 kb DNA region adjacent to the penicillin gene cluster, showing no conservation between these two fungi.
Fierro
Francisco
F
Instituto de Biotecnología de León (INBIOTEC) Science Park, Spain.
García-Estrada
Carlos
C
Castillo
Nancy I
NI
Rodríguez
Raquel
R
Velasco-Conde
Tania
T
Martín
Juan-Francisco
JF
D000595:Amino Acid Sequence; D001233:Aspergillus nidulans / Q000235:genetics; D001483:Base Sequence; D019295:Computational Biology; D004251:DNA Transposable Elements; D005784:Gene Amplification; D023061:Gene Order; D005800:Genes, Fungal; D008239:Lysine / Q000096:biosynthesis; D008969:Molecular Sequence Data; D005810:Multigene Family; D016366:Open Reading Frames / Q000235:genetics; D010406:Penicillins / Q000096:biosynthesis*; D010408:Penicillium chrysogenum / Q000235:genetics* / Q000254:growth & development; D010802:Phylogeny; D012443:Saccharopine Dehydrogenases / Q000235:genetics; D020080:Tandem Repeat Sequences; D014158:Transcription, Genetic
D004251:DNA Transposable Elements; D010406:Penicillins; D012443:Saccharopine Dehydrogenases; D008239:Lysine