Mutants blocked in penicillin biosynthesis show a deletion of the entire penicillin gene cluster at a specific site within a conserved hexanucleotide sequence

Fierro, Francisco; Montenegro, Eduardo José Nepomuceno; Gutiérrez, Santiago; Martı́n, Juan F.

doi:10.1007/bf00172491

Cited by 87 publications

(51 citation statements)

References 22 publications

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“…Spores of P. chrysogenum Wis 54-1255 were collected from plates of PW medium (Fierro et al 1996b) grown for 5 days at 28°C. Mycelia of this strain grown on MPPY (Fierro et al 1993) were collected by ®ltration of Nytal ®lters.…”

Section: Strains and Culture Conditionsmentioning

confidence: 99%

Characterization of the lys2 gene of Penicillium chrysogenum encoding α-aminoadipic acid reductase

et al. 1998

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A DNA fragment containing a gene homologous to LYS2 gene of Saccharomyces cerevisiae was cloned from a genomic DNA library of Penicillium chrysogenum AS-P-78. It encodes a protein of 1409 amino acids (Mr 154859) with strong similarity to the S. cerevisiae (49.9% identity) Schizosaccharomyces pombe (51.3% identity) and Candida albicans (48.12% identity) alpha-aminoadipate reductases and a lesser degree of identity to the amino acid-activating domains of the non-ribosomal peptide synthetases, including the alpha-aminoadipate-activating domain of the alpha-aminoadipyl-cysteinyl-valine synthetase of P. chrysogenum (12.4% identical amino acids). The lys2 gene contained one intron in the 5'-region and other in the 3'-region, as shown by comparing the nucleotide sequences of the cDNA and genomic DNA, and was transcribed as a 4.7-kb monocistronic mRNA. The lys2 gene was localized on chromosome III (7.5 Mb) in P. chrysogenum AS-P-78 and on chromosome IV (5.6 Mb) in strain P2, whereas the penicillin gene cluster is known to be located in chromosome I in both strains. The lys2-encoded protein is a member of the aminoacyladenylate-forming enzyme family with a reductase domain in its C-terminal region.

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Section: Strains and Culture Conditionsmentioning

confidence: 99%

Characterization of the lys2 gene of Penicillium chrysogenum encoding α-aminoadipic acid reductase

et al. 1998

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“…Escherichia coli strain LE-392 was used to obtain EMBL3 recombinant derivatives, and also served as a host for subcloning in plasmid pBluescript (Stratagene). Standard conditions for culture of A. chrysogenum and P. chrysogenum were as described elsewhere (Fierro et al 1996;GutieÂ rrez et al 1997;Casqueiro et al 1999). Sporulation of Acremonium strains was achieved in LPE solid medium.…”

Section: Strains and Culture Conditionsmentioning

confidence: 99%

Characterization of the lys2 gene of Acremonium chrysogenum encoding a functional α-aminoadipate activating and reducing enzyme

et al. 2001

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A 5.2-kb NotI DNA fragment isolated from a genomic library of Acremonium chrysogenum by hybridization with a probe internal to the Penicillium chrysogenum lys2 gene, was able to complement an alpha-aminoadipate reductase-deficient mutant of P. chrysogenum (lysine auxotroph L-G-). Enzyme assays showed that the alpha-aminoadipate reductase activity was restored in all the transformants tested. The lys2-encoded enzyme catalyzed both the activation and reduction of alpha-aminoadipic acid to its semialdehyde, as shown by reaction of the product with p-dimethylaminobenzaldehyde. The reaction required NADPH, and was not observed in the presence of NADH. Sequence analysis revealed that the gene encodes a protein with relatively high similarity to members of the superfamily of acyladenylate-forming enzymes. The Lys2 protein contained all nine motifs that are conserved in the adenylating domain of this enzyme family, a peptidyl carrier domain, and a reduction domain. In addition, a new NADP-binding motif located at the N-terminus of the reduction domain that may form a Rossmann-like betaalphabeta-fold has been identified and found to be shared by all known Lys2 proteins. The lys2 gene was mapped to chromosome I (2.2 Mb, the smallest chromosome) of A. chrysogenum C10 (the chromosome that contains the "late" cephalosporin cluster) and is transcribed as a monocistronic 4.5-kb mRNA although at relatively low levels compared with the beta-actin gene.

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“…chrysogenum spores were obtained from plates of PW medium (Fierro et al 1996) incubated for 5 days at 28°C. Seed cultures were initiated by inoculating fresh spores into DP medium (Casqueiro et al 1999a) without phenylacetate, and incubating for 30 h. Cultures in DP medium (100 ml in 500-ml¯asks) were inoculated with 10% of seed cultures and incubated in an orbital shaker (250 rpm, 25°C).…”

Section: Strains and Culture Conditionsmentioning

confidence: 99%

Subcellular localization of the homocitrate synthase in Penicillium chrysogenum

et al. 2002

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There are conflicting reports regarding the cellular localization in Saccharomyces cerevisiae and filamentous fungi of homocitrate synthase, the first enzyme in the lysine biosynthetic pathway. The homocitrate synthase (HS) gene (lys1) of Penicillium chrysogenum was disrupted in three transformants (HS(-)) of the Wis 54-1255 pyrG strain. The three mutants named HS1(-), HS2(-) and HS3(-) all lacked homocitrate synthase activity and showed lysine auxotrophy, indicating that there is a single gene for homocitrate synthase in P. chrysogenum. The lys1 ORF was fused in frame to the gene for the green fluorescent protein (GFP) gene of the jellyfish Aequorea victoria. Homocitrate synthase-deficient mutants transformed with a plasmid containing the lys1-GFP fusion recovered prototrophy and showed similar levels of homocitrate synthase activity to the parental strain Wis 54-1255, indicating that the hybrid protein retains the biological function of wild-type homocitrate synthase. Immunoblotting analysis revealed that the HS-GFP fusion protein is maintained intact and does not release the GFP moiety. Fluorescence microscopy analysis of the transformants showed that homocitrate synthase was mainly located in the cytoplasm in P. chrysogenum; in S. cerevisiae the enzyme is targeted to the nucleus. The control nuclear protein StuA was properly targeted to the nucleus when the StuA (targeting domain)-GFP hybrid protein was expressed in P. chrysogenum. The difference in localization of homocitrate synthase between P. chrysogenum and S. cerevisiae suggests that this protein may play a regulatory function, in addition to its catalytic function, in S. cerevisiae but not in P. chrysogenum.

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Mutants blocked in penicillin biosynthesis show a deletion of the entire penicillin gene cluster at a specific site within a conserved hexanucleotide sequence

Cited by 87 publications

References 22 publications

Characterization of the lys2 gene of Penicillium chrysogenum encoding α-aminoadipic acid reductase

Characterization of the lys2 gene of Penicillium chrysogenum encoding α-aminoadipic acid reductase

Characterization of the lys2 gene of Acremonium chrysogenum encoding a functional α-aminoadipate activating and reducing enzyme

Subcellular localization of the homocitrate synthase in Penicillium chrysogenum

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