Intrachromosomal recombination between direct repeats in Penicillium chrysogenum: gene conversion and deletion events

Casqueiro, Javier; Bañuelos, Óscar; Gutiérrez, Santiago; Hijarrubia, Maria Jose; Martı́n, Juan-Francisco

doi:10.1007/s004380051048

Cited by 35 publications

(28 citation statements)

References 33 publications

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“…The following strains are all Wis 54-1255 derivatives: P. chrysogenum L2, a lysine auxotroph, was obtained by selection of individual clones from a lysine bradytroph, L1 (Masurekar et al, 1972;Luengo et al, 1980); P. chrysogenum TD10-195 is a lysine auxotroph (Table 1) obtained by targeted inactivation of the lys2 gene, encoding a-aminoadipate reductase (Casqueiro et al, 1999a); P. chrysogenum HS 2 is a lysine auxotroph defective in homocitrate synthase obtained by targeted inactivation of the lys1 gene (Bañuelos et al, 2002). All fungal strains were maintained on Power medium (Casqueiro et al, 1999b) and spores were collected from cultures grown for 5 days at 28 uC. Mycelia of these strains grown in MPPY medium (Fierro et al, 1993) were collected by filtration through Nytal filters.…”

Section: Methodsmentioning

confidence: 99%

Transcriptional upregulation of four genes of the lysine biosynthetic pathway by homocitrate accumulation in Penicillium chrysogenum: homocitrate as a sensor of lysine-pathway distress

et al. 2009

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The lysine biosynthetic pathway has to supply large amounts of α-aminoadipic acid for penicillin biosynthesis in Penicillium chrysogenum. In this study, we have characterized the P. chrysogenum L2 mutant, a lysine auxotroph that shows highly increased expression of several lysine biosynthesis genes (lys1, lys2, lys3, lys7). The L2 mutant was found to be deficient in homoaconitase activity since it was complemented by the Aspergillus nidulans lysF gene. We have cloned a gene (named lys3) that complements the L2 mutation by transformation with a P. chrysogenum genomic library, constructed in an autonomous replicating plasmid. The lys3-encoded protein showed high identity to homoaconitases. In addition, we cloned the mutant lys3 allele from the L2 strain that showed a G1534 to A1534 point mutation resulting in a Gly495 to Asp495 substitution. This mutation is located in a highly conserved region adjacent to two of the three cysteine residues that act as ligands to bind the iron–sulfur cluster required for homoaconitase activity. The L2 mutant accumulates homocitrate. Deletion of the lys1 gene (homocitrate synthase) in the L2 strain prevented homocitrate accumulation and reverted expression levels of the four lysine biosynthesis genes tested to those of the parental prototrophic strain. Homocitrate accumulation seems to act as a sensor of lysine-pathway distress, triggering overexpression of four of the lysine biosynthesis genes.

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Section: Methodsmentioning

confidence: 99%

Transcriptional upregulation of four genes of the lysine biosynthetic pathway by homocitrate accumulation in Penicillium chrysogenum: homocitrate as a sensor of lysine-pathway distress

et al. 2009

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“…Czapek minimal medium was used in phenotype assays. The lysine auxotroph mutants were grown in the presence of 1.75 mM lysine (8).…”

Section: Methodsmentioning

confidence: 99%

“…To prove that pipecolic acid is synthesized from ␣-aminoadipic acid through P6C in P. chrysogenum, the intracellular accumulation of pipecolic acid in P. chrysogenum SR1 Ϫ (lacking saccharopine reductase) was compared to that in P. chrysogenum TDX195 (lacking ␣-aminoadipate reductase, the enzyme involved in the conversion of ␣-aminoadipic acid to ␣-aminoadipate-␦-semialdehyde). Strain TDX195 is unable to form P6C from ␣-aminoadipic acid (7)(8)(9). HPLC analysis of the intracellular concentration of pipecolic acid showed (Fig.…”

Section: Pipecolic Acid Is Formed In Mutant Sr1mentioning

confidence: 99%

Inactivation of the lys7 Gene, Encoding Saccharopine Reductase in Penicillium chrysogenum , Leads to Accumulation of the Secondary Metabolite Precursors Piperideine-6-Carboxylic Acid and Pipecolic Acid from α-Aminoadipic Acid

Naranjo

Valmaseda

Casqueiro

et al. 2004

Appl Environ Microbiol

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Pipecolic acid serves as a precursor of the biosynthesis of the alkaloids slaframine and swainsonine (an antitumor agent) in some fungi. It is not known whether other fungi are able to synthesize pipecolic acid. Penicillium chrysogenum has a very active ␣-aminoadipic acid pathway that is used for the synthesis of this precursor of penicillin. The lys7 gene, encoding saccharopine reductase in P. chrysogenum, was target inactivated by the double-recombination method. Analysis of a disrupted strain (named P. chrysogenum SR1 ؊ ) showed the presence of a mutant lys7 gene lacking about 1,000 bp in the 3-end region. P. chrysogenum SR1 ؊ lacked saccharopine reductase activity, which was recovered after transformation of this mutant with the intact lys7 gene in an autonomously replicating plasmid. P. chrysogenum SR1 ؊ was a lysine auxotroph and accumulated piperideine-6-carboxylic acid. When mutant P. chrysogenum SR1 ؊ was grown with L-lysine as the sole nitrogen source and supplemented with DL-␣-aminoadipic acid, a high level of pipecolic acid accumulated intracellularly. A comparison of strain SR1؊ with a lys2-defective mutant provided evidence showing that P. chrysogenum synthesizes pipecolic acid from ␣-aminoadipic acid and not from L-lysine catabolism.

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“…The genomic DNA of P. chrysogenum was obtained as described previously (10). For plasmid rescue, genomic DNA (1 g of each transformant) was used to electroporate electrocompetent E. coli DH10B cells (39) in Bio-Rad Gene Pulser cuvettes (0.2-cm gap).…”

Section: Methodsmentioning

confidence: 99%

Conversion of Pipecolic Acid into Lysine in Penicillium chrysogenum Requires Pipecolate Oxidase and Saccharopine Reductase: Characterization of the lys7 Gene Encoding Saccharopine Reductase

et al. 2001

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Pipecolic acid is a component of several secondary metabolites in plants and fungi. This compound is useful as a precursor of nonribosomal peptides with novel pharmacological activities. In Penicillium chrysogenum pipecolic acid is converted into lysine and complements the lysine requirement of three different lysine auxotrophs with mutations in the lys1, lys2, or lys3 genes allowing a slow growth of these auxotrophs. We have isolated two P. chrysogenum mutants, named 7.2 and 10.25, that are unable to convert pipecolic acid into lysine. These mutants lacked, respectively, the pipecolate oxidase that converts pipecolic acid into piperideine-6-carboxylic acid and the saccharopine reductase that catalyzes the transformation of piperideine-6-carboxylic acid into saccharopine. The 10.25 mutant was unable to grow in Czapek medium supplemented with ␣-aminoadipic acid. A DNA fragment complementing the 10.25 mutation has been cloned; sequence analysis of the cloned gene (named lys7) revealed that it encoded a protein with high similarity to the saccharopine reductase from Neurospora crassa, Magnaporthe grisea, Saccharomyces cerevisiae, and Schizosaccharomyces pombe. Complementation of the 10.25 mutant with the cloned gene restored saccharopine reductase activity, confirming that lys7 encodes a functional saccharopine reductase. Our data suggest that in P. chrysogenum the conversion of pipecolic acid into lysine proceeds through the transformation of pipecolic acid into piperideine-6-carboxylic acid, saccharopine, and lysine by the consecutive action of pipecolate oxidase, saccharopine reductase, and saccharopine dehydrogenase.Many secondary metabolites contain L-lysine, D-lysine, or pipecolic acid moieties (29,36). In filamentous fungi, the biosynthesis of pipecolic acid is related to lysine metabolism. In Metarhizium anisopliae and Rhizoctonia leguminicola pipecolic acid is an intermediate in the biosynthesis of alkaloid compounds, such as swansonine and slaframine. In these fungi pipecolic acid is formed by catabolism of lysine (40-42). However, using 14 C-and 15 N-labeled ␣-aminoadipic acid and [ 14 C]lysine, Aspen and Meister (2) showed in Aspergillus nidulans that the carbon chain of ␣-aminoadipic rather than that of lysine was the major precursor of pipecolic acid and the nitrogen atom of ␣-aminoadipic acid becomes the nitrogen atom of pipecolic acid. Furthermore, in Neurospora crassa kinetic studies with radioactively labeled D-lysine showed that pipecolic acid was an intermediate in the conversion of D-lysine into L-lysine (17).In Penicillium chrysogenum the biosynthetic pathway of lysine and penicillin have several steps in common (reviewed in references 1 and 12) (Fig. 1). ␣-Aminoadipic acid is the intermediate where both branching routes diverge (11). ␣-Aminoadipic acid has a key function in penicillin biosynthesis, since addition of exogenous ␣-aminoadipic acid (21) or genetic modifications that increase the internal ␣-aminoadipic acid pool (11,22,23) lead to a stimulation of the rate of penicillin biosynthes...

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Intrachromosomal recombination between direct repeats in Penicillium chrysogenum: gene conversion and deletion events

Cited by 35 publications

References 33 publications

Transcriptional upregulation of four genes of the lysine biosynthetic pathway by homocitrate accumulation in Penicillium chrysogenum: homocitrate as a sensor of lysine-pathway distress

Transcriptional upregulation of four genes of the lysine biosynthetic pathway by homocitrate accumulation in Penicillium chrysogenum: homocitrate as a sensor of lysine-pathway distress

Inactivation of the lys7 Gene, Encoding Saccharopine Reductase in Penicillium chrysogenum , Leads to Accumulation of the Secondary Metabolite Precursors Piperideine-6-Carboxylic Acid and Pipecolic Acid from α-Aminoadipic Acid

Conversion of Pipecolic Acid into Lysine in Penicillium chrysogenum Requires Pipecolate Oxidase and Saccharopine Reductase: Characterization of the lys7 Gene Encoding Saccharopine Reductase

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