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

Abstract

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...

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

“…To clone the P. chrysogenum homoaconitase gene, protoplasts of the L2 mutant were transformed with a genomic library constructed in the autonomous replicating (AMA1-based) plasmid pAM9L (Fierro et al, 1996;Naranjo et al, 2001). After protoplast regeneration, ten complemented transformants were obtained.…”

“…These results suggest that the protein encoded by the cloned gene corresponds to the P. chrysogenum homoaconitase. The gene was named lys3 according to the standard nomenclature proposed for the P. chrysogenum lys genes (Naranjo et al, 2001). …”

Lysine is catabolized to 2-aminoadipic acid in Penicillium chrysogenum by an omega-aminotransferase and to saccharopine by a lysine 2-ketoglutarate reductase. Characterization of the omega-aminotransferase

“…A similar pathway is probably active in P. chrysogenum since the second part of this conversion, i.e. the transformation of pipecolic acid into L-lysine, has been reported in this fungus (Naranjo et al 2001). When P. chrysogenum was grown on D-lysine, lysine and 2-aminoadipic acid accumulated intracellularly.…”

Characterization of the oat1 gene of Penicillium chrysogenum encoding an ω-aminotransferase: induction by L-lysine, L-ornithine and L-arginine and repression by ammonium

“…The double-marker selection technique (Liu et al 2001;Naranjo et al 2001;2004) allowed us to select a large number of oat1 disruption mutants. All the tested oat1 mutants were defective in the ornithine aminotransferase activity, and consequently were incapable of growing in Czapek minimal medium with ornithine as sole nitrogen source.…”