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...

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.