Characterization of pipecolic acid metabolism in Penicillium chrysogenum. Disruption of the lys7 gene leads to accumulation of the secondary metabolite precursors piperideine-6-carboxylic acid and pipecolic acid
Naranjo, L.; Martín de Valmaseda, E.; Casqueiro, J.; V. Ullán, R.V.; Lamas-Maceiras, M.; Bañuelos, O.; Vaca, I.; Campoy, S. y Martín, J.F.
Área
de Microbiología, Facultad de Biología, Universidad de León,
24071 León,
Instituto de biotecnología (INBIOTEC), Avda. del Real Nº1,
24006 León.
Pipecolic acid is an important compound that serves as substrate of some non-ribosomal peptide and polyketide synthetases, resulting in the formation of secondary metabolites with interesting pharmacological activities, e.g. immunosuppressors and antitumor agents. The formation of this compound is related to lysine metabolism in various organisms including plants, mammals, fungi and bacteria. In this work we showed that, in P. chrysogenum, pipecolic acid is converted into lysine. The conversion of pipecolic acid into lysine proceeds through the conversion of pipecolic acid into piperideine-6-carboxylic acid, saccharopine and lysine, by the consecutive action of pipecolate oxidase, saccharopine reductase and saccharopine dehydrogenase (Naranjo et al., 2001).
On other hand, we showed that P. chrysogenum is able to synthesize pipecolic acid (Naranjo et al., 2003). The lys7 gene encoding saccharopine reductase of P. chrysogenum was target-inactivated by the double recombination method. Analysis of disrupted strain (named P. chrysogenum SR1-) showed the presence of a mutant lys7 gene lacking about 1000 bp in the 3´-end region. The P. chrysogenum SR1- strain lacked saccharopine reductase activity, was a lysine auxotroph and accumulated P6C. The saccharopine reductase activity of this mutant was recovered after transformation with the intact lys7 gene in an autonomous replicating plasmid. When the P. chrysogenum SR1- mutant was grown with L-lysine and D,L-a-aminoadipic acid as nitrogen sources, a high levels of P6C and pipecolic acid were accumulated intracellularly. A comparison of the SR1- strain with a lys2 defective mutant (TDX195) provided evidence showing that P. chrysogenum synthesizes pipecolic acid from a-aminoadipic acid and not from lysine catabolism.
In this work we report for the first time that disruption of the lys7 gene encoding saccharopine reductase leads to the accumulation of P6C a cyclic form of a-aminoadipic acid semialdehyde, that is later converted into pipecolic acid. This recombinant strain may have important potential industrial application for the production of pipecolate-derived products with interesting pharmacological properties.
