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Pipecolate synthesis essay - Mellow Kad
As a first approach to study the relationship between pipecolic acid and lysine metabolism in P. chrysogenum, we tested if pipecolic acid could be converted into lysine. Three P. chrysogenum lysine auxotrophic mutants blocked (i) in the synthesis of α-aminoadipic, including P. chrysogenum HS1− (with a targeted inactivation of the homocitrate synthase) and P. chrysogenum L2 (defective in the homoaconitase) or (ii) in the conversion of α-aminoadipic acid to α-aminoadipic semialdehyde, e.g., P. chrysogenum TDX195 (disrupted in the lys2 gene encoding α-aminoadipate reductase), were tested for their ability to grow in Czapek minimal medium supplemented with pipecolic acid. Results showed (Fig. ) that all three different lysine auxotrophs were able to grow either with lysine or with pipecolic acid although growth on pipecolic acid was very slow, indicating that in P. chrysogenum, pipecolic acid is converted into lysine.
We observed incorporation of two different sulfur-containing analogs of -pipecolate into the rapamycin molecule. The analogs 1,4-thiazane-(3S)-carboxylic acid and 1,3-thiazane-(4S)-carboxylic acid were both successfully incorporated in fermentations containing 0.5% (±)-nipecotic acid, yielding the new rapamycin analogs 20-thiarapamycin and 15-deoxo-19-sulfoxylrapamycin, respectively. The biosynthetic differences in the incorporation of these two structurally similar analogs are particularly interesting. 1,4-Thiazane-(3S)-carboxylic acid was incorporated directly into the rapamycin molecule in place of -pipecolate. There was no apparent modification to the analog, and the downstream tailoring enzymes of the biosynthetic pathway were not disturbed. As a result, the structure of the product, 20-thiarapamycin, matched the predicted structure and was identical to the structure of rapamycin except for the presence of a sulfur atom on the pipecolate/thiazane ring. 1,3-Thiazane-(4S)-carboxylic acid was incorporated at a 10-fold-lower yield than 1,4-thiazane-(3S)-carboxylic acid, and the structure of the product, 15-deoxo-19-sulfoxylrapamycin, differed significantly from the predicted structure. The thiazane sulfur of 15-deoxo-19-sulfoxylrapamycin is oxidized to a sulfoxide, presumably after incorporation of 1,3-thiazane-(4S)-carboxylic acid. Also, the final product lacks the C-15 carbonyl that is present in both rapamycin and 20-thiarapamycin, which indicates that the usual activity of the downstream cytochrome P-450 monooxygenase is somehow disrupted. Perhaps, because of the modified structure of this compound, this cytochrome P-450 now acts on the thiazane sulfur instead of the C-15 carbon, but this hypothesis has not been confirmed experimentally.
Pipecolic acid biosynthesis in Rhizoctonia leguminicola
P. chrysogenum HS1− (a strain defective in the homocitrate synthase) spores (106/ml) were incubated in 0.1 M Tris-maleate buffer, pH 9.0, for 12 to 15 h. To induce synchronous spore germination, which correlates with DNA synthesis (), 1.75 mM lysine, glucose (15 g/liter), and (NH4)2SO4 (1 g/liter) were added to the spore suspension. The mutation was carried out during 90 min with 0.5 mM nitrosoguanidine in the same Tris-maleate buffer, pH 9.0, to obtain a mortality rate of 90%. Serial dilutions of mutated spores were plated in Power medium plus lysine (1.75 mM). Lysine auxotrophs unable to grow on pipecolic acid (lys− pip−) were screened by replicating each colony in Czapek minimal medium with pipecolic acid (100 mM) and Czapek minimal medium without pipecolic acid. Reversion rates of the mutations were determined for all the mutants by plating spore suspensions (with known viable spore concentrations) on Czapek minimal medium or Czapek medium plus pipecolic acid and counting the revertant colonies.
To study whether pipecolic acid is converted directly into lysine or whether it is transformed in some intermediate of the lysine biosynthesis pathway, the isolated mutants were plated in Czapek medium plus α-aminoadipic acid to test if they have a functional conversion of α-aminoadipic acid into lysine. Results showed (Table ) that mutant 10.25 was unable to grow in Czapek medium plus α-aminoadipic acid, indicating that the mutation of this strain alters one of the enzymes of the second half of the lysine biosynthetic pathway (i.e., the α-aminoadipate reductase, saccharopine reductase, or saccharopine dehydrogenase). The conversion of pipecolic acid into lysine proceeds, therefore, through the action of some of these enzymes. On the other hand mutant 7.2 was able to grow on α-aminoadipic acid (see below).
Amino Acid Synthesis and Metabolism
During rapamycin biosynthesis, the conversion of -lysine to pipecolate is catalyzed by the enzyme lysine cyclodeaminase. The gene encoding this enzyme, rapL, is in the rapamycin biosynthetic gene cluster (, ). It has been demonstrated previously that elimination of lysine cyclodeaminase activity, either by rapL gene knockout () or by inhibition of the enzyme itself with certain analogs of -proline (), results in a reduction in the rapamycin titer and an increase in the prolyl-rapamycin titer. It has also been shown that this disruption of pipecolate biosynthesis can be used to enhance the incorporation of certain -proline analogs into the rapamycin molecule (, ).
Pipecolic acid serves as a substrate of some nonribosomal peptide and polyketide synthetases, resulting in the formation of secondary metabolites with interesting novel pharmacological activities, i.e., the immunosuppressors rapamycin and immunomycin (, ). It seems, therefore, possible to use P. chrysogenum as a host for producing pipecolate-containing secondary metabolites. Because of the ability of P. chrysogenum to accumulate α-aminoadipic acid, it was of interest to study the interconversion of lysine and pipecolic acid in this industrially important filamentous fungus.
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Many secondary metabolites contain -lysine, -lysine, or pipecolic acid moieties (, ). 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 (–). However, using 14C- and 15N-labeled α-aminoadipic acid and [14C]lysine, Aspen and Meister () showed in Aspergillus nidulans that the carbon chain of α-aminoadipic rather than that of lysine was the major precursor of pipecolic acid and the nitrogen atom of α-aminoadipic acid becomes the nitrogen atom of pipecolic acid. Furthermore, in Neurospora crassa kinetic studies with radioactively labeled -lysine showed that pipecolic acid was an intermediate in the conversion of -lysine into -lysine ().
Biosynthesis of the immunosuppressant immunomycin: …
S. hygroscopicus was routinely cultivated at 28°C on an agar medium containing 0.5% dextrose, 0.25% yeast extract (Difco), 1% soluble starch (Difco), 0.25% NZ-Amine A (Quest), 0.05% CaCO3, and 1.5% agar. The seed culture medium and production medium used were the media described previously by Sehgal et al. (). Seed cultures were initiated by inoculating a loopful of culture from agar into 10 ml of seed inoculum broth. Seed cultures were incubated at 28°C with shaking at 200 rpm with a 2-in. throw for 3 days. For production of rapamycin and 21-nor-rapamycin (prolyl-rapamycin), 0.1 ml of seed culture broth was inoculated into a 50-ml Erlenmeyer shake flask containing 5 ml of production medium. For the directed biosynthesis of rapamycin analogs, the production medium was made without lysine or pipecolic acid and was supplemented with 5% (±)-nipecotic acid (Sigma). The unusual pipecolate analogs 1,4-thiazane-(3S)-carboxylic acid and 1,3-thiazane-(4S)-carboxylic acid were added to this modified production medium at a final concentration of 0.2%. Fermentation was performed as described above, and cultures were harvested after 6 days.
Structural enzymology of biotin biosynthesis
Pipecolic acid is formed by the catabolism of lysine in humans. Although this pathway is considered to be secondary in most tissues, pipecolic acid is the main lysine catabolism product in the brain (, ), and its accumulation was one of the first biochemical abnormalities detected in the Zellweger syndrome. In the filamentous fungi M. anisopliae and R. leguminicola, pipecolic acid, an intermediate in the biosynthesis of alkaloid compounds, comes directly from lysine catabolism, but through a different pathway than in mammals, involving the intermediates saccharopine and P6C (–).
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