Biosynthesis of Two Dihydropyrrole-Polyketides from a Marine-Derived Penicillium citrinum

Experimentos de incorporação com precursores marcados com C foram realizados objetivando-se estabelecer a biossíntese de dois dihidropirróis N-acilados, (8E)-1-(2,3-dihidro1H-pirrol-1-il)-2-metildec-8-eno-1,3-diona (1) e 1-(2,3-dihidro-1H-pirrol-1-il)-2-metildecano1,3-diona (2), isolados de culturas de Penicillium citrinum de origem marinha. A biossíntese de ambos, 1 e 2, procede através da incorporação de acetato, metionina e ornitina.


Introduction
Pyrrole, dihydropyrrole (pyrroline) and tetrahydropyrrole (pyrrolidine) alkaloids constitute a unique class of secondary metabolites, which include several bioactive members such as the tropane alkaloids, 1 tambjamines [2][3][4] and prodigiosin, 5 anatoxin-a, 6 as well as 2-alkyl or 2,5-dialkyl-branched pyrrolidine and pyrroline ant chemical defenses. 7However, such alkaloids are extremely rare in fungi, the only examples found in literature 8 include the dihydropyrrole-bearing peptide brevigellin isolated from Penicillium brevicompactum, 9 the dihydropyrrole b-keto amides 1 and 2 also isolated from P. brevicompactum, [10][11][12] the mildly antibiotic scalusamides A−C (3-5), which have been isolated from a marine-derived P. citrinum, 13 as well as the tetrahydropyrrole-citrinin conjugate perinadine A, isolated from P. citrinum of marine origin. 14ompounds 1 and 2 display insecticidal, fungicidal activities and induce precocious metamorphosis in the milkweed bug Oncopeltus fasciatus (Insecta). 12We have recently isolated the same compounds 1 and 2 from a marine-derived P. citrinum, 15 and became interested to establish the actual biosynthetic pathway leading to 1 and 2. This is because while pyrrole, dihydropyrrole or pyrrolidine alkaloids isolated from non-fungal sources are typically derived from proline, 16,17 ornithine, 1 or even from acetate, 16 the biosynthesis of the above mentioned dihydropyrroles isolated from fungi have not yet been investigated.Particularly, we aimed to verify if the dihydropyrrole moiety in 1 and 2 is derived from proline or ornithine, and if the branched moiety is derived from propionate or from acetate plus methionine.To the best of our knowledge, this is the first report on the biosynthesis of fungal dihydropyrrole-bearing secondary metabolites.

Results and Discussion
After toxicity evaluation with unlabeled sodium acetate, added to the growth medium of P. citrinum (up to 5 g L -1 ), for which no morphology alterations of the fungus in the growth medium or even alterations in the production yield of 1 and 2 were observed, experiments with sodium[1- 13 13 C 5 ]ornithine and [U- 13 C 5 ]proline were performed. 13C-Labeled precursors were added at the fourth day of growth, at a final concentration of 1 mg mL -1 for each precursor, using previously optimized growth conditions. 15Separate growth experiments were performed without the addition of labeled precursors as control.Cultures were harvested after 14 days for each growth experiment using distinct 13 C-labeled precursors.After growth media filtration and solid phase extraction, fractions were evaporated and subjected to HPLC-UV-ESI-MS analysis.
Preliminary small-scale experiments in 50 mL of growth media for each 13 C-labeled precursor gave, after the growth media clean-up, fractions containing both 1 and 2 which clearly showed the incorporation of sodium[1- 13 C]acetate, sodium[1,2-13 C 2 ]acetate, [methyl- 13 C]methionine and of [U- 13 C 5 ]ornithine by HPLC-UV-MS analyses.Clusters at m/z 250 for the [M+H] + ion and at m/z 272 for the [M+Na] + ion were observed (see Supplementary Information).The preliminary experiments with sodium[2,3-13 C 2 ]propionate and [U- 13 C 5 ]proline showed no incorporation in both 1 and 2. Therefore, large-scale feeding experiments with 13 C-labeled acetate, ornithine and methionine were performed in order to obtain sufficient amounts of labeled 1 and 2 for 13 C NMR analyses.
Separate P. citrinum growth experiments in a total volume of approximately 1.4 L were performed using sodium[1- 13 C]acetate, sodium[1,2-13 C 2 ]acetate, [methyl- 13 C]methionine and [U- 13 C 5 ]ornithine (see Experimental).After growth in the presence of the labeled precursors, solid-phase extraction of the growth media and extensive purification of both 1 and 2 by HPLC, 13 C NMR analyses clearly showed the incorporation of the labeled precursors.P. citrinum grown in medium enriched with sodium[1- 13 C]acetate gave, after isolation, compound 1 with significant incorporation at C-6 (d 166.2),C-8 (d 206.3),C-10 (d 22.5), C-12 (d 31.7) and C-14 (d 124.5), in agreement with a pattern expected for an acetate-derived polyketide chain (Table 1 and Figure S3 in Supplementary Information).An additional feeding experiment with sodium[1,2-13 C 2 ]acetate confirmed this result, indicating that the b-keto-amide chains in 1 and 2 are derived from acetate.The incorporation ratio of sodium[1,2-13 C 2 ] acetate into 1 was calculated as previously proposed by Kubanek and Andersen. 18We measured the incorporation rate at the amide carbonyl group (C-6) and at the terminal methyl group of the alkyl chain (C-15) as 12.7 and 11.7%, respectively (Table 1; see also Figures S6 to S10).
Although propionate is considered as toxic for fungi, [19][20][21] in few instances it has been demonstrated that labeled propionate was incorporated into fungal polyketides. 22,23Therefore, in principle the incorporation of propionate into 1 and 2 could not be ruled out.We have not verified sodium[2,3-13 C 2 ]propionate incorporation into 1 and 2 (data not shown).The C-16 methyl group was clearly methionine-derived, confirmed after a feeding experiment with [methyl- 13 C]methionine that showed a very significative incorporation at C-16 (d 12.7; see Table 1 and Figure S12).The feeding experiment with [U- 13 C 5 ]proline showed no incorporation into 1 and 2. On the other hand, feeding with [U- 13 C 5 ]ornithine showed marked incorporation at the dihydropyrrole group, for which 13 C- 13 C coupling constants of the dihydropyrrole moiety could be unambiguously measured: 74 Hz for C-2/C-3, 36 for C-3/C-4, 36 for C-4/C-5 and 7 for C-2/C-5 (Figures S14 to S16).Since compound 2 has been isolated as a minor derivative, 15 some of the feeding experiments gave insufficient amounts of labeled 2 to obtain reliable 13 C NMR spectra to measure the incorporation rates of [1,2-13 C 2 ]acetate and of [U- 13 C 5 ]ornithine.The results obtained evidenced that both 1 and 2 are derived from acetate, ornithine and methionine.
The dihydropyrrole moiety of 1 and 2 did not show incorporation of labeled acetate.This result was surprising, considering that ornithine is biosynthetically derived from arginine, itself originary from a-ketoglutarate via glutamate in the urea cycle. 24An additional growth experiment with P. citrinum in starch and unlabeled sodium acetate was performed in order to double check this result.No production of 1 and 2 was observed, as expected, since fungi require exogenous nitrogen sources. 24Therefore, we assumed that the biosynthesis of the dihydropyrrole moiety in 1 and 2 needs ornithine, but not proline, as precursor.

Conclusions
We have unambiguously established the biosynthetic origin of dihydropyrroles 1 and 2 as derived from acetate, methionine and ornithine.However, the sequence of events leading to compounds 1 and 2 in Penicillium citrinum still has to be established.

General procedures
NMR spectra were recorded on a Bruker ARX 9.4T instrument, operating at 100.10 MHz for 13 C channels, at 25 o C using TMS as internal reference.Solvents used for extraction and chromatography were HPLC-grade solvents.HPLC separations were performed either with a Waters quaternary pump 600, double beam UV detector 2487, and C NMR spectra recorded in DMSO-d 6 at 100.1 MHz; nd: not detected.

N
O O acetate methionine ornithine data module 746, or with a Waters 600E system controller liquid chromatography attached to a Waters 996 photodiode array detector, on which the UV spectra have been recorded as well.HPLC-UV-MS analysis were performed using a Waters Alliance 2695 coupled on-line with a Waters 2996 photodiode array detector, followed by a Micromass ZQ2000 mass spectrometry detector with an electrospray interface.The photodiode array scanned the samples at l max 230 and 254 nm.The mass spectrometer detector was optimized using the following conditions: capillary voltage, 3.00 kV; source block temperature, 100 o C; desolvation temperature, 350 o C; voltage cone, 25 V; electrospray, positive mode; detection range, 200-550 Da with total ion count extracting acquisition.Cone and desolvation gas flow were at 50 and 350 L h -1 , respectively, using a Nitrogen Peak Scientific N110DR as a nitrogen source.Data acquisition and processing were performed using Empower 2.0.

Microbial strain
The P. citrinum strain was isolated from a seaweed (Caulerpa sp.). 11P. citrinum F53 strain have been deposited in the CBMAI collection under the accession number CBMAI 1186.