Production of 5-hydroxy-7-methoxy-4-methylphthalide in a culture of Penicillium crustosum

The chemical reactions carried out by microorganisms have been used as a tool in modern chemistry. This paper reports the production of mycophenolic acid and a new phthalide by the endophytic fungus Penicillium crustosum obtained from coffee seeds. The fungus was cultivated in a liquid medium for a period of seven days and after that the culture medium was divided into four treatments: A, B, C and D, to which different organic substances were added. Treatment A was maintained as the control to evaluate the occurrence of biotransformation. Organic acids were added to the culture media of treatments B (ferulic and quinic acids) and C [cinnamic and 3,4-(methylenedioxy) cinnamic acids], and caffeine was added in the treatment D. All these organic compounds were dissolved in DMSO, and the fermentation was maintained for more 13 days, totalizing 20 days. Mycophenolic acid was isolated from the culture with no added acids (treatment A). Mycophenolic acid and a new phthalide, 5-hydroxy-7-methoxy-4-methylphthalide were isolated from treatments B and C, and mycophenolic acid and caffeine (added to the culture medium) were isolated from treatment D. The structures were determined by NMR techniques and confi rmed by MS and MS/MS techniques.


INTRODUCTION
The plant kingdom is colonized by a diversity of endophytic microorganisms, resulting in benefi cial associations that can stimulate plant growth, and increased resistance to diseases and adverse environmental conditions.There are several studies showing that endophytes do not act alone or only at the level of the host, but rather in a complex network of interactions with the microbial fl ora and native plant metabolism (Silva et al. 2006).
The colonization of the host plant by endophytes may be mediated by interesting secondary metabolites of potential use to modern medicine, agriculture and industry (Strobel andDaisy 2003, Mitchell et al. 2008).Surveys of the endophytes have been carried out in dozens of plants of important agricultural commodities, but coffee has been practically unexplored (Vega et al. 2005).Various endophytic Penicillium species were isolated from Coffea arabica, including Penicillium crustosum.The role that these endophytes play in the biology of the coffee plant remains enigmatic, but the fact that no Penicillium species have been reported as pathogens of Coffea spp implies that these endophytes are not latent pathogens, suggesting either commensal or mutualistic relationships (Vega and Posada 2006).
The Penicillium genus is noted for producing a variety of bioactive metabolites possessing diverse biological properties including plant growth regulators (Arnold 2007).
Mycophenolic acid is produced by Byssochlamys nivea and mainly by several Penicillium species, such as P. brevi-compactum, P. roqueforti, P. roqueforti var.carneum, P. bialowiezense, P. chrysogenum, P. raciborkii, P. stoloniferum, and other Penicillium spp.It presents several biological activities, amongst which antibiotic activity, and it has also been successfully applied as an immunosuppressant after organ transplantation, administered in the form of the pro-drug Mycophenolate Mofetil (Grinyo 2001).Recently mycophenolic acid was obtained by the biotransformation of imbricatolic acid, obtained from a culture of Cunninghamella echinulata (Schmeda-Hirschmann et al. 2007).
The chemical reactions carried out by microorganisms have been used as a tool in modern chemistry, but the use of microorganisms in chemistry is not a new issue.Bacteria and fungi have been used to produce chemicals, pharmaceuticals and perfumes for decades.However their use for modifying the chemical structures of natural products is more recent, it is very well documented (Boaventura et al. 2004).
This study aimed to use the endophytic fungus Penicillium crustosum, isolated from coffee beans, to transform characteristic substances of the coffee, such as caffeine, cinnamic, ferulic and quinic acids, and 3,4-methylenedioxycinnamic acid, which is a semi-synthetic substance.

SAMPLING
Apparently healthy coffee (Coffea arabica L.) fruits Coffea arabica L.) fruits Coffea arabica from ripe berries (crown, peduncle, pulp and seeds) were harvested from coffee plantations in Águas da Prata, Brazil and taken to the Laboratory of Molecular Microbiology and Mass Spectroscopy of the Chemistry Department at the Universidade Federal de São Carlos, where the fungus was isolated.

FUNGAL ISOLATION
Half of the coffee fruits were fragmented and the other half was dried in the sun, simulating the process of normal drying for subsequent storage.The fragments (peel, pulp and beans) were plated onto potato dextrose agar (PDA), and after drying in the sun for one week, the whole fruits were peeled and inoculated without cutting.Both the fruit fragments and the fruit peel (not cut) were NEW PHTHALIDE FROM P. crustosum sterilized by a series of immersions in the following order: 1 min in 70% ethanol, 4 min in 11% sodium hypochlorite, 0.5 min in 70% alcohol and 1 min in distilled water.The beans seeds were inoculated onto PDA contained in Petri dishes, and 0.1% stock antibiotic solution was then added (stock: 0.02 g streptomycin in 10 mL sterile distilled water, fi lter sterilized; from this 1 mL was added per liter of medium).The cultures were incubated at room temperature (25°C).

SCANNING ELECTRON MICROSCOPE
The method used to prepare the material for scanning electron microscopy (SEM) was similar to the one described by Melo and Faull (2004).The samples were dried in a critical point (Dryer) (Emitech), sputtered (Emitech plating) with gold for 3 minutes at 30 mA and observed under a Field Emission Scanning Microscope (Gemini Leo 982 Zeiss + Leica) at the Environmental Microbiology Laboratory of the Brazilian Agriculture Research Corporation -Embrapa.

STRAIN TAXONOMY
The genomic DNA from the P. crustosum strain, Catl2.3, was extracted according to the method described by Raeder and Broda (1985).The fungi were identifi ed by comparing the rDNA from the internal transcribed spacer (ITS) regions with sequences deposited in the GenBank.The ITS1-5.8S-ITS2 region was amplifi ed by the polymerase chain reaction (PCR) using the ITS1 primer (5'-TCCGTAGGTGAACCTGCGG-3') and the ITS4 primer (5'-TCCTCCGCTTATTGATATGC-3') in a Peltier Thermal Cycler 200, MJ Research.The conditions for DNA amplifi cation included an initial denaturation step of 5 min at 94°C, followed by 24 cycles of 30 s at 94°C (denaturation), 30 s at 55°C (annealing) and 30 s at 72°C (elongation), plus a fi nal elongation step of 7 min at 72°C.The amplicons were purifi ed using GFX columns (GE Healthcare UK Ltd., Buckinghamshire, England), and then cycle-sequenced using the BigDye ® Terminator Cycle Sequencing Kit (Applied Biosystems, Nieuwerkerk, The Netherlands).The PCR products were sequenced by using the same primers used for amplifi cation.Each sequence was obtained in duplicate from each of two separate PCR amplifi cations.The ITS sequence of the strain found in this study matched the ITS sequence of Penicillium crustosum (GenBank Accession No. GU723443), and displayed 99% similarity.The ITS sequence of the Penicillium crustosum isolated was aligned with different Penicillium isolates available in the GenBank nucleotide data base, using the MEGA 4.0 program (Tamura et al. 2007).The phylogenetic analysis was carried out by a neighbor-joining method to infer the relationships between the Penicillium crustosum isolate and sequences available for Penicillium in the GenBank.For this analysis, 1,000 bootstrap replicates were carried out to assess the statistical support for each tree.The fungus was deposited and preserved in the collection of the Environmental Microbiology Laboratory of the Brazilian Agriculture Research Corporation -Embrapa.

CULTURE (FUNGUS CATL2.3)
After fi ve days of growth, 3 -5 pieces of the PDA culture containing mycelium, each 5 mm in diameter, were inoculated into 40 Erlenmeyer fl asks (500 mL) containing 250 mL of the following medium: 3.0 g NaNO 3 , 1.0 g K 2 HPO 4 , 0.5 g MgSO 4 .7H 2 O, 0.5 g KCl, 0.01 g FeSO 4 .7H 2 O, 10.0 g glucose, and 8.0 g yeast extract in 1.0 liter of distilled water.This culture was autoclaved for 15 minutes at 120°C and incubated at room temperature (28°C) in the static mode for seven days.
On the seventh day, 10 fl asks were maintained as the control (A), 1 mL of quinic plus ferulic acids added to another 10 fl asks (B), 1 mL cinnamic acid plus methylenedioxycinnamic acid to another 10 (C) and 2 mL of caffeine added to the fi nal 10 (D).These solutions were sterilized using Millipore fi lters, and the cultivation continued for 13 days in the static mode.
NMR DATA All the NMR data were recorded at 298 K using a Bruker DRX400 spectrometer operating at 9.4 T ( 1 H at 400.2 and 13 C at 100.6 MHz).Tetramethylsilane (TMS) was employed as the internal reference for chemical shifts (δ 0.0 ppm).Mycophenolic acid was dissolved in 600 μL of CDCl3 and 5-hydroxy-7-methoxy-4methylphthalide in 600 μL of DMSO-d 6 . 1 H, 13 C, gHSQC and gHMBC NMR spectra were acquired using standard pulse sequences.The TopSpin software was used for data processing.

MASS SPECTROSCOPY DATA
Low-resolution ESI-MS and ESI-MS/MS data were acquired in the negative and positive ion modes, using a QuattroLC mass spectrometer (Micromass, triple-quadrupole, ESI/APCI).Mycophenolic acid and 5-hydroxy-7-methoxy-4-methylphthalide, dissolved in methanol, were constantly introduced by direct infusion using the equipment syringe pump at 5 mL.min -1 .The Masslynx 3.5 software was used for data processing.

RESULTS AND DISCUSSION
From 175 fragments of fresh fruits including pulp, skin and seeds, 64 strains of endophytic fungi were isolated.However, only one endophytic fungus (Catl2.3)was isolated from the seeds of the fruits NEW PHTHALIDE FROM P. crustosum dried in the sun.This strain was identifi ed as belonging to the genus Penicillium using Scanning Electron Microscope (SEM) methodology, based on the morphological structures.
The fungus was taxonomically assigned as Penicillium crustosum on the basis of the molecular techniques.The ITS sequence of the strain isolated in this study matched the ITS sequence of P. crustosum (GenBank Accession Nº GU723443), displaying 99% similarity.
P. crustosum produced mycophenolic acid in the four treatments evaluated (A, B, C and D).However, in the treatments B and C in which acids were added, P. crustosum produced mycophenolic acid and 5-hydroxy-7-methoxy-4-methylphthalide (Figure 1).
In treatment A (with no additional substances added) this fungus produced only mycophenolic acid (102.0 mg) with a high degree of purity.
In the treatments in which organic acids were added (treatments B and C), the biotransformation of the acids (B -ferulic and quinic acids; C -cinnamic and 3,4-methylenedioxycinnamic acid) did not occur, as expected in this assay.The acids added to the culture medium were completely metabolized by the fungus, since these compounds were not detected in the fractions.However, two compounds were isolated, mycophenolic acid [B -83.2 mg and C -85.3 mg] and 5-hydroxy-7-methoxy-4-methylphthalide [B -22.3 mg and C -27.1 mg].
Mycophenolic acid was isolated in treatments A, B and C due to its production in the fermentation medium before supplementation with the acids.These results were confi rmed by new cultures at several periods (Valente et al. 2007).
The synthesis of 5-hydroxy-7-methoxy-4methylptalide only occurred after the addition of the organic acids, since it was not isolated in treatment A. In experiment D (supplemented with caffeine), P. crustosun only produced mycophenolic acid (3.25 mg), and the caffeine was completely recovered (70.1 mg).This change in the metabolite produced in these treatments is probably related to the chemical composition of the culture medium, confi rming the data in the literature that reports that the production of mycophenolic acid is highly dependent on the chemical composition of the culture medium, since there is no specifi c enzyme involved in the process (Demain 1968).
The characterization of the compounds produced by P. crustosun is described and discussed.
Mycophenolic acid is a white crystalline solid -chemical formula: C 17 H 20 O 6 , molecular weight (M.W.): 320.3, soluble in methanol and ANGELA M.M.P. VALENTE et al. methylene chloride.The structure of the compound was determined by 1D ( 1 H and 13 C) and 2D ( 1 H-13 C gHMBC) NMR experiments (Table I) and gHMBC) NMR experiments (Table I) and g confi rmed by comparing the data with those from the literature (Rovirosa et al. 2006).
In the MS/MS experiment (ESI -negative mode, with a collision energy of 5 eV) a fragment of m/z 319.3 was obtained for the ion with a m/z 319.3 was obtained for the ion with a m/z m/z of m/z of m/z 639.5.This fragment was exactly half of the mass, suggesting that the ion with a m/z of 639.5 was m/z of 639.5 was m/z formed by two molecules of mycophenolic acid linked by hydrogen bonds (dimmer), with lose a hydrogen [H+] [M+M-H]-.The mass of 320.0 Da was coherent with the NMR results.The MS/MS experiment for the ion with a m/z of 319.3 showed m/z of 319.3 showed m/z the following fragments: m/z 191.1 (100%), m/z 191.1 (100%), m/z referring to the loss of a terpenic unit with a methyl arrangement; m/z 275.3, referring to the loss of a m/z 275.3, referring to the loss of a m/z carbon dioxide unit.H, 13 C and gHMBC NMR data for the compounds mycophenolic acid and 5-hydroxy-7methoxy-4methylphthalide.
gHMBC NMR data for the compounds mycophenolic acid and 5-hydroxy-7methoxy-4methylphthalide.g  I) and confi rmed by comparing the data with that of the NMR data for mycophenolic acid, especially using the gHMBC experiment.
From the results obtained, it was concluded that the production of mycophenolic acid was related to the acidic or basic nature of the substances added to the culture medium.

ACKNOWLEDGMENTS
The authors are thankful to the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), the Conselho Nacional de Desenvolvimento Científi co e Tecnológico (CNPq), the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and to the Financiadora de Estudos e Projetos (FINEP) for their fi nancial support.

TABLE I 1
gHMBC data set: the numbers correspond to the correlated carbons.gHMBC data set: the numbers correspond to the correlated carbons.g *