Lovastatin Production by Pleurotus Ostreatus: Effects of The C:N Ratio

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Lovastatin Production by Pleurotus ostreatus: Effects of the C:N Ratio

Julio Alarcon* and Sergio Aguila


Departamento de Ciencias Basicas, Facultad de Ciencias, Universidad del Bo Bo, Chillan, Chile. Fax: +56-42-20 30 46. E-mail: [email protected] * Author for correspondence and reprint requests Z. Naturforsch. 61 c, 9598 (2006); received May 27/July 19, 2005 The types of carbon source and nitrogen source used as well as the C:N ratio in the medium influenced lovastatin production by Pleurotus ostreatus. The maximum value of the lovastatin yield was obtained in a medium that contained organic nitrogen. Key words: Pleurotus ostreatus, Statins, Lovastatin

Introduction In fungi, the biosynthesis of a secondary metabolite with a complex chemical structure is performed through the polyketide route (Pfeifer and Khosla, 2001). Lovastatin is produced as a secondary metabolite by the fungi Aspergillus terreus, Monascus ruber, Penicillium brevicompactum and Pleurotus ostreatus. Lovastatin (C24H36O5, mevicolin, monacolin K) is a potent drug for lowering blood cholesterol. It acts by competitively inhibiting the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA) (Endo, 1992; Bobek et al., 1997). As with any fermentation product, the culture medium has a significant influence on the lovastatin yield and its rate of production. Therefore, selection and composition optimization of a suitable medium is important when establishing a process for lovastatin production. Of the principal culture nutrients, carbon and nitrogen sources generally play a dominant role in fermentation productivity because these nutrients are directly linked with biomass and metabolite formation. In previous studies (Alarcon et al., 2003), lovastatin was obtained from cultures in the middle liquid using Hagen medium from a native and commercial strain of P. ostreatus. In the present study, the effect of the C:N ratio in the lovastatin biosynthesis was studied using the native strain PLUBB-127 of P. ostreatus. Materials and Methods Organism collection Fruiting bodies of P. ostreatus were collected from forests in the VIII Region of Chile, growing
09395075/2006/01000095 $ 06.00

on Nothofagus sp. during autumn and spring 2001. Mycelial cultures of the strain were derived from the spore print of the fruiting body. A voucher specimen of the mushroom was deposited in the herbarium of the Departamento de Ciencias Basi cas de la Universidad del Bo Bo, Chillan, Chile. Fungal strain and culture conditions The strains of P. ostreatus (PLUBB-127) were kept on potato dextrose agar (PDA), and incubated for 710 d, and then stored at room temperature. Fermentation was carried out in Hagen medium containing the following (per liter of distilled water): 0.05 g CaCl2 2H2O (Merck), 0.025 g KH2PO4 (Merck), 0.25 g (NH4)2HPO4 (Merck), 0.15 g MgSO4 7H2O (Merck), 1.3 ml FeCl3 1% (Merck), 3.0 g malt extract (Merck) and 10 g glucose (Merck). In a 500 ml Erlenmeyer flask containing 250 ml of medium with aeration and agitation (150 rpm), the fermentation was performed; 125 ml of well-grown culture (7 d) in the same medium were used as inoculum. The fermentation was stopped after 10 and 30 d. The pH value of the medium was adjusted to 6.5 with HCl (2 m) or KOH (2 m). Eleven culture media were used. Table I indicates the composition of the chemically defined fermentation media developed in this study. Statins isolation Culture filtrate (250 ml) obtained by filtration was acidified to pH 3 with 0.01 m HCl and extracted with ethylacetate (3 100 ml). The combined extracts were dried (Na2SO4) and concentrated to a final volume of 5 ml.

2006 Verlag der Zeitschrift fr Naturforschung, Tbingen http://www.znaturforsch.com D

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J. Alarcon and S. Aguila Lovastatin by Pleurotus ostreatus: Effects of the C:N Ratio

Table I. Medium composition for screening of C- and N-sources.


Experimental media
Component
CaCl2 2H2O KH2PO4 (NH4)2HPO4 NH4Cl NaNO3 MgSO4 7H2O FeCl3 (1%) Malt extract Glucose Yeast extract Peptone water

Hagen
0.06 0.025 0.25 0 0 0.15 1.2 3 10 0 0

M1
0.06 0.025 0.25 0 0 0.15 1.2 13 10 0 0

M2
0.06 0.025 0.25 0 0 0.15 1.2 3 10 4 0

M3
0.06 0 0 0 0 0.15 0 10 4 4 0

M4
0.06 0.025 0.25 0 0 0.15 1.2 13 10 4 0

M5
0.06 0.025 0.25 0 0 0.15 1.2 13 10 4 4

M6
0.06 0.025 0.25 0 0 0.15 1.2 13 10 0 4

M7
0.06 0.025 0.25 0 0 0.15 1.2 13 10 4 8

M8
0.06 0.025 0.25 0 0 0.15 1.2 13 10 0 8

M9
0.06 0.025 4.12 0 0 0.15 1.2 0 10 4 0

M10
0.06 0.025 0 3.21 0 0.15 1.2 0 10 4 0

M11
0.06 0.025 0 0 5.41 0.15 1.2 0 10 4 0

The mycelial mass was washed with 0.05 m HCl and stirred at room temperature for 1 h, then filtered, and after acidification extracted with methylene chloride (3 100 ml) and ethylacetate (2 200 ml) for 1 h at 40 C under stirring. The extract was dried (Na2SO4) and concentrated to a final volume of 5 ml. The lovastatin structure was established based on the spectroscopic and GC-MS studies, and the spectral data was compared to data from the literature or from authentic samples. Analytical determinations Lovastatin identification and quantification were performed on the filtrated culture and extract by HPLC, using a Merck LiChrospher

100 RP18 reverse phase column with a diode array detector eluted at the flow rate 2 ml/min. Solvent A was 0.05% H3PO4 in water, and solvent B was acetonitrile. The separation gradient was linear, starting with 95% solvent A and 5% solvent B, reaching 50% solvent A and 50% solvent B in 45 min, 30% solvent A and 70% solvent B in 46 min, 10% solvent A and 90% solvent B in 48 min, 0% solvent A and 100% solvent B in 60 min, and finally continuing with an isocratic run for 5 min. Initial conditions were maintained for 6 min to re-equilibrate the column. Results and Discussion In mushrooms, the biosynthesis of secondary metabolites is subject to complex regulation. This

Table II. Media compositions for screening of C- and N-sources.


Experimental run
Hagen M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11

C-source concentration [g/l]


13.00 23.00 17.00 18.00 27.00 27.00 23.00 35.00 23.00 14.00 14.00 14.00

Medium [g C/l]
4.96 8.16 5.12 3.36 8.32 8.32 8.16 8.32 8.32 4.16 4.16 4.16

N-source concentration [g/l]


0.25 0.25 4.25 4.00 4.00 8.00 4.00 12.00 8.25 4.12 3.21 5.41

Medium [g N/l]
0.24 0.87 2.84 3.23 3.47 3.95 1.35 4.43 4.43 3.47 3.47 3.47

C:N ratio
20.67 9.43 1.80 1.04 2.40 2.11 6.07 1.88 1.88 1.20 1.20 1.20

Lovastatin content* (mg%)


0.415 0.062 4.817 0.356 9.591 0.815 13.433 2.01 14.975 1.24 7.517 0.761 25.497 1.842 21.809 1.912 11.893 1.643 3.305 0.382 3.569 0.216 2.001 0.165

* Means standard error.

J. Alarcon and S. Aguila Lovastatin by Pleurotus ostreatus: Effects of the C:N Ratio
C:N ratio

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lovastatin mg%

30 25 20 15 10 5 0 1 2 3 4 5 6 7 8 9 10 11 Experimental run*

Fig. 1. Relationship between lovastatin content and C:N ratio. * Experimental run, corresponding to the type medium culture used in this study.

study on the potential use of Chilean strains of P. ostreatus for lovastatin production in a liquid medium specifically analyzed the influence of N and C sources on the regulation of lovastatin biosynthesis in P. ostreatus. Under each cultivation condition, P. ostreatus produced lovastatin in a different content. The results show that the wild strain PLUBB127 presents a smaller capacity to produce lovastatin (4.15 mg/l) in comparison with the commercial strain PL-136, which produces 43 mg/l of lovastatin (Table II). In all the performed experiments, with the exception of experiment M3, glucose was used as the carbon source in a constant amount (10 g/l). Malt extract, which contains 1% of nitrogen and 80% carbohydrates, was used as an additional carbon source. Two nitrogen sources, an organic and an inorganic one, were used. The organic source used were yeast extract (Merck) and peptone water (Merck). The inorganic sources used were (NH4)3PO4 (Merck), NH4Cl (Merck) and NaNO3

(Merck). It can be observed that the lovastatin content increased in all the measured media as the contribution of carbon and nitrogen increased. The results (Fig. 1) show an important increase in the lovastatin content in relation to increased nitrogen concentration in the media. This increase is significant in those media that use an organic nitrogen source (peptone water or yeast extract). Furthermore, it can be observed that inorganic nitrogen did not significantly influence lovastatin production. Similar results have been reported in studies performed with Aspergillus terreus (Hajjaj et al., 2001). The incorporation of yeast extract and/or peptone water contributes glutamic acid, histidine and glycine among other amino acids to culture medium. Previous studies have demonstrated that glutamic acid, histidine and to a lesser extent glycine are necessary for the biosynthesis of lovastatin. Additionally, histidine and glutamic acid play a key role in generation of ideophase conditions through the formation of -ketoglutarate, which stimulates aflatoxin synthesis through tricarboxylic acid cycle inhibition (Bhatnagar et al., 1986). Different works have demonstrated the influence of nitrogen in the biosynthesis of secondary metabolites in fungi (Shim and Woloshuk, 1999; Luchese and Harrigan, 1993; Pfeifer and Khosla, 2001). Our results coincide with the results from studies performed with Aspergillus terreus or other filamentous fungi. Acknowledgements This work has been completed thanks to a DIUBB financial grant from University of Bo Bo.

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J. Alarcon and S. Aguila Lovastatin by Pleurotus ostreatus: Effects of the C:N Ratio Hajjaj H., Niederberger P., and Duboc Ph. (2001), Lovastatin biosynthesis by Aspergillus terreus in a chemically defined medium. Appl. Environ. Microbiol. 67, 25962602. Luchese R. H. and Harrigan W.F (1993), Biosynthesis of aflatoxin the role of nutritional factors. J. Appl. Bacteriol. 74, 514. Pfeifer B. A. and Khosla Ch. (2001), Biosynthesis of polyketides in heterologous hosts. Microb. Molec. Biol. Rev. 65, 106118. Shim W. B. and Woloshuk C. P.(1999), Nitrogen repression of fumonisin B1 biosynthesis in Gibberella fujikuroi. FEMS Microbiol. Lett. 177, 109116.

Alarcon J., Aguila S., Arancibia-Avila P., Fuentes O., Zamorano-Ponce E., and Hernandez M. (2003), Production and purification of statins from Pleurotus ostreatus (Basidiomycetes) strains. Z. Naturforsch. 58c, 6264. Bhatnagar R. K., Ahmad S., Mukerji K. G., and Venkitasubramanian T. A. (1986), Nitrogen metabolism in Aspergillus parasiticus NRRL 3240 and A. flavus NRRL 3557 in relation to aflotoxin production. J Appl. Bacteriol. 60, 203211. Bobek P., Ozdin L., Kuniak L., and Hromadova M. (1997), Regulation of cholesterol metabolism with dietary addition of oyster mushroom (Pleurotus ostreatus) in rats with hypercholesterolemia. Cas Lek Cesk 136, 186190. Endo A. (1992), The discovery and development of HMG-CoA reductase inhibitors. J. Lipid Res. 33, 15691582.

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