Elsevier

Journal of Biotechnology

Volume 159, Issue 4, 30 June 2012, Pages 329-335
Journal of Biotechnology

Bioconversion of car-3-ene by a dioxygenase of Pleurotus sapidus

https://doi.org/10.1016/j.jbiotec.2011.06.007Get rights and content

Abstract

Mycelium of the basidiomycete Pleurotus sapidus known to contain a novel dioxygenase was used for the bioconversion of car-3-ene [I]. After 4 h of incubation 25.3 mg L−1 car-3-en-5-one [V], 5.4 mg L−1 car-3-en-2-one [VII], and 7.3 mg L−1 car-2-en-4-one [XV] accumulated as major oxidation products. The identity of the respective carenones and their corresponding alcohols was confirmed by comparison with MS and NMR spectral data obtained for synthesized authentic compounds. The peak areas of oxidation products were at least five times higher as compared with autoxidation. A radical mechanism similar to lipoxygenase catalysis was proposed and substantiated with detailed product analyses. The reduction of assumed car-3-ene hydroperoxides to the corresponding alcohols evidenced the radical initiated formation of hydroperoxides and confirmed the regio- and stereo-selectivity of the dioxygenase. The introduction of molecular oxygen into the bicyclic car-3-ene [I] molecule occurred at allylic positions of a cyclic isopentenyl moiety with a pronounced preference for the position adjacent to the non-substituted carbon atom of the C–C-double bond. This co-factor independent selective oxygenation presents an alternative to P450 mono-oxygenase based approaches for the production of terpene derived flavor compounds, pharmaceuticals and other fine chemicals.

Introduction

Terpenoids (isoprenoids) encompass more than 40,000 structures and form the largest class of plant metabolites. They are widely used as industrially important chemicals, including pharmaceuticals, flavors, fragrances, pesticides and disinfectants, and as large-volume feedstock for chemical industries (Bohlmann and Keeling, 2008). The bicyclic monoterpene (+)-car-3-ene (3,7,7-trimethyl bicyclo [4.1.0] hept-3-ene, [I] is a constituent of the tropical pine trees Pinus roxburghii and Pinus logifolia (55–65%) (Pattekhan et al., 1997), some juniper species, and citrus trees. The (−)-car-3-ene enantiomer is found in the essential oil of the Scots pine (Pinus silvestris) (Breitmaier, 2005). Both enantiomers are natural, inexpensive and widely available raw materials and serve as appropriate synthons in the chemical synthesis of pharmaceuticals, agrochemicals, flavors and fragrances (Kuriata et al., 2010, Macaev and Malkov, 2006, Moreira and Nascimento, 2007). Terpene hydrocarbons present suitable precursors for a sustainable biotechnological production of antibiotics, hormones, vitamins, amino acids, and for flavor compounds (Antranikian and Heiden, 2006, Krings and Berger, 2010, Sijbesma and Schepens, 2003, Ulber and Sell, 2007).

As early as in 1962 car-3-ene [I] was used as a precursor in bioconversion studies with Aspergillus niger that resulted in the formation of low yields of a hydroxyketone (Prema and Bhattacharyya, 1962a, Prema and Bhattacharyya, 1962b). Another study described the microbial oxidation of (+)-car-3-ene [I] using Mycobacterium smegmatis, with (+)-chaminic acid, car-3-en-5-one [V] and 2-(3′-methylcyclohexa-3′,5′-dienyl)propane-2-ol as the products (Stumpf et al., 1990). The epoxidation of the cyclic double occurred during incubation of Nicotiana tabacum and Catharanthus roseus with car-3-ene [I] (Hirata et al., 1994, Miyazawa and Kano, 2010).

During the last decade the bioconversion of terpenes using higher fungi has attracted notice with a special emphasis on the production of natural flavors. The basidiomycete Pleurotus sapidus was shown to convert bicyclic terpene hydrocarbons regio- and stereo-specifically to valuable flavor compounds: α-pinene to verbenone (Krings et al., 2009) and valencene to the grapefruit aroma impact nootkatone (Kaspera et al., 2005, Kruegener et al., 2010). This study presents the regio- and stereo-selective oxidation of the bicyclic monoterpene car-3-ene [I] using a dioxygenase previously isolated and sequenced from mycelium homogenate of P. sapidus (Fraatz et al., 2009).

Section snippets

Chemicals

(+)-Car-3-ene [I] (90%, Sigma Aldrich, Germany) and azeotropic pentane/diethyl ether (1:1.12) were distilled (>99% purity) before use. In (+)-car-3-ene no oxidations products were detectable. BHT (3,5-di-tert-butyl-4-hydroxytoluene, 99%) was from Fluka (Seelze, Germany). All other chemicals used were analytical grade.

Fungi

Pleurotus sapidus (DSMZ 8266) was obtained from the culture collections of DSMZ, Braunschweig. For maintenance on agar slants and submerged culture, the fungus was grown on glucose/

Results

Homogenates of P. sapidus were incubated with 300 mg L−1 of (+)-car-3-ene [I]. After 4 h of agitating several car-3-ene [I] derived oxidation products were detected in concentration up to 25.3 mg L−1 for the major product car-3-en-5-one [V], 5.4 mg L−1 car-3-en-2-one [VII], and 7.3 mg L−1 car-2-en-4-one [XV]. It is known that, after contact with molecular oxygen, (+)-car-3-ene [I] is prone to autoxidation and readily polymerizes above 40 °C (Rothenberg and Sasson, 1998). In fact, longer incubation times

Discussion

The first introduction of oxygen is usually the rate limiting step in the catabolism of non-polar compounds, such as terpenes. Especially cytochrome P450 systems, constitutive of all living beings and representing one of the largest and oldest gene super-families, may act as terminal mono-oxygenases in a range of reactions. However, for an industrial application of co-factor dependent enzymes, several crucial obstacles have to be surmounted. Therefore, co-factor independent specific oxygenation

Acknowledgements

Supported by Forschungskreis der Ernährungsindustrie e.V. (Bonn) through AIF and BMWi (AIF 299 ZN). H. Zorn and M.A. Fraatz are thanked for their kind cooperation.

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