Cometabolic Isoterpinolene Formation from Isolimonene by Denitrifying

FEMS Microbiology Letters 169 (1998) 67^71

Cometabolic isoterpinolene formation from isolimonene by denitrifying Alcaligenes defragrans Downloaded from https://academic.oup.com/femsle/article/169/1/67/508117 by guest on 20 January 2021 Udo Heyen, Jens Harder *

Abteilung Mikrobiologie, Max-Planck-Institut fuër marine Mikrobiologie, Celsiusstr. 1, D-28359 Bremen, Germany

Received 29 June 1998; received in revised form 30 September 1998; accepted 1 October 1998

Abstract

Alcaligenes defragrans strains denitrify on monoterpenes with an unsaturated hydrocarbon structure. A new cometabolic reaction, the formation of isoterpinolene from isolimonene, was detected in cultures that grew on a monoterpene. The biotransformation of isolimonene, a monocyclic monoterpene with a sp3-hybridized C1 atom of the menthane skeleton, contrasts with the complete mineralization of monoterpenes with a sp2-hybridized C1 atom. This selectivity indicates a demand for a sp2-hybridized C1 atom as structural property for monoterpenes that can be oxidized by A. defragrans. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords: Anaerobic degradation; Denitri¢cation; Monoterpene; Biotransformation

1. Introduction zymes [8,9] and raises the question how the mineralization of alkanes and alkenes is commenced. Environmental concerns have led to intensive re- Isoprene and monoterpenes are the largest natural search on anaerobic degradation of hydrocarbons, source of alkenes. Estimated annual emission rates which resulted in the isolation of new bacteria capa- are in the order of 5U1014 g year31 for each class of ble of degrading alkanes [1,2] and aromatic com- compounds [10]. We have recently shown that mono- pounds [3,4]. So far, only the biochemistry of the terpenes were microbially degradable in the absence initial activation reaction of toluene has been re- of molecular oxygen [11]. The isolation of denitrify- vealed. A carbon-hydrogen bond of the methyl ing microbes on (+)-p-menth-1-ene, (3)-K-phellan- group is added to a fumarate molecule by the action drene, (+)-2-carene and (3)-K-pinene yielded four of benzylsuccinate synthase, a putative glycine radi- strains that were described as Alcaligenes defragrans cal enzyme [5^7]. This unprecedented observation is sp. nov. [12]. In this study, neutral metabolites of considerable interest for our understanding of en- formed during monoterpene consumption were ana- lyzed and identi¢ed. We report the stoichiometric formation of isoterpinolene (2,4(8)-p-menthadiene, 6-methyl-3-(1-methylethylidene)-cyclohexene) from * Corresponding author. Tel.: +49 (421) 2028-750; isolimonene and propose a structural requirement Fax: +49 (421) 2028-580; E-mail: [email protected] for the central hydrocarbon activation reaction.

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2. Materials and methods of K-phellandrene (70.6%), K-terpinene (23.0%), Q- terpinene (3.3%) and isoterpinolene (3.2%) and was A. defragrans strains 51Men, 54PinT, 62Car and used as isoterpinolene standard. 65Phen and Thauera terpenica strain 58EuT were cultivated anaerobically on monoterpene and nitrate as sole electron donor and acceptor, respectively, on 3. Results and discussion a bicarbonate-bu¡ered de¢ned medium with a 2,2,4,4,6,8,8-heptamethylnonane (HMN) phase as The monoterpenes supporting denitrifying growth described [11^13]. Typically, 30 Wmol monoterpene of A. defragrans strains contain a common structural and 150 Wmol nitrate were present in 1.5 ml HMN motif: the methyl group-carrying carbon atom (C1 phase and 15 ml freshwater medium before inocula- in the menthane skeleton nomenclature [15]) is sp2- tion. Turbidimetric determinations at 660 nm were hybridized. For example, limonene, (3)-K-pinene Downloaded from https://academic.oup.com/femsle/article/169/1/67/508117 by guest on 20 January 2021 performed to measure biomass formation. Samples and (3)-L-pinene were utilized, but not isolimonene of the aqueous and the HMN phases were with- or (3)-trans-pinane [12]. Therefore we investigated drawn with sterile, N2-£ushed plastic and glass sy- the fate of isolimonene, a monocyclic compound ringes, respectively. Nitrite and nitrate were quanti- with a sp3-hybridized C1 atom, in cultures of A. ¢ed by anion exchange HPLC as described [11]. defragrans strains and T. terpenica 58EuT, which is Concentrations of monoterpenes relate to the aque- also capable of growing anaerobically on a limited ous phase. Monoterpenes were determined by dual- range of unsaturated monoterpenes [13]. Isolimonene column capillary-column gas chromatography [14] does not support microbial growth or denitri¢cation and by gas chromatography-mass spectroscopy [11]. of the strains [12,13]. Compounds were identi¢ed by mass spectroscopy In cultures of A. defragrans strain 54PinT on limo- and retention time analysis (Kovaéts indices). The nene and nitrate, isolimonene disappeared and a new commercially not available isoterpinolene was syn- neutral metabolite accumulated corresponding to the thesized by acid-catalyzed isomerization of K-phel- decline of isolimonene (Fig. 1). A comparison of the landrene according to the literature [15]. A 1.5-ml mass spectrum obtained by GC-MS (Fig. 2A) with portion of 100 mM K-phellandrene in HMN was published spectra [16] identi¢ed the metabolite as mixed once with 20 ml of 60% (w/w) aqueous sulfu- isoterpinolene. Because the di¡erentiation from other ric acid and then incubated for 30 min at 67³C. The structural isomers, e.g. terpinolene, was predomi- reaction yielded according to GC analysis a mixture nantly based on di¡erences in the signal intensity

Fig. 1. Formation of a new metabolite (R) during growth (a)ofA. defragrans 54PinT on limonene (8), isolimonene (S) and nitrate (W). A culture tube contained 3 ml HMN and 17 ml freshwater medium.

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Fig. 2. A: Mass spectrum of the new metabolite. B: GC analysis of the metabolite (trace a), isoterpinolene (b), terpinolene (c), and standard additions of the metabolite and isoterpinolene (d) and the metabolite and terpinolene (e). of mass ions, a retention time analysis was per- isolimonene as isoterpinolene attests that these com- formed and yielded a Kovaéts retention index (RI) pounds are not e¤ciently metabolized further during of 1091 for the metabolite. The published RI values an incubation time of 3 weeks. Analysis of the elec- of isoterpinolene and terpinolene, 1085 and 1088 re- tron balances indicates that more electrons became spectively [17], were close to the determined value. available from the complete oxidation of the disap- Therefore an identi¢cation by coinjection with iso- peared amount of the growth-supporting monoter- terpinolene or terpinolene standards was performed pene to carbon dioxide than are required for the (Fig. 2B). The new metabolite comigrated with iso- reduction of the consumed amounts of nitrate. Elec- terpinolene, thus corroborating the GC-MS analysis. tron ratios of 1.1^1.5 resemble earlier experiments The microbial synthesis of isoterpinolene from iso- [11^14] and sustain the hypothesis that isoterpino- limonene required the presence of a physiologically lene is a dead-end product. active culture and the presence of a monoterpene as Surprisingly, the growth-supporting monoterpene growth substrate (Table 1). The concentration of was not completely consumed. A comparison of all 1 mM of limonene in the presence of 10 mM of strains indicated a toxic e¡ect. All four A. defragrans nitrate represents a carbon limitation. Hence isoli- strains catalyzed the allylic isomerization, but not T. monene and isoterpinolene could be oxidized when terpenica 58EuT isolated on eucalyptol. Microbial the bacteria had the required physiological traits. growth of this strain on limonene (1 mM) was com- However, the recovery of 64^98% of the consumed pletely suppressed in the presence of isolimonene

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Table 1 Quanti¢cation of monoterpene metabolism by nitrate-reducing A. defragrans strains and T. terpenica 58EuT Strain and substrate Bacterial Amount of Amount of Amount of Amount of growth nitrate consumed growth terpene isolimonene isoterpinolene

(vOD660nm) (Wmol, consumed (Wmol, consumed (Wmol, formed % consumption) % consumption) % consumption) (Wmol) Alcaligenes defragrans 54PinT Limonene (2 mM) 0.222 154 (100) 17.0 (59) 0.0 0.0 Isolimonene (2 mM) 0.017 15 (10) ^ 0.2 0.0 Limonene (1 mM)+isolimonene (1 mM) 0.156 105 (76) 12.9 (86) 2.3 (15) 2.1 K-Pinene (1 mM)+isolimonene (1 mM) 0.139 101 (66) 13.2 (88) 10.1 (67) 9.5 Alcaligenes defragrans 51Men Limonene (1 mM)+isolimonene (1 mM) 0.204 122 (85) 14.2 (95) 1.7 (11) 1.1

Alcaligenes defragrans 62Car Downloaded from https://academic.oup.com/femsle/article/169/1/67/508117 by guest on 20 January 2021 Limonene (1 mM)+isolimonene (1 mM) 0.053 77 (51) 7.8 (52) 1.4 (9) 0.9 Alcaligenes defragrans 65Phen Limonene (1 mM)+isolimonene (1 mM) 0.122 125 (81) 12.3 (82) 2.9 (19) 2.3 K-Phellandrene (1 mM)+isolimonene (1 mM) 0.151 118 (79) 13.7 (91) 5.6 (37) 5.5 Thauera terpenica 58EuT Limonene (2 mM) 0.190 138 (95) 14.1 (47) 0.0 0.0 Limonene (1 mM)+isolimonene (1 mM) 0.019 26 (18) 0.0 0.0 0.0

(1 mM), but could be restored by the addition of In enrichment cultures, trace amounts of micro- 5 mM acetate. Thus isolimonene causes a speci¢c bially formed monoterpenes were noticed [11]. Sim- inhibition of the limonene degradation. ilar observations were now made with pure cultures Isoterpinolene formation from isolimonene oc- of A. defragrans. Neutral monoterpenes accumulated curred with all monoterpenes tested as substrate for to concentrations of up to 30 WM during growth on growth ((3)-K-pinene, limonene, (3)-K-phellandrene di¡erent monoterpenes. GC-MS analyses of HMN or a mixture of K-terpinene and Q-terpinene), but phases identi¢ed verbenene, 2,3-bis(methylene)-bicy- could not be detected in cultures utilizing either glu- clo[3.2.1]octane, 3-carene, eucalyptol, limonene, L- tamate (5 mM) or gluconate (5 mM). This di¡erence phellandrene, K-terpinene, Q-terpinene, p-cymene might be caused by either the absence of monoter- and terpinolene. These substances may be perceived pene degrading enzymes in these cultures or the low either as fortuitously formed dead-end metabolites solubility of monoterpenes (ca. 50 WM for alkenes) (p-cymene) or as intermediates of the degradation that restricts the rates of turnover in comparison to pathway (menthadienes). the fast growth rate on a highly water-soluble substrate. Besides isolimonene, further monoterpenes with a sp3-hybridized C1 atom were tested to identify stoichiometric biotransformations. A. defragrans strains 54PinT and 65Phen grow on (+)-2-carene and on (+)-3-carene [12]. cis-4-Carene and trans-4-carene could not serve as growth substrate and were not cometabolically transformed during growth on 2-carene. Myrcene was utilized by A. defragrans 65Phen, whereas (3)-L-citronellene was neither transformed nor mineralized. A delocalized Z-system is present in p-cymene. This compound was not at- tacked as sole carbon source [12] or cometabolically during growth on (3)-K-phellandrene. Fig. 3. Biotransformation of isolimonene to isoterpinolene.

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In summary, the accumulation of isoterpinolene as [6] Coschigano, P.W., Wehrman, T.S. and Young, L.Y. (1998) the product of an allylic rearrangement of isolimo- Identi¢cation and analysis of genes involved in anaerobic toluene metabolism by strain T1: putative role of a glycine free nene (Fig. 3) manifests the capacity of A. defragrans radical. Appl. Environ. Microbiol. 64, 1650^1656. to activate alkene bonds of unsaturated monoter- [7] Leuthner, B., Leutwein, C., Schulz, H., Hoërth, P., Haehnel, penes and the necessity of a sp2-hybridized C1 W., Schlitz, E., Schaëgger, H. and Heider, J. (1998) Biochem- atom with localized bonds for the further metabo- ical and genetic characterization of benzylsuccinate synthase lism in which the monoterpene may be transformed from Thauera aromatica: a new glycyl radical enzyme catalys- ing the ¢rst step in anaerobic toluene metabolism. Mol. Mi- into an ionic compound that stays intracellular as crobiol. 28, 615^628. substrate. [8] Harwood, C.S. and Gibson, J. (1997) Shedding light on anaerobic benzene ring degradation: a process unique to prokar- yotes? J. Bacteriol. 179, 301^309. Acknowledgments [9] Heider, J. and Fuchs, G. (1997) Anaerobic metabolism of aromatic compounds. Eur. J. Biochem. 243, 577^596. Downloaded from https://academic.oup.com/femsle/article/169/1/67/508117 by guest on 20 January 2021 [10] Zimmermann, P.R., Chat¢eld, R.B., Fishman, J., Crutzen, We like to thank Sabine FoM for critical discus- P.J. and Hanst, P.L. (1978) Estimates of the production of sions. This work was supported by the Deutsche CO and H2 from the oxidation of hydrocarbon emissions Forschungsgemeinschaft (to J.H.) and the Max- from vegetation. Geophys. Res. Lett. 5, 679^682. Planck-Gesellschaft. [11] Harder, J. and Probian, C. (1995) Microbial degradation of monoterpenes in the absence of molecular oxygen. Appl. En- viron. Microbiol. 61, 3804^3808. [12] FoM, S., Heyen, U. and Harder, J. (1998) Alcaligenes defra- References grans sp. nov., description of four strains isolated on alkenoic monoterpenes ((+)-menthene, K-pinene, 2- carene and K-phel- [1] Aeckersberg, F., Bak, F. and Widdel, F. (1991) Anaerobic landrene) and nitrate. Syst. Appl. Microbiol. 21, 237^244. [13] FoM, S. and Harder, J.(1998) Thauera linaloolentis sp. nov. oxidation of saturated hydrocarbons to CO2 by a new type of sulfate-reducing bacterium. Arch. Microbiol. 156, 5^14. and Thauera terpenica sp. nov., isolated on oxygen-containing [2] Rueter, P., Rabus, R., Wilkes, H., Aeckersberg, F., Rainey, monoterpenes (linalool, menthol and eucalyptol) and nitrate. F.A., Jannasch, H. and Widdel, F. (1994) Anaerobic oxida- Syst. Appl. Microbiol. (in press). tion of hydrocarbons in crude oil by new types of sulfate- [14] FoM, S. and Harder, J. (1997) Microbial transformation of a reducing bacteria. Nature 372, 455^458. tertiary allylalcohol: regioselective isomerisation of linalool to [3] Dol¢ng, J., Zeyer, J., Binder-Eicher, P. and Schwarzenbach, geraniol without nerol formation. FEMS Microbiol. Lett. 149, R.P. (1990) Isolation and characterization of a bacterium that 71^75. mineralizes toluene in the absence of molecular oxygen. Arch. [15] Erman, W.F. (1985) Chemistry of the Monoterpenes. Marcel Microbiol. 154, 336^341. Dekker, New York. [4] Lovley, D.R., Baedecker, M.J., Lonergan, D.J., Cozarelli, [16] McLa¡erty, F.W. and Stau¡er, D.B. (1989) The Wiley/NBS I.M., Phillips, E.J.P. and Siegel, D.I. (1989) Oxidation of ar- Registry of Mass Spectral Data. Wiley and Sons, New York. omatic contaminants coupled to microbial iron reduction. Na- [17] Bestmann, H.J., Kobold, U. and Vostrowsky, O. (1985) Gas- ture 339, 297^299. phasen-Dehydratisierung von p-Menthenolen durch Reak- [5] Biegert, T., Fuchs, G. and Heider, J. (1996) Evidence that tionsgaschromatographie. Korrelation der Eliminierungspro- anaerobic oxidation of toluene in the denitrifying bacterium dukte mit Bildungsenthalpien aus Kraftfeldberechnungen. Thauera aromatica is initiated by formation of benzylsuccinate Liebigs Ann. Chem. 1986, 234^241. from toluene and fumarate. Eur. J. Biochem. 238, 661^668.

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