Microbial monoterpene transformations—a review

Isoprene and monoterpenes constitute a significant fraction of new plant biomass. Emission rates into the atmosphere alone are estimated to be over 500 Tg per year. These natural hydrocarbons are mineralized annually in similar quantities. In the atmosphere, abiotic photochemical processes cause lifetimes of minutes to hours. Microorganisms encounter isoprene, monoterpenes, and other volatiles of plant origin while living in and on plants, in the soil and in aquatic habitats. Below toxic concentrations, the compounds can serve as carbon and energy source for aerobic and anaerobic microorganisms. Besides these catabolic reactions, transformations may occur as part of detoxification processes. Initial transformations of monoterpenes involve the introduction of functional groups, oxidation reactions, and molecular rearrangements catalyzed by various enzymes. Pseudomonas and Rhodococcus strains and members of the genera Castellaniella and Thauera have become model organisms for the elucidation of biochemical pathways. We review here the enzymes and their genes together with microorganisms known for a monoterpene metabolism, with a strong focus on microorganisms that are taxonomically validly described and currently available from culture collections. Metagenomes of microbiomes with a monoterpene-rich diet confirmed the ecological relevance of monoterpene metabolism and raised concerns on the quality of our insights based on the limited biochemical knowledge.


INTRODUCTION
Annually about 120 Pg of carbon dioxide are assimilated by plants. A part is transformed into chemically complex molecules and released into the environment by emission or excretion (Ghirardo et al., 2011). Volatile organic compounds (VOCs) comprise a large number of molecules, including various hydrocarbons, single carbon compounds (e.g. methane), isoprene and terpenes (e.g. mono-and sesquiterpenes). The atmosphere is loaded with an estimated VOC emission rate of about 1150 Tg C yr −1 (Stotzky and Schenck, 1976;Guenther et al., 1995;Atkinson and Arey, 2003). These estimates included only nonmethane VOCs of biogenic origin (BVOCs); a second source are anthropogenic VOCs. Among the BVOCs, isoprene and monoterpenes dominate with estimated emission rates of about 500 Tg C yr −1 and 127 Tg C yr −1 , respectively (Guenther et al., 1995). Monoterpenes (C 10 H 16 ) consist of two linked isoprene (C 5 H 8 ) units and include in the strict sense only hydrocarbons. Often the term monoterpene is applied including monoterpenoids which are characterized by oxygen-containing functional groups. Structural isomers-acyclic, mono-, and bicyclic monoterpenes-, stereoisomers as well as a variety of substitutions result in a large diversity of molecules. Today, more than 55,000 different isoprenoids are known (Ajikumar et al., 2008). Monoterpenes are not only emitted as cooling substances (Sharkey et al., 2008), but can also be stored intracellularly serving mainly as deterrent or infochemical (Dudareva et al., 2013). Wood plants mainly accumulate pinene and other pure hydrocarbon monoterpenes as constituents of their resins, whereas citrus plants are the major source of limonene. Flowers, however, produce and emit a variety of oxygenated monoterpenes (e.g. linalool) (Kesselmeier and Staudt, 1999 and references therein, Sharkey and Yeh, 2001;Bicas et al., 2009).
In the atmosphere, monoterpenes are transformed in purely chemical reactions within hours. Photolysis and reactions with molecular oxygen, ozone, hydroxyl radicals, NO x species, and chlorine atoms result in carbonyls, alcohols, esters, halogenated hydrocarbons, and peroxynitrates. These products condense and lead to the formation of secondary aerosols. Rain or precipitation transports them to soils (Atkinson and Arey, 2003;Fu et al., 2009;Ziemann and Atkinson, 2012). Monoterpenes reach the surface layers of soils by leaf fall and excreted resins. Also roots emit monoterpenes into the rhizosphere (Wilt et al., 1993;Kainulainen and Holopainen, 2002). Deeper soil layers do contain significant less monoterpenes than the surface soil layer. Emission into the atmosphere and biotransformations in the surface layer mainly by microorganisms are the major sinks. An alternative, abiotic photoreactions like in the atmosphere, is limited by light availability in soil (Kainulainen and Holopainen, 2002;Insam and Seewald, 2010).
Bacteria encountering monoterpenes have to deal with their toxic effects (reviewed by Bakkali et al., 2008). In order to prevent the accumulation of monoterpenes in the cell and cytoplasmatic membrane, bacteria modify their membrane lipids, transform monoterpenes and use active transport by efflux pumps (Papadopoulos et al., 2008;Martinez et al., 2009). Below toxic concentrations monoterpenes are used by microorganisms as sole carbon and energy source. The mineralization of the hydrocarbons requires the introduction of functional groups to access beta-oxidation like fragmentation reactions yielding central metabolites, e.g. acetyl-CoA. In many aerobic microorganisms molecular oxygen serves as reactive agent to functionalize the monoterpenes (Figure 1). Strains of Pseudomonas and Rhodococcus have become model organisms for the elucidation of pathways in aerobic bacteria. Nearly 40 years after the first reports on aerobic mineralization (Seubert, 1960;Seubert and Fass, 1964;, the mineralization of monoterpenes in denitrifying bacteria and methanogenic communities was discovered (Harder and Probian, 1995;Harder and Foss, 1999). Betaproteobacterial strains of the genera Castellaniella and Thauera are the study objects for the elucidation of anaerobic pathways. All these bacteria were obtained in single-fed batch enrichments with high substrate concentrations (mmol * L −1 ), in contrast to low concentrations in nature (μmol * L −1 ). Consequently, in batch enrichments isolated strains exhibit often a solvent tolerance; they grow in the presence of a pure monoterpene phase. Cultivation was rarely attempted by physical separation followed by single-fed batch cultivations. Such dilution-to-extinction series performed in replicates-also known as most-probable-number (MPN) method-revealed a frequent presence of the degradative capacities in natural populations: denitrifying communities in sewage sludge and forest soil yielded 10 6 -10 7 monoterpene-utilizing cells ml −1 , representing 0.7-100% of the total cultivable nitrate-reducing microorganisms . MPN cultivations for aerobic bacteria have not been reported so far, and for both cases the highly abundant bacteria with the capacity to grow on monoterpenes have not been identified.
Over the last 50 years, many monoterpene transformations have been reported for microbial cultures, but the biochemical pathways were rarely disclosed. More important for the maintenance of our knowledge, only a small portion of the investigated strains were deposited in culture collections. Without detailed knowledge of genes or the availability of strains, the observations of biotransformation experiments are of limited value for future studies. Therefore, this review on the transformation of monoterpenes focusses on enzymes for which the gene and protein sequences are available in public databases as well as on microorganisms that at least have been deposited in a public culture collection and ideally are validly described (Table 1). A broad overview on microbial biotransformations is also provided by a number of older review articles (Trudgill, 1990(Trudgill, , 1994van der Werf et al., 1997;Hylemon and Harder, 1998;Duetz et al., 2003;Ishida, 2005;Li et al., 2006;Bicas et al., 2009;Li and Lan, 2011;Schewe et al., 2011;Tong, 2013). KEGG and MetaCyc, two widely used reference datasets of metabolic pathways (reviewed by Altman et al., 2013), include degradation pathways of limonene, pinene, geraniol, and citronellol. Single reactions of p-cymene and p-cumate degradation are covered. MetaCyc additionally covers the metabolism of myrcene, camphor, eucalyptol, and carveol.
The most abundant bicyclic monoterpene is pinene with the isomers α-pinene [3] and β-pinene [4] (C 10 H 16 ), a main constituent of wood resins (e.g. conifers). Pseudomonas rhodesiae (CIP 107491) and P. fluorescens (NCIMB 11671) grew on α-pinene as sole carbon source. α-pinene is oxidized to αpinene oxide [5] by a NADH-dependent α-pinene oxygenase (EC 1.14.12.155) and undergoes ring cleavage by action of a specific α-pinene oxide lyase (EC 5.5.1.10), forming apparently isonovalal as first product which is isomerized to novalal (Best et al., 1987;Bicas et al., 2008;Linares et al., 2009). The cleavage reaction of α-pinene oxide was also described for a Nocardia sp. strain P18.3 (Griffiths et al., 1987;Trudgill, 1990Trudgill, , 1994. An alternative route for pinene degradation via a monocyclic p-menthene derivate has been described for Pseudomonas sp. strain PIN (Yoo and Day, 2002). Bacillus pallidus BR425 degrades αand β-pinene apparently via limonene [6] and pinocarveol. While α-pinene is transformed into limonene and pinocarveol, β-pinene yields pinocarveol only. Both intermediates may be further transformed into carveol [7] and carvone. The activity of a specific monooxygenases has been suggested, but experimental evidence is lacking (Savithiry et al., 1998). Serratia marcescens uses α-pinene as sole carbon source. Trans-verbenol [8] was a detectable metabolite. In glucose and nitrogen supplemented medium, this strain formed αterpineol [9]. The two oxidation products were considered to be dead-end products as they accumulated in cultures (Wright et al., 1986). A general precaution has to be mentioned here for many biotransformation studies: monoterpenes contain often impurities and oxidation products which may be utilized as substrates resulting in traces of monoterpene and monoterpenoid transformation products that are not further metabolized. Stoichiometric experiments have to show that the amount of metabolite is larger than the amount of impurity in the substrate. Only such careful stoichiometric experiments, mutants in functional genes or the characterization of enzymes in vitro can provide a proof of the presence of a biotransformation.
Pseudomonas putida (MTCC 1072) converts limonene to pmenth-1-ene-6,8-diol [19] and perillyl alcohol (Chatterjee and Bhattacharyya, 2001). No sequence information was found in public databases. Two other strains of Pseudomonas putida (F1 and GS1) have been found to convert (+)-limonene to perillic acid in co-substrate fed-batch cultures (Speelmans et al., 1998). Experimental results indicated the participation of the p-cymene pathway (CYM) (Mars et al., 2001). Castellaniella defragrans grows anaerobically on cyclic monoterpenes as sole carbon and energy source under denitrifying conditions . Recent experiments suggested an oxygen-independent hydroxylation on the methyl group of limonene to perillyl alcohol as the initial activation step, followed by subsequent oxidation to perillic acid (Petasch et al., 2014).
Thauera terpenica 21 Mol utilizes menthol [22] as sole carbon source. The proposed degradation mechanism involves two initial oxidation reactions leading to menth-2-enone, followed by a hydration and an additional oxidation step. Finally, ring cleavage may occur and the molecule is attached to coenzyme A to yield 3,7-dimethyl-5-oxo-octyl-CoA Hylemon and Harder, 1998).

ACYCLIC MONOTERPENES
First studies on acyclic monoterpenoids in the early sixties by Seubert and colleagues described the degradation of citronellol [23], geraniol [24], and nerol via an oxidation of the alcohol to an acid, followed by the formation of a CoA-thioester and subsequent beta-oxidation in Pseudomonas citronellolis (ATCC 13674) (Seubert, 1960;Seubert and Fass, 1964). This knowledge has been extended toward other Pseudomonas strains (Cantwell et al., 1978). The complete degradation pathway has been classified as the acyclic terpene utilization and leucine utilization (ATU/LIU) pathway involving the genes atuABCDEFGH and   (Cantwell et al., 1978;Prakash et al., 2007;Förster-Fromme and Jendrossek, 2010). Among the few reactions described in detail is a molybdenum dependent dehydrogenase responsible for the geranial oxidation to geranylate in P. aeruginosa PAO1 . The acyclic monoterpene β-myrcene [25] (C 10 H 16 ) is transformed by Pseudomonas aeruginosa (PTCC 1074) into dihydrolinalool, 2,6-dimethyloctane and α-terpineol. Limonene has been proposed as possible intermediate in α-terpineol formation but was not detected in the culture broth (Esmaeili and Hashemi, 2011). Pseudomonas sp. M1 accomplishes degradation by hydroxylation on the C8 position to myrcene-8-ol, which is further oxidized, linked to coenzyme A and metabolized in a beta-oxidation like manner (Iurescia et al., 1999). The formation of geraniol from β-myrcene has been observed with resting cells of Rhodococcus erythropolis MLT1, regardless of the presence of a cytochrome P450 inhibitor. The reaction was dependent on aerobic conditions, however it remains unclear if a monooxygenase or lyase system is involved (Thompson et al., 2010).

MONOTERPENE TRANSFORMATION BY FUNGI
Fungi excrete laccases which are copper-containing oxidases. Utilizing molecular oxygen as a cosubstrate, an unspecific oxidation of organic molecules is initiated by these enzymes. Additionally, fungi express a variety of cytochrome P450 monoand di-oxygenases. Thus, several fungi were described to transform monoterpenes during growth in rich medium (reviewed by Farooq et al., 2004). Species with a reported capacity to transform monoterpenes are Aspergillus niger, Botrytis cinerea, Diplodia gossypina, Mucor circinelloides, Penicillium italicum, Penicillium digitatum, Corynespora cassiicola, and Glomerella cingulata. For a long time, no species have been described to use monoterpenes as sole carbon and energy source for growth (Trudgill, 1994 and references therein). Recently, Grosmannia clavigera, a bark beetle-associated fungal pathogen of pine trees, was shown to grow on a mono-and diterpene mixture, containing α/βpinene and 3-carene (Diguistini et al., 2011). ABC efflux transporter and cytochrome P450 enzymes confer a monoterpene resistance to the blue-stain fungi (Lah et al., 2013;Wang et al., 2013).

MONOTERPENES IN THE CARBON CYCLE
Habitats with a dense vegetation of wood and flowers are expected to contain larger populations of monoterpene transforming microorganisms. Whereas coniferous forests emit up to 6.7 g carbon * m −2 * yr −1 , broadleaf evergreen forest and grassland emit only 3.5 and 2.5 g carbon * m −2 * yr −1 , respectively (Tanaka et al., 2012). Monoterpene emission rates between 0.3 and 7 g carbon * m −2 * yr −1 for the United States-mainly αand β-pinene, limonene and β-myrcene (Geron et al., 2000)-can support the aerobic growth of 0.15-3.5 g bacteria * m −2 * yr −1 , assuming 50% of carbon incorporated into biomass. This is a significant potential, considering the presence of around 10 g microbial biomass in the top centimeter of soil per square meter.
In marine systems, isoprene and monoterpenes (mainly αpinene) are produced by phytoplankton and algae and partially emitted into the atmosphere (reviewed by Yassaa et al., 2008;Shaw et al., 2010). Isoprene emission was estimated to 0.2-1.2 Tg carbon * yr −1 (Palmer and Shaw, 2005;Gantt et al., 2009;Shaw et al., 2010). For the ocean surface area this results in an emission rate of 0.0025 g carbon * m −2 * yr −1 . Current uncertainties in the size of emission based on shipborne measurements in comparison to satellite data (Luo and Yu, 2010) may be resolved by incorporating an export from the continental atmosphere to the oceanic atmosphere (Hu et al., 2013). Isoprene-amended samples from marine habitats were enriched in bacteria affiliating with Actinobacteria, Alphaproteobacteria, and Bacteroidetes and first strains were shown to degrade isoprene and aliphatic hydrocarbons (Acuña Alvarez et al., 2009).
In summary, these findings indicate a higher abundance of monoterpene transforming and mineralizing bacteria in soils than in the ocean. Indeed, most monoterpene transforming bacteria have been enriched or isolated from soil and freshwater samples in habitats with monoterpene emitting vegetation.

DATABASES FOR PATHWAY ANALYSIS AND A LOOK AT METAGENOMES
Databases are nowadays available for the analysis of enzymatic reactions and metabolic pathways in metagenomic and genomic sequence datasets. The most relevant are the Kyoto Encyclopedia of Genes and Genomes (KEGG), MetaCyc and the Biocatalysis/Biodegradation database of the University of Minnesota.
First studies used KEGG to identify monoterpene-related genes in metagenomes of microbiomes in insects and nematodes feeding on a monoterpene-rich diet. Pine beetles encounter the high terpenoid concentrations of conifers and may take advantage of detoxification processes catalyzed by their symbionts/microbiomes (Adams et al., 2013). The KEGG pathway for limonene and pinene degradation (ko00903) was used to identify genes encoding enzymes putatively involved in monoterpene degradation. Five enzymes were present and more abundant in the metagenomes than in a combined metagenomic set of plant biomass-degrading communities. These enzymes were an aldehyde dehydrogenase, an oxidoreductase, an enoyl-CoA hydratase and two hydratases/epimerases. Whether these genes are truly involved in monoterpene metabolism or the degradation of cyclic compounds, e.g. related aromatic lignin monomers, is an open question. Taxonomically, these genes affiliated with the genera Pseudomonas, Rahnella, Serratia, and Stenotrophomonas.
The pinewood nematode Bursaphlenchus xylophilus transcribes cytochrome P450 genes as main metabolic pathway for xenobiotics detoxification, but not all enzymes needed for terpenoid metabolism were detected by transcriptomic analysis. Metagenomic data of nematode bacterial symbionts included the complete α-pinene degradation pathway (Cheng et al., 2013). Annotation based on KEGG revealed that the degradation pathways for limonene and pinene (map00903) and for geraniol (map00281) accounted for 2.5% of mapped metagenes. The majority of these genes affiliated to Pseudomonas, Achromobacter, and Agrobacterium. Strains isolated from the nematode and capable of growth on α-pinene affiliated to Pseudomonas, Achromobacter, Agrobacterium, Cytophaga, Herbaspirillum, and Stenotrophomonas.

CONCLUSION
The synthesis and transformation of BVOCs, especially terpenoids, by plants is well studied (Kesselmeier and Staudt, 1999). Corresponding pathways have been elucidated and a variety of corresponding enzymes have been isolated and characterized (Mahmoud and Croteau, 2002;Yu and Utsumi, 2009). In contrast, the exploration of the microbial transformation and mineralization of monoterpenes has accumulated a small coverage of the field. Simply, over the last 50 years, research on bacterial monoterpene metabolism had only found the interest of very few principal investigators. Now, large sequence datasets of organisms and biological communities provide an unprecedented insight into the diversity of pathways and provide us with challenging hypotheses. However, the basis for the annotation is the biochemical characterization of enzymes which is only available for few monoterpenes. Only three pathways are completely known on the genetic and enzymatic level: the ones for camphor (CAM), p-cymene (CYM/CMT), and citronellol/geraniol (ATU/LIU). For pinene, the gene for a key enzyme, the α-pinene oxide lyase (EC 5.5.1.10), is still unknown. The lack of such a key enzyme sequence for a KEGG pathway (map00903) illustrates our uncertainty in the interpretation of metagenomic and genomic datasets. Progress in proteomic and metabolomic analyses in the last years support now biochemical and genetic experiments which will swiftly reveal the desired identification of key enzymes in the monoterpene metabolism.