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FEBS Letters 585 (2011) 1031–1036

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Molecular characterization of an oxidosqualene cyclase that yields shionone, a unique tetracyclic triterpene ketone of Aster tataricus ⇑ Satoru Sawai 1, Hiroshi Uchiyama, Syuhei Mizuno, Toshio Aoki, Tomoyoshi Akashi, Shin-ichi Ayabe , Takeyoshi Takahashi

Department of Applied Biological Sciences, Nihon University, Fujisawa, Kanagawa 252-0880, Japan

article info abstract

Article history: Shionone is the major triterpenoid component of Aster tataricus possessing a unique all six-mem- Received 31 January 2011 bered tetracyclic skeleton and 3-oxo-4-monomethyl structure. To clarify its biosynthetic process, an Revised 27 February 2011 oxidosqualene cyclase cDNA was isolated from A. tataricus, and the function of the was Accepted 28 February 2011 determined in -deficient yeast. The cyclase yielded ca. 90% shionone and small Available online 4 March 2011 amounts of b-amyrin, friedelin, dammara-20,24-dienol, and 4-epishionone and was designated as Edited by Ulf-Ingo Flügge a shionone synthase (SHS). Transcripts of SHS were detected in A. tataricus organs, confirming its involvement in shionone biosynthesis. SHS was shown to have evolved in the Asteraceae from b- amyrin synthase lineages and acquired characteristic species- and product-specificities. Keywords: Asteraceae Ó 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Evolution Oxidosqualene cyclase Shionone Triterpene Aster tataricus

1. Introduction 3-oxo structure, the molecular formula (C30H50O) of shionone as well as of friedelin is identical to that of many other cyclic 3- Plant triterpenes display a remarkable variety of skeletons. They hydroxytriterpenes (triterpene monoalcohols), which are biosyn- contain mono- to pentacyclic structures, but the most frequently thesized directly from 2,3-oxidosqualene in single oxidosqualene occurring are a tetracyclic system with three six- and one five- cyclase (OSC) reactions. In the classical structural study of shio- membered rings (designated hereafter as the 6/6/6/5 system) and none done in the 1960s by Ourisson, Takahashi (an author of the both pentacyclic 6/6/6/6/6 and 6/6/6/6/5 systems [1,2]. Shionone present paper), and coworkers, the biogenetic consideration was is the main triterpenoid component of an Asteraceae plant Aster adopted to elucidate a skeleton conforming to the friedelin type

tataricus L. (Japanese name: ‘shion’), the dried roots (Asteris Radix) A/B ring and a C6 side chain attached to a tertiary carbon atom of which have been used in a traditional oriental medicine to [4,5]: i.e., a unique intermediate, the baccharenyl cation [2], was eliminate phlegm and relieve cough [3]. Shionone has a rare 6/6/ hypothesized for the first time. The structure of shionone was then 6/6 tetracyclic skeleton and a C-3 carbonyl carbon (Fig. 1). The unambiguously established by chemico-physical methods [5,6]. structure of the A/B/C ring portion of shionone is the same as that However, no biochemical or molecular biological study of shionone of friedelin, another triterpene ketone with a 6/6/6/6/6 pentacyclic biosynthesis has yet been published, and thus whether a single system widely distributed in the plant kingdom. In spite of the enzyme carries out the complex steps of shionone formation or multiple are involved in its biosynthesis remains unclear. Since the first molecular characterization of Arabidopsis thaliana Abbreviations: BARS, baruol synthase; BAS, b-amyrin synthase; BOS, baccharis cycloartenol synthase (CAS) [7], several dozen plant OSC cDNAs oxide synthase; CAS, cycloartenol synthase; FRS, friedelin synthase; LAS, lanosterol synthase; LUP, ; OSC, oxidosqualene cyclase; SHS, shionone have been reported [1]. Single plants have multiple OSCs with synthase various product specificities: for example, A. thaliana has 13 OSC ⇑ Corresponding author. Address: Department of Applied Biological Sciences, genes in its genome, and their very diverse functions have now Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-0880, Japan. Fax: +81 all been characterized [8]. The structural diversity of plant 466 84 3704. triterpenoid skeletons is attributed to both the presence of diverse E-mail address: [email protected] (S. Ayabe). OSC genes (proteins) and multiple products often observed in 1 Present address: RIKEN Plant Science Center, Yokohama, Kanagawa 230-0045, Japan. single OSC reactions. Molecular phylogenetic analysis has catego- Deceased. rized most of the plant OSCs so far identified into four families:

0014-5793/$36.00 Ó 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2011.02.037 1032 S. Sawai et al. / FEBS Letters 585 (2011) 1031–1036

Fig. 1. The predicted pathway for the SHS reaction (A) and biosynthesis of baccharis oxide and baruol from baccharenyl cation (B). lanosterol synthase (LAS) and CAS families of the sterol biosyn- mRNA isolation system (Novagen). The preparation of cDNA tem- thetic pathway, lupeol synthase (LUP) family, and b-amyrin syn- plates and RACE PCR were performed using a Marathon cDNA thase (BAS) family [9,10]. The BAS family contains the most Amplification Kit (Clontech). The Zw1 cDNA was obtained by a ser- diverse OSCs, yielding mainly the pentacycles and multiple prod- ies of nested degenerate PCR, 50- and 30-RACE PCRs and a final ucts. Until recently, the only plant OSC reported to yield a triter- amplification of the full ORF. The PCR primers and reaction condi- pene with a 6/6/6/6 tetracyclic system was A. thaliana At4g15370 tions are listed in Supplementary Table S1. Briefly, after the two (PEN2), which gives baruol as the major product together with rounds of degenerate PCRs with the primer sets d111S/d610As 22 minor products (see Fig. 1B) [11], except that recombinant and d158S/d561AS, the PCR product was cloned into a pT7-Blue Pisum sativum BAS produced bacchar-12-en-3b-ol from the T vector (Novagen). The RACE PCRs with the primer sets ZwAS2/ modified substrate 22,23-dihydro-2,3-oxidosqualene [12]. Subse- AP1 (50-RACE) and ZwS2/AP1 (30-RACE), respectively, were then quently, while our study on A. tataricus OSCs was in progress, bac- done, and the nested RACE PCRs with ZwAS1/AP2 (50-RACE) and charis oxide synthase (BOS) was cloned from Stevia rebaudiana, ZwS1/AP2 (30-RACE) were followed. The full ORF of Zw1 was ampli- also as a multiproduct enzyme [13]. On the other hand, very re- fied from the cDNA template, and cloned into the pT7-Blue T vec- cently, characterization of friedelin synthase (FRS) from Kalanchoe tor. This Zw1 ORF was transferred to HindIII and XhoI sites of the daigremontiana was reported [14]. In the FRS reaction, up to 10 pYES2 (Invitrogen) by PCR subcloning with KOD plus DNA poly- rearrangement steps are presumed to yield friedelin from 2,3- merase (Toyobo, Osaka, Japan) with a set of primers containing oxidosqualene. Shionone should also be synthesized by a very either a HindIII (Zw1_FUH1) or XhoI site (Zw1_FLX1). similar reaction mechanism, but the formation of shionone among the products of any OSC has never been documented. 2.2. Functional expression of Zw1 cDNA in yeast and analysis of We have identified an A. tataricus OSC that yields shionone as triterpene products the major product and report it here. The evolutionary aspects of this and related plant OSCs are discussed based on the mechanistic Saccharomyces cerevisiae GIL77 (gal2, hem3-6, erg7, ura3-167) consideration and phylogenetic analysis. strain [15] was transformed with Zw1 cDNA cloned into pYES2 using Frozen-EZ Yeast Transformation II (Zymo Research). The 2. Materials and methods transformant was cultured in synthetic complete medium without uracil supplemented with 20 lg/ml ergosterol, 5 mg/ml Tween 80 2.1. Cloning of Zw1 cDNA and 13 lg/ml hemin for 2 days at 28 °C. The collected cells were resuspended in the same medium containing 2% galactose instead A. tataricus plants were grown in our University garden. mRNA of glucose to induce the recombinant cDNA and cultured for 2 was isolated from its underground parts using the Straight A’s more days. Finally, the cells were incubated in 0.1 M K-Pi buffer S. Sawai et al. / FEBS Letters 585 (2011) 1031–1036 1033

(pH 7.0) supplemented with 3% glucose for 1 day before they were an unusual partial sequence around the conserved DCTAE motif harvested. For the pilot experiment, 400 ml medium at each stage (including the catalytic Asp) of OSCs [18]: i.e., it showed the was employed, and for the identification of the Zw1 reaction prod- amino acid sequence of LSDCTTI in contrast to the [V/I]SDCTAE ucts, a large-scale culture using 4.8 l of medium was performed. of the common plant OSCs (the changed amino acid residues The cells harvested were refluxed with 20% KOH in 50% (v/v) EtOH are underlined). Because the catalytic Asp at the of for 1 h and extracted with n-hexane. The n-hexane extract from OSC proteins is located proximal to the oxygen atom of 2,3- 70.3 g of transformed yeast cells was separated by silica gel (Wako oxidosqualene, the unusual sequence near this residue could gel C-200) column chromatography with n-hexane:ethyl acetate affect the product structure around the A-ring. Given the charac- (9:1 v/v) as an eluent. The fractions were monitored by TLC using teristic 3-oxo-4-monomethyl A-ring structure of shionone, it a silica gel 60 F254 TLC plate (Merck) with n-hexane:ethyl acetate seemed reasonable to assume that this sequence is part of the

(9:1 v/v) and visualized by spraying with 10% H2SO4 and heating at target OSC. Thus, we concentrated on this fragment and carried 100 °C. The major triterpene ketone fraction containing shionone out further 50- and 30-RACE PCRs. As a result, a full length Zw1 and a minor fraction composed of 4-epishionone were purified cDNA of 2286 bp long encoding a polypeptide of 761 amino acid by silica gel TLC. The triterpene monoalcohol fraction was purified residues was cloned (Supplementary Fig. S1; database ID, by silica gel TLC and HPLC using a TSK-GEL ODS-80TM column AB609123 (AtaSHS); Zw named after ‘Ziwan,’ which is Chinese (Tosoh, Tokyo, Japan) with 85% (v/v) acetonitrile for the eluent. for the drug Asteris Radix). The isolation yields of the triterpene ketone fraction containing For the functional analysis of Zw1, the cells of yeast GIL77 [15] shionone, the other ketone fraction containing 4-epishionone, devoid of LAS were transformed with an expression vector harbor- and the triterpene monoalcohol fraction after the preparative TLC ing Zw1, and the constituents produced instead of lanosterol from were 13.6, 0.2, and 0.2 mg, respectively. 2,3-oxidosqualene by Zw1 protein were analyzed. Thin-layer chro- matograms of the n-hexane extract of the transformed GIL77 after 2.3. Expression analysis of SHS (shionone synthase) and BAS-like2 saponification showed a major spot of products that are less polar genes in A. tataricus organs than triterpene monoalcohols and other minor spots including the spot of triterpene monoalcohols (Fig. 2A and Supplementary Total RNA from roots, stolons, rhizomes, radical leaves, stems, Fig. S2). The fraction (Fr. 1) from the silica gel column chromatog- leaves, and flower heads of A. tataricus were isolated by a CTAB raphy of the extract from the large-scale (4.8 l) culture of Zw1/ method and purified using an RNeasy Plant Mini Kit (QIAGEN), GIL77 giving the major spot was composed substantially of a single and contaminated genomic DNA was digested using an RNase- compound according to HPLC analysis (Fig. 2B). This product was Free DNase Set (Qiagen). First strand cDNA synthesis was per- identified as shionone by comparison with an authentic sample formed using a SuperScript III First-Strand Synthesis System (Life as well as by its electron ionization-MS [m/z (relative intensity) Technologies). Semi-quantitative RT-PCR was done with GoTaq 426 (M+, 100), 411 (11), 341 (40), 273 (13)] [19], 1H NMR, and Green Master Mix (Promega). For expression analysis of SHS, 13C NMR data (Supplementary Table S3). two different sets of gene-specific primers from the 30-UTR re- The triterpene monoalcohol fraction (Fr. 3 in Fig. 2B) contained gion (AstSHS3Uf1/AstSHS3Ur1) and from the coding region two major compounds at roughly equal levels by HPLC analysis, (AstSHSf2/AstSHSr2) were designed (for the primer sequences and they were identified as b-amyrin and dammara-20,24-dienol and the PCR conditions, see Supplementary Table S1). Expression through comparison of spectral data of a standard b-amyrin sam- analysis of the BAS-like2 gene was also performed, and as an ple and the record in the literature (Supplementary Table S3) internal control, the actin gene was amplified. [20,21]. The fraction eluted between the triterpene ketone and monoalcohol fractions (Fr. 2 in Fig. 2B) displayed a 1H NMR spec- 2.4. Phylogenetic analysis trum almost identical to the spectrum of shionone except that a

secondary-CH3 at C-4 (CH3-23) appeared at d 1.12 and the CH3-24 Forty-seven nucleotide sequences of BAS and LUP (as out- group) family genes of which the catalytic functions have been characterized were used for phylogenetic analysis (Supplemen- tary Table S2). Phylogenetic and molecular evolutionary analyses were conducted using MEGA ver. 4 [16] and ver. 5-Beta. Multiple alignment of nucleotide sequences was generated from aligned amino acid sequences with Muscle [17] using default parame- ters. A neighbor-joining (NJ) tree was constructed using the maximum composite likelihood model and pairwise deletion of alignment gaps. The estimated reliability of the phylogenetic tree was tested by the bootstrap method (1000 replications).

3. Results

3.1. Cloning and characterization of Zw1 (SHS cDNA) from A. tataricus

By PCR using nested degenerate primer sets designed from common sequences of known plant OSCs with the cDNA pool prepared from A. tataricus roots as the template, ca. 1.2 kb frag- ments were amplified, and cloned into the vector. Twenty trans- Fig. 2. TLC of the hexane extract of yeast GIL77 transformed with Zw1 (A) and HPLC formed Escherichia coli colonies were picked up, and the inserted charts of the silica gel column chromatography fractions (B). Fr. 1 contained shionone, Fr. 2 eluted between Frs. 1 and 3 and consisted of 4-epishionone, and Fr. 3 cDNA fragments were sequenced. They were composed of three was composed of triterpene monoalcohols. The HPLC conditions were as follows: Fr. BAS-like and one CAS-like sequences. The BAS-like sequence that 1, 100% acetonitrile at a flow rate of 1 ml/min; Fr. 2, 90% acetonitrile at a flow rate of was the most abundantly amplified (found in 17 colonies) had 1.5 ml/min; Fr. 3, 85% acetonitrile at a flow rate of 1.5 ml/min. 1034 S. Sawai et al. / FEBS Letters 585 (2011) 1031–1036 at C-5 was at d 0.92. These downfield shifts can be explained if the 3.2. Phylogenetic relationship between SHS and other OSCs secondary-CH3 is located at 4a with an axial orientation, and this configuration was supported by the assignments of 13C and 1H sig- The SHS cDNA sequence exhibited the highest identity, 83% and nals for C-2 to C-4 parts by 2D-NMR techniques and particularly by 80% at the nucleotide and amino acid levels, with BOS cDNA of S. the observation of NOE between CH3-23 and H-2a (ax) [d 2.46 (1H, rebaudiana (Asteraceae) [13]. Also, it was almost equally identical, m)] (Supplementary Fig. S3). Therefore, the product was deter- i.e., 79% and 77% at the nucleotide and amino acid levels, respec- mined to be 4-epishionone, which is not known as a natural prod- tively, to BAS cDNAs of two other Asteraceae plants, Aster sedifolius uct [22]. [23] and Artemisia annua [24]. In contrast, only low identities In addition, when examined carefully, the HPLC chromatogram (<70%) were demonstrated between SHS cDNA and A. thaliana of the triterpene ketone fraction (Fig. 2B, Fr. 1) exhibited a tiny baruol synthase (BARS1) cDNA [11] or K. daigremontiana FRS cDNA peak at the same retention time (18.27 min) as the standard friede- [14] (Supplementary Table S4). lin sample, and the 1H NMR spectrum of Fr. 1 displayed small Fig. 3A shows the molecular phylogenetic tree based on the signals assignable to protons of CH3-24, CH3-26, CH3-27, CH3-28, nucleotide sequences of SHS and related OSC genes from dicotyle- CH3-29, and CH3-30 of friedelin (Supplementary Fig. S4). Therefore, donous plants using LUP family genes as the outgroup (see also although it was not isolated, the Zw1/GIL77 very likely produced a Supplementary Fig. S5). SHS and BOS form a distinct clade together small amount of a triterpene ketone friedelin together with a pre- with two BASs of the Asteraceae plants. BASs from two other aste- dominant amount of shionone. rid plants, Panax ginseng and Gentiana straminea, are also posi- Deduced from the isolation yields of the column chromatogra- tioned closely and form a monophyletic clade with Asteraceae phy and HPLC fractions from a large scale culture of the yeast, OSCs. FRS is among the four OSCs cloned from K. daigremontiana, the ratio of the products was roughly estimated as shionone:b- all of which are clustered with BASs and multifunctional OSCs of amyrin:friedelin:dammara-20,24-dienol:4-epishionone = 90%:2%: various plants (although none of the Kalanchoe OSCs produce 2%:1.5%:1.5%. None of these products was detected in the control b-amyrin as the main product). BARS1 (PEN2) is in the A. yeast harboring the empty vector (Supplementary Fig. S2). Thus, thaliana-specific branch composed of PENs, which are a unique the enzyme encoded by Zw1 was designated as a SHS that gives group of enzymes of a rapidly evolving lineage containing those rise to small amounts of byproducts. that yield tricyclic and A- and C-ring cleaved triterpenes.

Fig. 3. Molecular phylogenetic trees of SHS and related plant OSCs. (A) A tree of selected full length nucleotide sequences. (B) A tree of the nucleotide sequences of the degenerate RT-PCR products (ca. 1 kb) from A. tataricus roots and corresponding regions of known sequences. S. Sawai et al. / FEBS Letters 585 (2011) 1031–1036 1035

Fig. 5. Comparison of active-site residues (shaded) in human LAS [34] and corresponding residues of SHS and related OSCs (after [8]). Magenta and green shaded residues in human LAS indicate residues that interact with A- and B-rings, Fig. 4. RT-PCR detection of SHS and BAS-like2 sequences in different organs of A. and C- and D-rings, respectively. tataricus. The patterns of band intensities from the two primer sets for SHS were almost identical, indicating that the fragments originating from the same SHS mRNA were amplified. Almost equal intensities of actin bands of all organs guarantees the semi-quantitative detection of the cDNA originating from each Shionone is the main triterpenoid constituent of dried A. mRNA existed in the organs. tataricus roots, and very limited information regarding its occur- rence in plants other than A. tataricus is available [25–27]. The observed accumulation of SHS transcript in the A. tataricus organs The nucleotide sequences (ca. 1 kb) of the fragments amplified including the roots (Fig. 4) indicates that the SHS cDNA encodes by the degenerate RT-PCR on the mRNA of A. tataricus roots were the functional enzyme of this plant species. Baccharis oxide, also analyzed (Fig. 3B). In addition to the partial SHS sequence which should be produced by the same pathway to shionone un- (SHS), two BAS-like sequences were obtained. One of these frag- til two steps before the C-4 cation (Fig. 1B), has been reported ments (BAS-like1) was more closely related to SHS, while the other from the Baccharis species (Asteraceae) [28,29], and a few other (BAS-like2) was more similar to A. sedifolius BAS, and the latter was Asteraceae plants including S. rebaudiana appear to be its pro- estimated to be the partial sequence of an A. tataricus BAS. ducer [13,30]. SHS and BOS should have evolved specifically in the Asteraceae, and SHS must have acquired a high product spec- 3.3. Expression of SHS gene in A. tataricus organs ificity, due to a rather strict controls of the reaction pathway at the branching points (see Fig. 1), yielding ca. 90% shionone. Semi-quantitative RT-PCR was performed to examine the Baruol is also the product of the same pathway as SHS until the expression of SHS and the tentative A. tataricus BAS (BAS-like2) formation of the C-5 cation (Fig. 1B), and BARS1 was character- genes in different organs of mature A. tataricus. Two sets of primers ized among the OSCs of A. thaliana [11], but production of baruol for the SHS coding region and the 30-UTR, respectively, were used in A. thaliana has not yet been reported. On the other hand, in order to exclude the possibility of amplification of homologous friedelin has been known since the 19th century as a constituent gene transcripts. As shown in Fig. 4, transcripts of both SHS and of a very wide variety of monocotyledonous and dicotyledonous BAS-like2 were detected in all organs, but the expression magni- plants with bryophytes and gymnosperms [31,32], and therefore, tudes were characteristically different. SHS was highly expressed a ubiquitous occurrence of the OSC that yields friedelin can be in the aerial parts (stems, leaves, and flowers) except radical predicted. Based on the proposed intrinsic versatility of the prod- leaves, and moderately in the roots, whereas lower expression uct formation in OSC reactions, i.e., the multiple products of OSC was demonstrated in stolons, rhizomes, and radical leaves. In con- reactions due to the mechanistic diversity are the default state of trast, the expression pattern of BAS-like2 was almost the same in all triterpene synthesis [11], it may be reasonable to presume that organs examined (see also Supplementary Fig. S6). the K. daigremontiana FRS is the first reported of a diverse array of OSCs that have acquired, or more likely retained, the activity 4. Discussion of friedelin production [14]. One of the characteristic features of the SHS reaction, shared The foregoing results show that a single OSC, SHS, constructs with BOS and BARS1, is the predominant formation of a baccharane the shionone structure from 2,3-oxidosqualene, clearly confirming skeleton rather than moving on to pentacyclic systems, and an- the biosynthetic scheme for shionone more than 40 years after it other, in common with FRS, is the extensive methyl and hydride was first proposed [4,5]. In the reaction catalyzed by SHS, an early rearrangements that are not terminated by trapping the intermedi- intermediate 6/6/6/5 dammarenyl cation is converted to a 6/6/6/6 ate cations until formation of the characteristic A-ring structure. baccharenyl cation, from which extensive rearrangement steps, i.e., When the amino acid sequences of these OSCs are compared, the

1,2-shifts of three hydrides and four methyls (H-13b to C-18, CH3- conserved Val (most BAS family OSCs and LAS) or Ile (CAS) at the 14a to C-13, CH3-8b to C-14, H-9a to C-8, CH3-10b to C-9, H-5a to position two amino acids upstream to the catalytic Asp (included C-10, and CH3-4b to C-5), yield the direct shionone precursor with in the DCTAE motif) is changed to Leu in SHS and FRS or Thr in a positive charge at C-4, tentatively named the ‘C-4 cation’. Depro- BOS (Fig. 5). While the hydrophilic Thr residue in BOS is implicated tonation of the C-4 cation from H-3a results in the enol form of in flipping the A-ring of the immediate precursor of baccharis oxide shionone, and it may be isomerized non-enzymatically into the up to enable the formation of an ether linkage between C-3–C-10 keto forms, shionone and 4-epishionone. While a hydride shift [13], the bulkiness and the shape of the residue here can be an (H-3a to C-4) in the C-4 cation and deprotonation from the 3- important factor for the characteristic product formation [33]. Also, hydroxyl group can produce shionone, the observation of 4- conserved Y237 and F521 (human LAS numbering) near the prod- epishionone among the reaction products suggests that the shio- uct C–D ring portion based on the human LAS stereostructure [34] none enol form is the direct enzyme product. The very high ratio in many BAS-like OSCs are changed into Phe and Ile in both SHS of shionone compared to 4-epishionone should be due to the unfa- and BARS1. However, phylogenetically closer SHS and BOS do not vorable diaxial interaction of CH3-4a and H-10a of 4-epishionone share these characteristic amino acid substitutions, and, thus, sub- caused by the deformation of the A ring that is brought about by tle conformational changes of the proteins would also dynamically another 1,3-diaxial CH3-5b and CH3-9b (see Supplementary affect the reaction course in these versatile multiproduct OSCs. Fig. S3) [22]. Further mechanistic studies by site-directed mutagenesis as well 1036 S. Sawai et al. / FEBS Letters 585 (2011) 1031–1036 as in planta expressions of the wild type and mutated gene should mechanisms: a challenge to prevailing concepts of triterpene biosynthesis. J. be interesting from the viewpoints of the versatility of secondary Am. Chem. Soc. 129, 11213–11222. [12] Abe, I., Sakano, Y., Tanaka, H., Lou, W., Noguchi, H., Shibuya, M. and Ebizuka, Y. metabolism and its possible ecological functions. 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