Biochemical Conservation and Evolution of Germacrene A Oxidase in Asteraceae

Abstract

Sesquiterpene lactones are characteristic natural products in Asteraceae, which constitutes ∼8% of all plant species. Despite their physiological and pharmaceutical importance, the biochemistry and evolution of sesquiterpene lactones remain unexplored. Here we show that germacrene A oxidase (GAO), evolutionarily conserved in all major subfamilies of Asteraceae, catalyzes three consecutive oxidations of germacrene A to yield germacrene A acid. Furthermore, it is also capable of oxidizing non-natural substrate amorphadiene. Co-expression of lettuce GAO with germacrene synthase in engineered yeast synthesized aberrant products, costic acids and ilicic acid, in an acidic condition. However, cultivation in a neutral condition allowed the de novo synthesis of a single novel compound that was identified as germacrene A acid by gas and liquid chromatography and NMR analyses. To trace the evolutionary lineage of GAO in Asteraceae, homologous genes were further isolated from the representative species of three major subfamilies of Asteraceae (sunflower, chicory, and costus from Asteroideae, Cichorioideae, and Carduoideae, respectively) and also from the phylogenetically basal species, Barnadesia spinosa, from Barnadesioideae. The recombinant GAOs from these genes clearly showed germacrene A oxidase activities, suggesting that GAO activity is widely conserved in Asteraceae including the basal lineage. All GAOs could catalyze the three-step oxidation of non-natural substrate amorphadiene to artemisinic acid, whereas amorphadiene oxidase diverged from GAO displayed negligible activity for germacrene A oxidation. The observed amorphadiene oxidase activity in GAOs suggests that the catalytic plasticity is embedded in ancestral GAO enzymes that may contribute to the chemical and catalytic diversity in nature.

Introduction

Terpenoids, derived from isopentenyl diphosphate, are structurally the most diverse class of natural products with many known biological functions and commercial applications (1). One subclass of terpenoids is sesquiterpene lactone (STL)³, characterized by its α-methylene γ-lactone moiety on the 15-carbon core backbone (2). Although STLs are found in several plant families including Cupressaceae (3), liverwort (4, 5), and even fungus (6, 7), their occurrence in nature is by far the most frequent among Asteraceae (or Compositae) plants, the second largest plant family, after Orchidaceae, composed of 23,000 plant species (8, 9). Despite the structural complexities of STLs, the basic backbones of all STLs are constrained to several core skeletons, such as germacranolide, eudesmanolide, guaianolide, and helenanolide (2). This may suggest that STL biosynthesis is governed by a limited number of biochemical rules.

Apparently, the chemical diversification of STLs is interlocked with plant speciation in Asteraceae, and hence, the structural features of STLs have been routinely used as chemosystematic markers in Asteraceae (10–12). Reported allelochemical, deterrent, dermal allergic, and insecticidal properties of STLs suggest important eco-physiological roles of STLs in interorganism interactions (3). Thus, STLs are likely to be in part attributable to the evolutionary success of Asteraceae plants. In addition, several STLs have benefited human health and wellness as anti-inflammatory (e.g. parthenolide), sedative and analgesic (e.g. lactucopicrin), anti-cancer (e.g. thapsigargin), and anti-malarial (e.g. artemisinin) medicines (13–18).

Although structural and bioactivity studies of STLs have been extensively performed, the biosynthetic route of STLs remains poorly understood at the molecular level. The proposed biosynthetic route of the simplest STL, costunolide, is depicted in Fig. 1 (19, 20). Farnesyl diphosphate is cyclized to C15 hydrocarbon germacrene A by germacrene A synthase (GAS), and subsequently its C12 methyl group undergoes a three-step oxidation reaction to yield the germacrene A acid (germacra-1(10),4,11(13)-trien-12-oic acid). An additional hydroxylation at the C6 position of germacrene A acid facilitates nonenzymatic lactonization of C6 hydroxyl and C12 carboxylic group, yielding costunolide. The costunolide in turn serves as a framework of guaianolides, eudesmanolides, germacranolides, and other STLs by unknown mechanisms. Last, elaborate chemical decorations of STL scaffolds are carried out by P450s and several other modifying enzymes to produce more complex and biologically active STL end products.

FIGURE 1.

Sesquiterpene lactone biosynthetic pathways in Asteraceae. Left, the proposed biosynthetic pathway of general sesquiterpene lactones in Asteraceae is shown. Right, the artemisinic acid biosynthetic pathway in A. annua is shown. FPP, farnesyl diphosphate.

A number of GAS genes have been isolated and characterized in Asteraceae (21–24); however, the biochemical mechanism of germacrene A C12 oxidation and other downstream modifications have not been fully understood. Based on cell-free assays in chicory, it has been proposed that three distinct enzymes, germacrene A C12 hydroxylase (P450), germacrene alcohol dehydrogenase, and germacrene aldehyde dehydrogenase, are involved in the biosynthesis of germacrene A acid in chicory (19). The P450-mediated biosynthesis of costunolide from germacrene A acid has also been demonstrated in chicory extract (20). Nonetheless, the corresponding genes responsible for these reactions have not been identified to date. Recently, the biochemical scenario of such three independent enzymes for germacrene A acid synthesis has been challenged by the discovery of multifunctional amorphadiene oxidase (AMO or CYP71AV1), which can catalyze three-step oxidations of amorphadiene to yield artemisinic acid in Asteraceae plant, Artemisia annua (Fig. 1) (25, 26).

From the perspective of biochemical evolution, the artemisinic acid biosynthesis is a specific evolutionary event that only occurred in a single modern species, A. annua. It can, therefore, be theorized that the biochemistry of artemisinic acid diverged from the more general germacrene A acid biosynthetic pathway. Comparative analysis of artemisinic acid and germacrene A acid biosynthesis would be an attractive model to understand the adaptive evolution of enzymes in terpenoid metabolism.

Germacrene A acid is certainly a necessary chemical for in-depth investigation of STL metabolism in Asteraceae. However, this compound is difficult to obtain because germacrene A acid is a low abundant, transient intermediate in the STL biosynthetic pathway (27), and the chemical synthesis of terpenoid is difficult to achieve. One report showed that a minute amount (2 mg) of germacrene A acid could be purified from 300 g of costus (Saussurea lappa) (27). Thus, microbial de novo production of germacrene A acid using cDNA clones can be a convenient alternative to acquire germacrene A acid.

In this report germacrene A oxidase (GAO) isolated from lettuce (Lactuca sativa) was expressed in an engineered yeast to synthesize germacrene A acid de novo. Furthermore, GAO cDNAs were isolated from several other Asteraceae plants representing major subfamilies, including the phylogenetically basal species Barnadesia spinosa. Resulting biochemical data provided evidence that GAO activity is highly conserved in Asteraceae. In addition, the cross-reactivity of GAOs toward amorphadiene highlighted the evolutionary significance of the catalytic plasticity encoded in GAOs.

EXPERIMENTAL PROCEDURES

Plasmid Construct for Gene Expression

Sequence information at the start and stop codons of LsGAO was obtained from the Compositae Genome Project Data base at the University of California Davis. LsGAO was amplified from the cDNA pool prepared from lettuce leaf by a forward primer, 5′-CGAGGTCTAGAATGGAGCTTTCAATAACCACC-3′, and a reverse primer, 5′-GCCCTCTAGAGCAAAACTCGGTACGAGTAACAAC-3′. The amplified product was digested by XbaI and ligated into the SpeI site of pESC-Ura plasmid. A. annua cytochrome P450 reductase (CPR) in a pESC-Ura plasmid (25) was digested by BamHI and NheI, and the digested fragment was ligated to the corresponding sites in LsGAO::pESC-Ura, resulting in a dual expression plasmid, LsGAO/CPR::pESC-Ura. This plasmid and the previously generated GAS::pESC-Leu plasmid (24) were co-transformed to the EPY300 strain (25, 28). For chemical purification purposes, plasmid stability was enhanced by coding three genes in a single plasmid. This was achieved by amplifying the expression cassette of GAS from the GAS::pESC-Leu plasmid by a forward primer, 5′-GTCAATCACTACGTGAGTACGGATTAGAAGCCGCCGA-3′, and a reverse primer, 5′-GTCAATGCCGGCCTTCGAGCGTCCCAAAACCT-3′. The amplified product was digested by DraIII and NaeI, and the digested fragment was ligated into the corresponding sites of an empty pESC-Leu2d. This DNA manipulation freed two multiple cloning sites for further cloning. Two expression cassettes for LsGAO and CPR were digested from the LsGAO/CPR::pESC-Ura by PacI and ScaI, and the digested fragment was ligated to the corresponding sites of the newly generated GAS::pESC-Leu2d, resulting in the triple expression plasmid named GAS/LsGAO/CPR::pESC-Leu2d. Bioinformatic analyses identified start and stop codons of chicory and sunflower, and their open reading frame sequences were used to design appropriate primers. The Barnadesia clone ordered from the Arizona Genomics Institute at the University of Arizona encoded a full-length cDNA (clone CCHS24399). For costus SlGAO clone isolation, a 1.4-kb fragment of SlGAO was first obtained from costus cDNA using primers designed at the highly conserved domains of other GAOs. The primer pair used was a forward primer, 5′-ACCGTGGCTCAAAGCTCTCAGTC-3′, and a reverse primer, 5′-GACTCCCCATAATCGGTCACATGC-3′. Both 5′- and 3′-rapid amplification of cDNA ends were conducted to determine start and stop codons of SlGAO followed by the recovery of a full-length cDNA. All the isolated GAOs were first cloned to pESC-Leu vector to make translational fusions to the FLAG epitope. For subcloning purposes, HaGAO was amplified using a forward primer, 5′-GCACTAGTATGGAAGTCTCCCTCACCACTTC-3′, and a reverse primer, 5′-CGATACTAGTGCAAAACTTGGTACAAGCATCAA-3′. SlGAO was amplified using a forward primer, 5′-TAATCTAGAATGGAACTCTCCTTCACCACTTCCATTGC-3′, and a reverse primer, 5′-TATTCTAGACGAAAACTAGGTACCAGTACCAAATGAGTC-3′. CiGAO was amplified using a forward primer, 5′-ACGTCTAGAATGGAGCTCTCACTCACTACTTCCA-3′, and a reverse primer, 5′-ACGTCTAGAGCAAAACTTGGTACGAGTATCAATTCGGT-3′. BsGAO was amplified using a forward primer, 5′-ATATCTAGAACCATGGAACTCACTCTCACCACTTCCC-3′, and a reverse primer, 5′-ATACTAGTCGAGCAGAGTTGTTAGCAGTCTTGTAAGCTG-3′. The amplified fragments were digested by XbaI or SpeI and cloned into the SpeI site of pESC-Leu vector. The entire open reading frames in fusion with the FLAG epitope were digested by NotI and PacI, and the LsGAO in the triple expression vector was removed and replaced with the NotI- and PacI-digested GAO open reading frames.

Yeast Culture and Metabolite Sample Preparation

For standard yeast culture, the transgenic yeast strain of interest was inoculated in 3 ml of Synthetic Complete (SC) medium omitting the appropriate amino acids with 2% Glc. The inocula were cultured overnight at 30 °C and 200 rpm. The start culture was diluted 100-fold in SC medium omitting the appropriate amino acids with 1.8% Gal and 0.2% Glc. 100 ml of medium was cultured for metabolite profiling, whereas 1.0–2.5 liters of medium was cultured for chemical purification and identification. Rearranged sesquiterpenoids (costic acids) were extracted and analyzed by GC-MS according to the published methods (28). For buffered neutral culture, culture medium was adjusted to have 100 mm HEPES/NaOH (pH 7.5). After cultivating yeast for 48–72 h at 30 °C, the culture medium was adjusted to pH 6.0 with 2 m HCl, and the medium was extracted with ethyl acetate twice. The ethyl acetate fractions were evaporated in N₂ gas to concentrate samples to 1 ml, and 1 μl was analyzed by GC-MS. For LC-MS analyses, the solvent was replaced with methanol.

Microsome Preparation, Immunoblot Analysis, and in Vitro Enzyme Assay

Microsomes were prepared according to the published protocol (29) except that a micro-beadbeater (Biospec Products, Bartlesville, OK) was used for 90 s with glass beads (500-μm diameter). For immunoblot analysis, microsomal proteins were separated on a 10% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane. The membrane was blocked by 5% nonfat milk in TBST buffer (25 mm Tris-HCl, pH 7.5, 150 mm NaCl, and 0.05% Tween 20) for at least 1 h, incubated with anti-FLAG M2 antibodies (Sigma) in 1:5000 dilution, washed 3 times with TBST, and incubated with goat anti-mouse secondary antibody (GE Healthcare) in a 1:5000 dilution. After washing the membrane three times with TBST, the bound secondary antibodies were detected with ECL Plus detection reagents (GE Healthcare). For in vitro enzyme assay, the protease-deficient Saccharomyces cerevisiae YPL 154C:Pep4 KO strain was transformed with LsGAO/CPR::pESC-Leu2d and CPR::pESC-Leu2d. After 15 h of cultivation in Glc, the yeast culture was shifted to fresh medium with 2% Gal, and the yeasts were further cultivated for 24 h. The in vitro enzyme reactions were carried out in 3 ml of 50 mm HEPES/NaOH (pH 7.5) buffer containing 3 mg of microsomal protein, 200 μm germacrene A, 500 μm NADPH, and an NADPH regeneration system (10 mm Glc 6-phosphate and 3 units of Glc-6-phosphate dehydrogenase). The reaction occurred at 23 °C for 4 h with gentle agitation. The reaction product was acidified with 2 m HCl to pH 6.0 and extracted with ethyl acetate. The extract solvent was then replaced with methanol for LC-MS analysis.

GC-MS Analysis of Sesquiterpenoids

Products obtained by the expression of the GAS/LsGAO/CPR::pESC-Leu2d plasmid were analyzed on an Agilent 6890N gas chromatography system coupled to an Agilent 5975B mass spectrometer. 1-μl samples were injected at a port temperature of 150 °C and analyzed on a DB5-MS column (30 m × 250-μm inner diameter × 0.25-μm film thickness). Helium was used as carrier gas with a constant flow rate of 1 ml min⁻¹. The temperature program was set to 40 °C for 1 min followed by a linear gradient of 10 °C min⁻¹ to 250 °C. For separating α- and β-costic acids, 2-μl samples were analyzed on an Agilent chiral Cyclodex-B column (30 m × 250-μm inner diameter × 0.25-μm film thickness). The temperature program used was 1 min hold at 40 °C, 10 °C min⁻¹ increments from 40 °C to 100 °C, and 3 °C min⁻¹ increments from 100 to 245 °C. Retention indices for methyl esters of α-, β-, and γ-costic acid and ilicic acid were 1807, 1805, 1788, and 1966, respectively, in the DB-5 column and 1914, 1920, 1889, and 2208, respectively, in the Cyclodex-B column. Retention indices values for native forms of α-, β-, and γ- costic acid and ilicic acid were 1873, 1870, 1852, and 2103, respectively, in DB5 column. Cope-rearranged product, elemenic acid, showed a retention index of 1762, and electron impact MS relative ion intensity as follows: M⁺ 234 (1), 81 (100), 67 (49), 91 (38), 105 (34), 53 (33), 121 (29), 133 (18), 147 (17), 177 (17), 189 (14), 219 (8), 161 (9), 207 (4).

NMR Analyses

For costic acids and ilicic acid, NMR spectra were recorded in 3-mm standard NMR tubes on a Varian Unity Inova 500 MHz spectrometer equipped with a 3-mm indirect detection pulsed field gradient probe. The ¹H and ¹³C NMR chemical shifts were referenced to solvent signals at δ_H/C 7.14/127.68 (C₆D₆) relative to tetramethylsilane. One- and two-dimensional homonuclear NMR spectra were measured with standard Varian pulse sequences, and the experiments performed included gCOSY, TOCSY, ROESY, gHSQCAD, and gHMBCAD. Adiabatic broadband and band-selective GHSQCAD and GHMBCAD spectra were recorded using CHEMPACK 4.0 pulse sequences (implemented in Varian Vnmrj 2.1B spectrometer software). For germacrene A acid, ¹H and ¹³C NMR spectra were acquired in 5-mm standard NMR tubes at 400.13 and 100.6 MHz on a Bruker AVANCE 400 Spectrometer equipped with 5-mm inverse probe with triple axis gradients. Chemical shifts (δ) were referenced to internal tetramethylsilane for both ¹³C and ¹H. Spectra were recorded with standard Bruker pulse sequences under Xwinnmr. Experiments performed included one-dimensional proton, one-dimensional ¹³C with proton decoupling, ¹³C attached proton test with proton decoupling, COSY with double quantum filter, TOSCY with 60-ms mixing time, heteronuclear multiple quantum coherence, and heteronuclear multiple bond coherence.

Isolation of Ilicic Acid (4)

The EtOAc extract was concentrated to 100 μl in a vacuum concentrator and separated by TLC on silica gel 60 F₂₅₄ (Merck) with hexane/EtOAC (1:4 v/v) as the solvent. UV 254 nm identified an absorbent band with an R_F value of 0.08 that was eluted from silica with EtOAC and subsequently identified to contain 4 (peak 4) in Fig. 2. Further purification was achieved by using an isocratic HPLC fractionation (P580 Dionex Liquid Chromatography system, Grom-Sil 120 ODS-5 ST column, 5 μm, 4.6 × 250 mm (Grom, Herrenberg, Germany). The mobile phase used was 40% acetonitrile acidified to pH 2.5 with trifluoroacetic acid, and compounds were detected with Dionex DAD UVD340S. Compound 4 eluted at 9.9 min.

FIGURE 2.

Analyses of the metabolites de novo synthesized from transgenic yeast. A, GC-MS chromatographs are shown for the sesquiterpenoids from yeast transformed with the indicated genes. Lines a and b are negative controls, and line c displays the metabolites unique to the yeast transformed with three genes (GAS, GAO, and CPR). Compounds 2 and 3 were not separated by DB-5 MS column but were clearly separated by the chiral column (Cyclodex-B column) as shown in the inset. B, the mass fragmentation patterns of compound 1 to 4 are given. C, proposed acid-induced rearrangements of germacrene A acid to α-, β-, and γ-costic acid and additional modification of costic acids to ilicic acid in yeast culture are shown. The speculated structure of peak 2 as α-costic acid was also given with a question mark. D, cope rearrangement of germacrene A acid to elemenic acid by heat is shown.

Isolation of Costic Acids (1 and 3)

The same TLC separation was conducted as described above. In addition to the ilicic acid band, another band on the TLC plate showing strong absorbance at UV254 with an R_F value of 0.62 was identified to contain 1 and 3 (peaks 1 and 3, respectively) in Fig. 2 by GC-MS analysis. This band was excised, eluted with EtOAC, and further separated by HPLC as described above. Compound 1 (γ-costic acid) was eluted at 9.6 min with a shoulder peak. A separate collection of the shoulder peak and subsequent NMR analysis afforded β-costic acid (3).

Isolation of Germacrene A Acid

For purification of germacrene A acid, the culture medium was extracted with EtOAc, concentrated to 100 μl, and fractionated by a reverse-phase column, Sep-Pak Plus C18 cartridge 55–105-μm column (Waters). Step gradients of elution solvents (water and acetonitrile) starting from 100% water with 10% increments of acetonitrile were used to isolate germacrene A acid. A total of 56 fractions (0.5 ml each) were collected, and the presence of germacrene A acid was visualized as a blue spot on TLC plate using the method described by de Kraker et al. (27). Fractions showing blue spots on TLC were further monitored by HPLC (Waters 279 Separation Module; Waters Nova-Pak C18 column 4 μm, 3.9 × 150 mm; Waters 2996 Photodiode Array Detector with UV wavelength at 194 nm) to distinguish germacrene A acid (13.3 min retention time) from minor amounts of ilicic acid (9.3 min) and costic acids (14.5 min). This separation was achieved with a solvent gradient of 95:5 (A:B) to 20:80 (A:B) over 20 min at 1 ml min⁻¹ and 40 °C column temperature (A: H₂O with 1% acetic acid; B: 100% acetonitrile).

LC-MS Analyses of Sesquiterpenoids

Metabolite mass profiles were generated by an Agilent 1200 Rapid Resolution LC system coupled with an Agilent 6410 MS using 10-μl injections of samples onto a reverse phase C₁₈ column (2.1 × 50 mm, 1.8 μm, Eclipse plus C18 Zorbax) with a solvent gradient of 80:20 (A:B) to 20:80 (A:B) over 12 min at 0.4 ml min⁻¹ at 40 °C column temperature (A: H₂O with 1% acetic acid; B: 100% acetonitrile). The initial 30 s of LC was operated at an isocratic mode with solvent composition 80:20 (A:B). Total ion scans were used in both negative and positive mode, and specific ion masses were selected for further mass analysis. For germacrene A acid detection in B. spinosa, 100 mg of ground fresh tissue (a mixture of flower and leaf that were used for the cDNA library preparation in the Compositae genomics project) was provided from the Center for Genomics and Bioinformatics, Indiana University. The sample was extracted with 5 ml of ethyl acetate, filtered, dried, and dissolved in methanol. The same Rapid Resolution LC program as above was employed using the Zorbax SB-C18 column (2.1 × 30 mm, 3.5 μm).

Phylogenetic Analysis

GAO and AMO amino acid sequences were aligned by ClustalW algorithm. Phylogenetic analysis was performed using the Phylogenetic Analysis Using Parsimony (PAUP) 4.0 software (Sinauer Associates Inc., Sunderland, MA). The first 21 amino acids corresponding to the membrane domain were excluded. Characters were reweighted according to the rescaled consistency index. Parsimony analysis was performed using the tree-bisection-reconnection (TBR) algorithm. 1000 replicates of the bootstrap analysis were performed to evaluate the statistical significance of each node.

Sequence Deposition

The DNA sequences for the GAO genes have been deposited in the GenBank^TM data library under the following accession numbers: GU198171 (L. sativa), GU256644 (C. intybus), GU256645 (S. lappa), GU256646 (Helianthus annuus), and GU256647 (B. spinosa).

RESULTS

Isolation of GAO and Pathway Reconstitution in Engineered Yeast

Lettuce (L. sativa) accumulates STLs inside its laticiferous cells in stems and leaves (30), and hence, we first aimed at functionally identifying a gene encoding the enzyme for germacrene A oxidation from lettuce. A bioinformatics screening of the lettuce express sequence tag data base using A. annua AMO (or CYP71AV1) identified a gene highly homologous to AMO (25, 26). PCR amplification using primers designed at the start and stop codons of the identified ESTs allowed us to isolate a full-length gene from lettuce leaf cDNA. The isolated cDNA encodes a polypeptide of 488 amino acids with a predicted molecular mass of 54.9 kDa. The deduced amino acid sequence from this gene showed 86.7% identity to that from A. annua AMO. This P450 gene was designated as germacrene A oxidase (GAO) based on its catalytic property (see below).

To assess the enzymatic activity of GAO, yeast strain EPY300 engineered to produce a markedly increased level of farnesyl diphosphate (an immediate precursor of germacrene A) served as a platform strain. The production of the hydrocarbon germacrene A in the EPY300 strain expressing previously characterized lettuce GAS has been demonstrated (21, 24). For in vivo catalytic coupling of GAS and GAO, open reading frames of GAO with a FLAG epitope tag and A. annua CPR with a cMyc epitope tag under Gal10 and Gal1 promoters, respectively, were placed in the pESC-URA dual expression vector. This plasmid and previously generated GAS::pESC-Leu were co-transformed in EPY300, resulting in simultaneous expression of GAS, GAO, and CPR in farnesyl diphosphate abundant cells. Upon induction of transgenes, the presence of GAO and CPR recombinant proteins was confirmed by immunoblot analysis with FLAG and cMyc antibodies (data not shown).

Terpenoids were extracted, derivatized as described previously (25), and subjected to the GC-MS analysis. Unexpectedly, four novel compounds that were not present in the yeast expressing either GAS or GAO/CPR were identified in the yeast expressing GAS, GAO, and CPR (Fig. 2A). The four compounds (1, 2, 3, and 4) showed the same parental mass of m/z 248 (methyl ester form) but distinct fragmentation patterns in electron impact MS (Fig. 2B). The electron impact MS analysis of their native (non-methylated) forms revealed an identical parent mass of m/z 234 in each case. The observation of m/z 248 (methyl ester) and 234 (native form) suggested that the hydrocarbon germacrene A (M_r 204) is sequentially oxidized three times by GAO as is the case for the biosynthesis of artemisinic acid by AMO (Fig. 1). (−/+)LC ESI-MS analyses of 1, 2, and 3 displayed their [M-H]⁻ ions at m/z 233 and their [M+H]⁺ ions at m/z 235, confirming the above findings. However, the (−)ESI-MS of 4 revealed a quasi-molecular ion [M-H]⁻ at m/z 251. In the (+)LC ESI-MS, compound 4 displayed an abundant ion at m/z 235 that appears to be derived from the loss of H₂O from the unobserved parent ion [M+H]⁺ at m/z 253. In summary, the expression of lettuce GAO with GAS and CPR in EPY300 led to the production of four new compounds, and their MS analyses showed that 1, 2, and 3 have parent masses of 234, but 4 has a mass of 252.

Structure Analyses of Products from Yeast Culture under Acidic Conditions

We pursued the chemical purification of the new compounds for structural analysis. Before purification, plasmid stabilities were enhanced by coding all three genes (GAS, GAO, and CPR) in a single plasmid in pESC-Leu2d vector. The EPY300 strain transgenic for this new construct was cultured in 2.5 liters, and the newly synthesized compounds were purified through TLC and HPLC. Compound 1, 3, and 4 were successfully purified and subjected to extensive one- and two-dimensional NMR studies (see “Experimental Procedures” for details). The structures of compounds 1 and 4 were unambiguously determined to be γ-costic acid and ilicic acid, respectively (supplemental Table 1, Fig. 2C). Due to a limited amount of 3, the ¹H NMR and indirectly detected ¹³C signals of peak 3 could only be partially assigned. Nevertheless, structure elucidation indicated this compound to be β-costic acid. Compounds 1, 3, and 4 represent cyclization products of germacrene A acid (Fig. 2C). Although purification of 2 was not achieved, one can assume that this compound could be α-costic acid, the third form of cyclized product from germacrene A acid, in accordance with the observed molecular mass of 234 atomic mass units. Compound 4 (ilicic acid) is believed to be derived from hydration of the double-bonds in costic acids.

Altered Metabolite Profile from the pH Adjusted Yeast Cultivation

We speculated that the cyclization of germacrene A acid occurs in the course of de novo germacrene A acid synthesis in an acidic culture condition. When pH values of the yeast culture were monitored, the yeast medium (initially pH 6) became very acidic after a 48-h cultivation (pH ∼ 3). To overcome the medium acidity, the pH was stabilized by supplementing the medium with 100 mm HEPES buffer (pH 7.5). In the buffered medium, the pH of the medium decreased to 6 after a 48-h cultivation. In the GC-MS analysis of the metabolites, the four peaks identified in the acidic condition almost disappeared, and instead a very broad, new peak with m/z 234 (without methylation) appeared (Fig. 3A, top). The broad peak was also observed previously when the hydrocarbon germacrene A was subjected to GC-MS analysis (27). This broadening was attributed to the on-column heat-induced Cope rearrangement of germacrene A into faster migrating β-elemene.

FIGURE 3.

GC-MS and LC-MS analyses of sesquiterpenoids from yeast expressing GAS, GAO, and CPR in buffered neutral culture. A, GC-MS chromatographs of the sesquiterpenoid products are shown. Inlet temperatures and chemical treatment are shown in front of the chromatograms. TCA, trichloroacetic acid; bracket, α-, β-, and γ-costic acids; asterisk, germacrene A acid; arrow, heat-induced rearranged product of germacrene A acid. B, shown are LC-MS ion traces from negative ions of m/z 171, 233, and 251. m/z 171 corresponds to the internal standard (IS), decanoic acid, with a retention time of 7.68 min; m/z 233 corresponds to germacrene A acid (*), with a retention time of 8.41 min and α-, β-, γ-costic acids (in the bracket) with a retention time of 9.02 min and 9.17 min (α- and β-costic acids co-migrate at 9.02 min); m/z 251 corresponds to ilicic acid (triangle), with retention time of 5.38 min. C, LC-MS chromatographs (negative m/z 233) of in vitro enzyme assay products are shown.

Before chemical purification, we evaluated if this new compound (broad peak) was indeed the precursor of the costic acids detected previously. To assess this possibility, the compound from neutral cultivation was treated with trichloroacetic acid. When the acidified samples were reanalyzed by GC-MS, the three early eluting peaks identified as costic acids were clearly detected with a complete disappearance of the broad peak (Fig. 3A, middle). In addition, when the compound from the neutral cultivation was injected at a 330 °C GC inlet temperature (previously 150 °C), the minute peak preceding the broad peak increased by 19-fold (Fig. 3A, arrow). GC-MS analysis suggested that the fast-migrating compound was β-elemenic acid (heat-induced product), in agreement with the published data (27) (Fig. 2D, see “Experimental Procedures” for the full MS spectrum). In LC-MS analysis, the samples prepared from non-buffered medium showed the presence of costic acids (m/z 233) and ilicic acid (m/z 251) with a negligible amount of putative germacrene A acid (m/z 233) (Fig. 3B, top). However, in the samples prepared from the buffered medium, the amount of costic acids and ilicic acid decreased to 15 and 34%, respectively, relative to their levels in non-buffered culture. Concomitantly, a new peak of m/z 233 at a slightly earlier retention time than costic acids increased by 44-fold (Fig. 3B, bottom). These GC- and LC-MS results showed that simply cultivating the transgenic yeast in buffered medium dramatically alters the sesquiterpenoid profile from aberrant rearranged products (costic acids and ilicic acid) to a new peak with m/z 233 in negative ions.

Structure Elucidation of Germacrene A Acid

4 mg of the new compound was purified from a 1-liter neutralized medium by solid-phase extraction column (C18 reverse phase). High-resolution Fourier transform ion cyclotron resonance MS showed [M-H]⁻ ions at m/z 233.1547 corresponding to the molecular mass for germacrene A acid (calculated mass for C₁₅H₂₁O₂⁻, 233.1547). Based on the complete signal assignment of the compound by standard one- and two-dimensional NMR experiments (see “Experimental Procedures,” supplemental Table 1), the compound was determined to be germacrene A acid. The extensive peak broadening observed in the ¹H NMR spectra was attributable to the presence of different conformers as were found in the NMR analysis of germacrene A (31). In this case, the broad signals are due to intermediate exchange on the NMR time scale by the various conformers. The in vitro activity of the recombinant GAO was further confirmed using the microsomes isolated from the yeast expressing GAO and CPR. In a carefully pH-controlled experiment (pH was >6.0 in all experimental conditions except for the 12-min HPLC run), (−)LC-MS analysis showed that 233 ions for germacrene A acid were clearly detectable, but the other 233 ions from costic acids could not be detected (Fig. 3C). The in vivo, in vitro, and chemical analysis data demonstrated that GAO encodes an enzyme for the conversion of germacrene A to germacrene A acid.

GAO Activity Is Highly Conserved in Asteraceae

The in vivo system was used to trace the advent of GAO in various Asteraceae plant species. To study the evolutionary lineage of GAO, a combination of express sequence tag-mining, PCR on conserved regions, and rapid amplification of cDNA end methods was used to isolate full-length clones of AMO/GAO homologs from selected Asteraceae plants. Sunflower (H. annuus), chicory (Cichorium intybus), and costus (S. lappa) were chosen as the representatives of Asteroideae, Cichorioideae, and Carduoideae, respectively; these are the core subfamilies that constitute 95% of all Asteraceae. B. spinosa was selected as the representative of the phylogenetic base lineage, subfamily Barnadesioideae (Fig. 4A) (9).

FIGURE 4.

Phylogenetic and sequence analyses of GAOs from various Asteraceae plants. A, the Asteraceae phylogeny simplified from the figure by Panero and Funk (9) is shown. Asterisks indicate the four subfamilies where GAOs were isolated, and the parentheses indicate specific species names. B, the phylogenetic tree of AMO/GAO in Asteraceae is shown. HPO and EAH used as outgroups are Hyoscyamus muticus premnaspirodiene oxygenase and tobacco 5-epi-aristolochene dihydroxylase, respectively. Bootstrap values were shown in percentage values from 1000 replicates. The bracket indicates the GAO clade that is clearly distinguished from BsGAO and AMO. C, alignment of deduced amino acids from GAOs and AMO is shown. Amino acid sequences were obtained from cDNAs deposited at the NCBI. AMO, amorphadiene oxidase from A. annua (DQ268763 or DQ315671); LsGAO, germacrene A oxidase from L. sativa (GU198171) or from C. intybus (Ci; GU256644), S. lappa (Sl; GU256645), H. annuus (Ha; GU256646), and B. spinosa (Bs; GU256647). Stars indicate the residues conserved in GAO but different in AMO. Circled marks indicate the residues unique in AMO but not conserved in GAO. The alignment is shaded to a 50% consensus. Dark and light shading indicate identical and similar residues, respectively.

The enzymatic activities of the isolated GAO clones were examined in the yeast in vivo system by co-expressing GAS and CPR. In the (−)LC-MS analyses, the 233 ions from the germacrene A acid were detected in all samples tested, and quantitative analyses showed that comparable amounts of germacrene A acid were synthesized in yeast expressing either a lettuce, chicory, costus, or Barnadesia clone (Fig. 5A). Although the germacrene A acid from the yeast-expressing sunflower clone was an order of magnitude lower (10–15-fold) than that of the others, semiquantitative immunoblot analysis of isolated microsomes revealed that the sunflower recombinant protein was at least 10-fold lower than that from the other clones (Fig. 5B). Hence, the sunflower enzyme also resulted in a comparable level of catalytic activity.

FIGURE 5.

Biochemical analyses of GAOs from various Asteraceae plants. A, shown is LC-MS chromatography at selective (−)233 ion for germacrene A acid and (−)171 ion for the internal standard (IS), decanoic acid. The arrow is germacrene A acid, and the arrowhead is the internal standard. Yields of germacrene A acid from four independent transformants were given at the start of the chromatographs (mean ± S.D.). B, immunoblot analysis of recombinant GAOs is shown. FLAG antibodies were used to detect the epitope tags at the C termini of GAOs. Loaded microsome amounts are indicated. Ls, L. sativa; Ci, C. intybus; Sl, S. lappa; Bs, B. spinosa; Ha, H. annuus. C, LC-MS chromatography of B. spinosa extract at (−)233 ion was shown in the top line. Standards for germacrene A acid (GAA) and costic acids are shown in the second and third line, respectively.

Although the involvement of germacrene A acid is apparent in sesquiterpenoid metabolisms of sunflower, costus, chicory, and lettuce on the basis of chemical structures and/or in vitro enzyme assays (19, 20, 24, 27), the biochemical relevance of germacrene A acid in B. spinosa has not been addressed. Thus, the presence of germacrene A acid was examined by (−)LC-SIM (selective ion mode) from the ethyl acetate extract of B. spinosa (leaf and flower). In the (−)233 ion monitoring, germacrene A acid together with costic acids was detected at the concentration of 0.013% (weight/fresh weight) (Fig. 5C). This result showed that germacrene A acid is present in B. spinosa most likely as an intermediate. Collectively, these results demonstrated that GAOs and their corresponding enzymatic activities (three consecutive oxidations of germacrene A C12) are highly conserved at the phylogenetic basal clade of Asteraceae (i.e. Barnadesioideae) and retained in three major subfamilies of Asteraceae.

Phylogenetic Analysis of GAO and AMO

The deduced amino acids from these clones shared significant sequence identities ranging from 78.4 to 97.3% (Fig. 4C). Interestingly, AMO shared a higher degree of homology to the GAOs from lettuce, chicory, sunflower, and costus (84.2–86.8%) than BsGAO did with these GAOs (79.6–82.6%). To obtain a better insight into the AMO and GAO evolution, a phylogenetic tree was reconstructed from AMO and five GAOs using two cytochrome P450s for sesquiterpene oxidations as outgroups (32, 33). The phylogenetic analysis showed that AMO forms a distinctive node from the major GAO clade within the lineage originated from the Barnadesia GAO (Fig. 4B). Although both sunflower and Artemisia belong to the same Asteroideae subfamily, sunflower GAO constitutes part of the major GAO clade (Fig. 4B, bracket) that can be distinguished from AMO by a strong statistical support. This data implied that AMO in A. annua recently underwent a specific biochemical microevolution that was not mirrored by the overall speciation patterns of the subfamily Asteroideae.

Cross-reactivities of AMO and GAO

In the sequence alignment, after excluding the membrane-bound domain, only 21 amino acids in AMO were different from the conserved residues in GAO, and an additional 7 amino acids in AMO were unique residues that were not present among the variable residues of other GAOs (Fig. 4C). Such high sequence homology between AMO and GAOs intrigued the substrate specificities of these two types of enzymes and the molecular evolution of AMO in Asteraceae. To address these questions, cross-reactivities of GAOs toward amorphadiene and AMO toward germacrene A were investigated using an in vivo system. Co-expression plasmids for the swapped gene pairs (GAS/AMO or ADS/GAOs) were constructed in the CPR::pESC-Leu2d plasmid and were transformed to the EPY300 strain. When co-expressed, the GAS and AMO gene pair displayed negligible activity (0.04%) for germacrene A acid synthesis, relative to the activity detected from the native enzyme pair, GAS and GAO (Table 1). However, when co-expressed with ADS, all five GAOs from various Asteraceae plants displayed ∼10²–10³-fold higher relative activities (5–40%) for artemisinic acid synthesis than that from the GAS and AMO pair. These results suggest that AMO appeared to have lost its ancestral capacity to oxidize germacrene A during its evolutionary path, but the GAOs (predecessors of AMO) from various Asteraceae species including B. spinosa possess the catalytic potential (or plasticity) to oxidize amorphadiene.

TABLE 1.

Germacrene A acid or artemisinic acid production from the transgenic yeast in which terpene synthase (ADS or GAS) and cytochrome P450 (AMO or GAO) were co-expressed

Sesquiterene synthase	Cytochrome P450	Germacrene A acid or artemisinic acid yield	Relative activitya
		μg ml−1	%
Lettuce GAS	Lettuce GAO	22.0 ± 5.3b	100
Lettuce GAS	Artemisia AMO	0.003 ± 0.001b	0.04 ± 0.02
Artemisia ADS	Artemisia AMO	27.0 ± 2.0c	100
Artemisia ADS	Lettuce GAO	9.3 ± 1.3c	40 ± 16
Artemisia ADS	Chicory GAO	11.4 ± 1.7c	36 ± 11
Artemisia ADS	Costus GAO	4.8 ± 0.8c	25 ± 13
Artemisia ADS	Sunflower GAO	0.4 ± 0.1c	7 ± 1
Artemisia ADS	Barnadesia GAO	3.7 ± 0.2c	5 ± 2

^a Natural pairs of sesquiterpene synthase and P450 (e.g., lettuce GAS and lettuce GAO or Artemisia ADS and Artemisia AMO) were set as 100% activities. The production levels of germacrene A acid or artemisinic acid were normalized by relative P450 abundance estimated by immunoblot analysis. Values (mean ± S.D.) were obtained from at least three independent transformants.

^b Germacrene A acid measurement.

^c Artemisinic acid measurement.

DISCUSSION

In this work GAO catalytic activity was unambiguously determined to be germacrene A oxidase using the pathway reconstitution in yeast system in vivo and a standard in vitro assay. Lack of substrates and chemical standards often interfere with the progress of natural product biochemistry. Moreover, the instability of transient intermediates complicates biochemical studies. Both germacrene A and germacrene A acid are unstable, transient intermediates in the STL metabolic pathway in plants, and thus, the acquisition of these compounds by purification from plant sources is difficult. In the absence of an authentic standard (germacrene A acid), the enzymatic production of germacrene A acid by in vitro assays and subsequent purification to the level adequate for standard NMR analyses would be technically challenging. The results presented here demonstrated that pathway reconstruction in yeast is a convenient alternative for the characterization of gene function, mass production of natural products for chemical analysis, and substrate preparation for further biochemical studies.

The β- and γ-costic acids identified in this study are known natural products that can be derived from germacrene A acid in vitro (27). It was, therefore, not surprising to detect these products in acidic yeast culture conditions, but the identification of ilicic acid was not expected from the yeast system. Ilicic acid and its hydroxyl derivatives have been reported as natural products in Asteraceae plants (34–36), and their anti-tumor and anti-inflammatory activities have been suggested (34, 37). The formation of ilicic acid from costic acids could be mediated by a nonspecific yeast enzyme(s) due to the stereospecific hydroxyl group at C4 position, but the nature of this chemical conversion was not further pursued in this study.

The origin and phylogeny of Asteraceae has been a subject of intensive systematic studies for the last 30 years. Based on fossil records and molecular systematic data, it is believed that Asteraceae originated about 30 million years ago in South America and radiated rapidly in all worlds except Antarctica (9, 38, 39). In 1987 it was discovered that a 22-kb DNA inversion in the chloroplastic genome shared by all Asteraceae plants is lacking in the Barnadesioideae group, and thereafter this inversion data has served as key molecular evidence to support an ancient split between Barnadesioideae and the rest of the Asteraceae (39).

The yeast in vivo reconstitution results in this study clearly demonstrated that GAO and its activity are highly conserved in five plant species representing the major subfamilies and the phylogenetic base of Asteraceae. In particular, GAO gene and its three sequential oxidation activity in the ancient surviving species B. spinosa indicate the occurrence of GAO at the beginning of Asteraceae evolution and subsequent retention of GAO and its enzyme activity in the major subfamilies for more than 30 million years. Considering that the genes in plant secondary metabolism, such as cytochrome P450s of the CYP71 family, are often rapidly diversified for neofunctionalization, the conservation of GAO in Asteraceae suggests that GAO offers increased fitness to plants in this family. Because various STLs have been isolated from sunflower, lettuce, chicory, and costus (30, 40, 41), it is tempting to speculate that STLs synthesized through GAO activity are the chemical entities responsible for the enhanced fitness of Asteraceae. However, phytochemical studies of Barnadesioideae have only been limited to flavonoids to date (42, 43), and further investigation is required to correlate GAO to STLs in Barnadesioideae.

A. annua is the only plant species known to produce artemisinic acid and artemisinin. Specific evolutionary events seem to occur to drive the advent of ADS in A. annua, and GAO may have subsequently evolved to accommodate a new sesquiterpene hydrocarbon (amorphadiene) for the biosynthesis of artemisinic acid. From this study, AMO compromised its activity for germacrene A oxidation on its way to acquire amorphadiene oxidation activity. However, all the GAOs displayed noticeable activities to oxidize amorphadiene even though these enzymes have not been exposed to amorphadiene in nature. The observed activity of GAOs for amorphadiene is in agreement with the general hypothesis that primitive enzymes have broader substrate specificities, thereby providing necessary plasticity to evolve into more specific present-day enzymes (44–46). According to this hypothesis, selective advantages may be provided by the pre-existing AMO activity in GAO, which allows a head start for GAO (evolutionary precursor of AMO) to more easily reach the catalytic threshold required for evolutionary selection.

In directed evolution studies in vitro, enzyme evolvability toward non-natural substrates with decreased activity for the native substrate has been shown a number of times (45). Although rare in nature, a similar enzyme evolution is seen in the homologous enzymes, deoxyhypusine synthase and homospermidine synthase (47). The primordial deoxyhypusine synthase conserved in all eukaryotes can utilize the substrate of homospermidine synthase, which has evolved from deoxyhypusine synthase in some plants for pyrrolizidine alkaloid biosynthesis. However, the later evolved enzyme, homospermidine synthase, cannot utilize deoxyhypusine synthase substrate, closely mirroring the results shown in this study.

In most plants in Asteraceae, diversification and specification of primitive GAO may not be favored due to the importance of the compounds derived from germacrene A acid unless GAO is pressured to evolve by alteration of its substrate structure (e.g. amorphadiene in A. annua). Therefore, the naturally occurring gene pairs, GAS/GAO and ADS/AMO, evolved for the optimal catalytic coupling, can be an excellent model system to infer the co-evolutionary process of the enzymes in a linear metabolic pathway.

Currently, the enzymatic conversion of amorphadiene to artemisinic acid is limited and incomplete in metabolically engineered E. coli and yeast (28, 48). In this respect, the several GAOs identified here and other undiscovered Asteraceae GAOs could serve as ideal molecular templates to create a catalytically more efficient amorphadiene oxidase through in vitro evolution and engineering methods.