Identification and Functional Characterization of Monofunctional ent-Copalyl Diphosphate and ent-Kaurene Synthases in White Spruce Reveal Different Patterns for Diterpene Synthase Evolution for Primary and Secondary Metabolism in Gymnosperms

The biosynthesis of the tetracyclic diterpene ent-kaurene is a critical step in the general (primary) metabolism of gibberellin hormones. ent-Kaurene is formed by a two-step cyclization of geranylgeranyl diphosphate via the intermediate ent-copalyl diphosphate. In a lower land plant, the moss Physcomitrella patens, a single bifunctional diterpene synthase (diTPS) catalyzes both steps. In contrast, in angiosperms, the two consecutive cyclizations are catalyzed by two distinct monofunctional enzymes, ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS). The enzyme, or enzymes, responsible for ent-kaurene biosynthesis in gymnosperms has been elusive. However, several bifunctional diTPS of specialized (secondary) metabolism have previously been characterized in gymnosperms, and all known diTPSs for resin acid biosynthesis in conifers are bifunctional. To further understand the evolution of ent-kaurene biosynthesis as well as the evolution of general and specialized diterpenoid metabolisms in gymnosperms, we set out to determine whether conifers use a single bifunctional diTPS or two monofunctional diTPSs in the ent-kaurene pathway. Using a combination of expressed sequence tag, full-length cDNA, genomic DNA, and targeted bacterial artificial chromosome sequencing, we identified two candidate CPS and KS genes from white spruce (Picea glauca) and their orthologs in Sitka spruce (Picea sitchensis). Functional characterization of the recombinant enzymes established that ent-kaurene biosynthesis in white spruce is catalyzed by two monofunctional diTPSs, PgCPS and PgKS. Comparative analysis of gene structures and enzyme functions highlights the molecular evolution of these diTPSs as conserved between gymnosperms and angiosperms. In contrast, diTPSs for specialized metabolism have evolved differently in angiosperms and gymnosperms.Conifers (Coniferophyta) are well known for producing an abundant and diverse assortment of oleoresin diterpenoids, predominantly in the form of diterpene resin acids from specialized (or secondary) metabolism, that play roles in conifer defense (Trapp and Croteau, 2001a; Keeling and Bohlmann, 2006a; Bohlmann, 2008) and are an important source of biomaterials (Bohlmann and Keeling, 2008). Several conifer diterpene synthases (diTPSs) that biosynthesize these compounds have been functionally characterized (Stofer Vogel et al., 1996; Peters et al., 2000; Martin et al., 2004; Keeling and Bohlmann, 2006b; Ro and Bohlmann, 2006). The formation of diterpene resin acids of conifer specialized metabolism parallels the formation of ent-kaurenoic acid in the biosynthesis of the gibberellin diterpenoid phytohormones (Fig. 1; Keeling and Bohlmann, 2006a; Yamaguchi, 2008). In gibberellin biosynthesis, geranylgeranyl diphosphate (GGPP) is cyclized by diTPS activity to ent-copalyl diphosphate (ent-CPP), and the ent-CPP is further cyclized by diTPS activity to ent-kaurene. A cytochrome P450 (P450)-dependent monooxygenase (CYP701) oxidizes ent-kaurene to ent-kaurenoic acid (Davidson et al., 2006), paralleling the activity of a P450 (CYP720B1) that oxidizes abietadiene to abietic acid in conifer diterpene resin acid biosynthesis (Ro et al., 2005). Other P450s further functionalize ent-kaurenoic acid to form the biologically active gibberellins. Surprisingly, no conifer diTPS involved in the general (or primary) metabolism of gibberellins has been reported to date, while metabolite profiles of gibberellins have been well characterized in conifers for their role in flowering (Moritz et al., 1990).Open in a separate windowFigure 1.Comparison of the biosynthesis of gibberellins, as it is known in angiosperm and lower plants, with the biosynthesis of diterpene resin acids in conifers, a large group of gymnosperm trees. In conifers, the formation of diterpene resin acids involves bifunctional diTPS (e.g. abietadiene synthase) for the stepwise cyclization of GGPP into diterpenes such as abietadiene via a copalyl diphosphate intermediate that moves between the two active sites of the bifunctional diTPS (Peters et al., 2001). The products of the diTPS are subsequently oxidized by P450 to the resin acids. In contrast, gibberellin biosynthesis in angiosperms requires two monofunctional diTPSs to convert GGPP into ent-kaurene, which is subsequently modified by P450s. The two monofunctional diTPSs in angiosperm gibberellin biosynthesis are CPS and KS. In the lower plant P. patens, the CPS and KS activities are combined in a bifunctional diTPS similar to the bifunctional diTPS in conifer diterpene resin acid biosynthesis. Prior to this work, to our knowledge, it was not known if the formation of gibberellins in a gymnosperm involves two monofunctional diTPSs, as in angiosperms, or a bifunctional diTPS, as in gymnosperm diterpene resin acid biosynthesis and in P. patens gibberellin biosynthesis. (Figure adapted from Keeling and Bohlmann [2006a].)In the fungi Gibberella fujikuroi (Toyomasu et al., 2000) and Phaeosphaeria species L487 (Kawaide et al., 1997) and in the primitive land plant Physcomitrella patens (Bryophyta; Hayashi et al., 2006; Anterola and Shanle, 2008), the formation of ent-kaurene from GGPP is catalyzed by bifunctional diTPS enzymes. These enzymes contain two active sites. The N-terminal active site domain harbors a conserved DXDD motif and catalyzes the protonation-initiated cyclization of GGPP to ent-CPP (Prisic et al., 2007). In the C-terminal active site domain, a conserved DDXXD motif is essential for the diphosphate ionization-initiated cyclization of ent-CPP to ent-kaurene (Christianson, 2006). The presence of two active sites with their characteristic DXDD and DDXXD motifs resembles the structure of conifer bifunctional diTPSs in specialized metabolism of diterpene resin acid biosynthesis (Fig. 1), such as the grand fir (Abies grandis) abietadiene synthase (AgAS) and Norway spruce (Picea abies) levopimaradiene/abietadiene synthases (PaLAS; Peters et al., 2001; Martin et al., 2004; Keeling and Bohlmann, 2006a). In contrast, the formation of ent-kaurene from GGPP in angiosperms is catalyzed by two separate monofunctional enzymes, one with only the DXDD motif and having ent-copalyl diphosphate synthase (ent-CPS) activity and the other with only the DDXXD motif and having ent-kaurene synthase (ent-KS) activity (Yamaguchi, 2008).A previously published model for the evolution of plant diTPS (Trapp and Croteau, 2001b) suggests that genes encoding the monofunctional CPS and KS enzymes known in angiosperms originated by gene duplication and subfunctionalization (Lynch and Force, 2000) of an ancestral bifunctional CPS/KS gene that may have been similar to the gene for the CPS/KS enzyme of the moss P. patens. The same model also suggests that genes for diTPSs of gymnosperm specialized diterpene resin acid metabolism arose from duplication and subsequent neofunctionalization of an ancestral bifunctional diTPS of the gibberellin pathway (Trapp and Croteau, 2001b). The pathways to specialized oleoresin diterpenes existed in ancient plants prior to the differentiation of gymnosperms and angiosperms (Bray and Anderson, 2009). Vascular plants split from nonvascular plants approximately 500 million years ago, and angiosperms split from gymnosperms approximately 300 million years ago (Palmer et al., 2004). As there has been no report to date of genes involved in gibberellin biosynthesis in gymnosperms, it remains unresolved and cannot be predicted whether conifers have a bifunctional CPS/KS for the formation of ent-kaurene similar to the primitive land plant P. patens and paralleling the diTPSs for conifer specialized diterpene resin acid biosynthesis or whether they have separate monofunctional CPS and KS enzymes, as is the case in angiosperms.In this study, we made use of the extensive EST resources for spruce species (Pavy et al., 2005; Ralph et al., 2008), combined with isolation and sequencing of full-length cDNAs, genomic (g)DNA, and targeted bacterial artificial chromosome (BAC) clones, as well as enzyme assays with recombinant proteins to search for, and functionally characterize, possible monofunctional or bifunctional diTPS for ent-kaurene biosynthesis in a gymnosperm. In summary, we successfully isolated and characterized monofunctional ent-CPS (PgCPS) and ent-KS (PgKS) from white spruce (Picea glauca) and isolated orthologous cDNAs from Sitka spruce (Picea sitchensis). Comparison of enzyme functions and gene structures support common ancestry but different routes of evolution of monofunctional and bifunctional diTPS in conifer general and specialized metabolism, respectively.     

Structural and mechanistic insights into the precise product synthesis by a bifunctional miltiradiene synthase

Selaginella moellendorffii miltiradiene synthase (SmMDS) is a unique bifunctional diterpene synthase (diTPS) that catalyses the successive cyclization of (E,E,E)-geranylgeranyl diphosphate (GGPP) via (+)-copalyl diphosphate (CPP) to miltiradiene, which is a crucial precursor of important medicinal compounds, such as triptolide, ecabet sodium and carnosol. Miltiradiene synthetic processes have been studied in monofunctional diTPSs, while the precise mechanism by which active site amino acids determine product simplicity and the experimental evidence for reaction intermediates remain elusive. In addition, how bifunctional diTPSs work compared to monofunctional enzymes is attractive for detailed research. Here, by mutagenesis studies of SmMDS, we confirmed that pimar-15-en-8-yl⁺ is an intermediate in miltiradiene synthesis. Moreover, we determined the apo-state and the GGPP-bound state crystal structures of SmMDS. By structure analysis and mutagenesis experiments, possible contributions of key residues both in class I and II active sites were suggested. Based on the structural and functional analyses, we confirmed the copal-15-yl⁺ intermediate and unveiled more details of the catalysis process in the SmMDS class I active site. Moreover, the structural and experimental results suggest an internal channel for (+)-CPP produced in the class II active site moving towards the class I active site. Our research is a good example for intermediate identification of diTPSs and provides new insights into the product specificity determinants and intermediate transport, which should greatly facilitate the precise controlled synthesis of various diterpenes.     

Mono and diterpene production in Escherichia coli

Mono- and diterpenoids are of great industrial and medical value as specialty chemicals and pharmaceuticals. Production of these compounds in microbial hosts, such as Escherichia coli, can be limited by intracellular levels of the polyprenyl diphosphate precursors, geranyl diphosphate (GPP), and geranylgeranyl diphosphate (GGPP). To alleviate this limitation, we constructed synthetic operons that express three key enzymes for biosynthesis of these precursors: (1). DXS,1-deoxy-d-xylulose-5-phosphate synthase; (2). IPPHp, IPP isomerase from Haematococcus pluvialis; and (3). one of two variants of IspA, FPP synthase that produces either GPP or GGPP. The reporter plasmids pAC-LYC and pACYC-IB, which encode enzymes that convert either FPP or GGPP, respectively, to the pigment lycopene, were used to demonstrate that at full induction, the operon encoding the wild-type FPP synthase and mutant GGPP synthase produced similar levels of lycopene. To synthesize di- or monoterpenes in E. coli using the GGPP and GPP encoding operons either a diterpene cyclase [casbene cyclase (Ricinus communis L) and ent-kaurene cyclase (Phaeosphaeria sp. L487)] or a monoterpene cyclase [3-carene cyclase (Picea abies)] was coexpressed with their respective precursor production operon. Analysis of culture extracts or headspace by gas chromatography-mass spectrometry confirmed the in vivo production of the diterpenes casbene, kaur-15-ene, and kaur-16-ene and the monoterpenes alpha-pinene, myrcene, sabinene, 3-carene, alpha-terpinene, limonene, beta-phellandrene, alpha-terpinene, and terpinolene. Construction and functional expression of GGPP and GPP operons provides an in vivo precursor platform host for the future engineering of di- and monoterpene cyclases and the overproduction of terpenes in bacteria.     

构建酿酒酵母工程菌合成香紫苏醇

香紫苏醇是一种来源于植物的双环二萜醇,常用于香味成分且具有重要生物学活性。为实现香紫苏醇的微生物生产,以酿酒酵母为宿主,表达焦磷酸赖百当烯二醇酯合酶和香紫苏醇合酶,构建香紫苏醇的人工生物合成途径。发现过表达前体代谢关键酶、蛋白质融合增强底物通道效应及去除异源蛋白信号肽等,有利于香紫苏醇合成。在摇瓶培养条件下,组合优化得到的工程菌株S6的香紫苏醇产量达到8.96 mg/L。研究结果对其他萜类化合物的异源生物合成具有参考价值。     

Molecular cloning and functional expression of geranylgeranyl pyrophosphate synthase from <Emphasis Type="Italic">Coleus forskohlii</Emphasis>Briq

Background  

Isopentenyl diphosphate (IPP), a common biosynthetic precursor to the labdane diterpene forskolin, has been biosynthesised via a non-mevalonate pathway. Geranylgeranyl diphosphate (GGPP) synthase is an important branch point enzyme in terpenoid biosynthesis. Therefore, GGPP synthase is thought to be a key enzyme in biosynthesis of forskolin. Herein we report the first confirmation of the GGPP synthase gene in Coleus forskohlii Briq.     

Mutational analysis of white spruce (Picea glauca) ent-kaurene synthase (PgKS) reveals common and distinct mechanisms of conifer diterpene synthases of general and specialized metabolism

Conifer diterpene synthases (diTPSs) catalyze the multi-step cycloisomerization of geranylgeranyl diphosphate, or copalyl diphosphate, to a variety of diterpenes in general (i.e., primary) and specialized (i.e., secondary) metabolism. Despite their functional diversity, the known conifer diTPSs are structurally closely related, with variations in three conserved domains, α, β and γ. The catalytic specificity of conifer class I and class I/II diTPSs is predominantly determined by the protein environment of the C-terminal class I active site through stabilization of common and unique carbocation intermediates. Using the crystal structure of Taxus brevifolia taxadiene synthase as template, comparative modeling and mutagenesis of the class I diTPS ent-kaurene synthase from Picea glauca (PgKS) was performed to elucidate the catalytic specificity of PgKS relative to spruce diTPSs of specialized metabolism. N-terminal truncations demonstrated a role for the βγ domain in class I enzyme activity for PgKS, facilitating the closure of the class I active site upon substrate binding. Based on position, Arg476 and Asp736 in the C-terminal α domain of PgKS may contribute to this conformational transition and appear critical for catalysis. Consistent with the mechanism of other diTPSs, the subsequent ionization of a copalyl diphosphate substrate and coordination of the diphosphate group is controlled by strictly conserved residues in the DDxxD and NDIQGCKRE motif of PgKS, such as Asn656 and Arg653. Furthermore, Lys478, Trp502, Met588, Ala615 and Ile619 control the enzymatic activity and specificity of PgKS via carbocation stabilization en route to ent-kaurene. These positions show a high level of amino acid variation, consistent with functional plasticity among conifer diTPSs of different functions in general or specialized metabolism.     

An intron-free methyl jasmonate inducible geranylgeranyl diphosphate synthase gene from Taxus media and its functional identification in yeast

Geranylgeranyl diphosphate synthase (GGPPS, EC: 2.5.1.29) catalyzes the biosynthesis of geranylgeranyl diphosphate (GGPP), which is a key precursor for diterpenes including Taxol, one of the most potent antitumor drugs. In order to investigate the role of GGPP synthase in taxol biosynthesis, we cloned, characterized and functionally expressed the GGPP synthase gene from Taxus media. A 3743-bp genomic sequence of T. media was isolated by genome walking strategy which contained an 1182-bp open reading frame (ORF) encoding a 393-amino acid polypeptide that showed high similarity to other plant GGPPSs. Subsequently the full-length cDNA of the GGPPS gene of T. media (designated TmGGPPS) was amplified by RACE. Bioinformatic analysis showed that TmGGPPS was an intron-free gene and its deduced polypeptide contained all the five conserved domains and functional aspartate-rich motifs of the prenyltransferases. By constructing the phylogenetic tree of plant GGPPSs, it was found that plant-derived GGPPSs could be divided into two classes, angiosperm and gymnosperm classes, which might have evolved in parallel from the same ancestor. To our knowledge this was the first report that the geranylgeranyl diphosphate synthase genes were free of intron and evolved in parallel between angiosperms and gymnosperms. The coding sequence of TmGGPPS was expressed in yeast mutant (SFNY368) lacking of GGPP synthase activity through functional complementation, and the transgenic yeast showed to have activity of GGPP synthase. This was also the first time to use SFNY368 to identify the function of plant-derived GGPPSs. Furthermore, investigation of the impact of methyl jasmonate (MeJA) on the expression of TmGGPPS revealed that MeJA-treated T. media cultured cells had much higher expression of TmGGPPS than untreated cells.     

真菌二萜环化酶的研究进展

二萜类化合物广泛存在于植物和真菌中,是一类具有重要商业价值的天然产物。二萜环化酶作为催化牻牛儿牻牛儿焦磷酸(geranylgeranyl diphosphate,GGPP)形成二萜的关键生物合成酶,在不同生物中的特异性决定了二萜化合物的结构多样性和生物活性多样性。对不同物种中二萜环化酶基因的分离、克隆和表达特征的分析有利于二萜类化合物的生物合成及调控研究。相比植物,真菌二萜化合物和二萜环化酶的研究刚刚起步。本文综述了近几年真菌二萜环化酶的研究进展,重点叙述了真菌二萜化合物的生物合成途径、二萜环化酶的特征及其克隆策略,并对二萜环化酶的代谢工程作了简要概述。     

Metabolic engineering of isoprenoid biosynthesis in Arabidopsis for the production of taxadiene, the first committed precursor of Taxol

Paclitaxel (Taxol) is a widely used anticancer isoprenoid produced by the secondary metabolism of yew (Taxus sp.) trees. However, only limited amounts of Taxol or related metabolites (taxoids) can be obtained from the currently available sources. In this work we have taken the first step toward genetically engineering the biosynthesis of taxoids in angiosperms. The first committed step in Taxol biosynthesis is the production of taxadiene from geranylgeranyl diphosphate (GGPP), catalyzed by the plastid-localized enzyme taxadiene synthase (TXS). A recombinant T. baccata TXS lacking the putative plastid targeting peptide and fused to a C-terminal histidine (His) tag was shown to be enzymatically active in Escherichia coli. Constitutive production of the full-length His-tagged enzyme in Arabidopsis thaliana plants led to the accumulation of taxadiene and concomitant growth retardation and decreased levels of photosynthetic pigment in transgenic plants. Although these phenotypes may derive from a toxic effect of taxadiene, the lower accumulation of endogenous plastid isoprenoid products such as carotenoids and chlorophylls in transgenic plants also suggests that the constitutive production of an active TXS enzyme might alter the balance of the GGPP pool. Induction of transgene expression using a glucocorticoid-mediated system consistently resulted in a more efficient recruitment of GGPP for the production of taxadiene, which reached levels 30-fold higher than those in plants constitutively expressing the transgene. This accomplishment illustrates the possibility of engineering the production of taxoids and other GGPP-derived isoprenoids in crop plants despite the constraints associated with limited knowledge with regard to regulation of GGPP availability.     

Functional analysis and subcellular localization of two geranylgeranyl diphosphate synthases from <Emphasis Type="Italic">Penicillium paxilli</Emphasis>

The filamentous fungus Penicillium paxilli contains two distinct geranylgeranyl diphosphate (GGPP) synthases, GgsA and GgsB (PaxG). PaxG and its homologues in Neotyphodium lolii and Fusarium fujikuroi are associated with diterpene secondary metabolite gene clusters. The genomes of other filamentous fungi including Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae and Fusarium graminearum also contain two or more copies of GGPP synthase genes, although the diterpene metabolite capability of these fungi is not known. The objective of this study was to understand the biological significance of the presence of two copies of GGPP synthases in P. paxilli by investigating their subcellular localization. Using a carotenoid complementation assay and gene deletion analysis, we show that P. paxilli GgsA and PaxG have GGPP synthase activities and that paxG is required for paxilline biosynthesis, respectively. In the ΔpaxG mutant background ggsA was unable to complement paxilline biosynthesis. A GgsA-EGFP fusion protein was localized to punctuate organelles and the EGFP-GRV fusion protein, containing the C-terminus tripeptide GRV of PaxG, was localized to peroxisomes. A truncated PaxG mutant lacking the C-terminus tripeptide GRV was unable to complement a ΔpaxG mutant demonstrating that the tripeptide is functionally important for paxilline biosynthesis. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Regulation of carotenoid biosynthesis in plants: evidence for a key role of hydroxymethylbutenyl diphosphate reductase in controlling the supply of plastidial isoprenoid precursors

Carotenoids are isoprenoid pigments that function as photoprotectors, precursors of the hormone abscisic acid (ABA), colorants and nutraceuticals. A major problem for the metabolic engineering of high carotenoid levels in plants is the limited supply of their isoprenoid precursor geranylgeranyl diphosphate (GGPP), formed by condensation of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) units usually synthesized by the methylerythritol phosphate (MEP) pathway in plastids. Our earlier work with three of the seven MEP pathway enzymes suggested that the first reaction of the pathway catalyzed by deoxyxylulose 5-phosphate synthase (DXS) is limiting for carotenoid biosynthesis during tomato (Lycopersicon esculentum) fruit ripening. Here we investigate the contribution of the enzyme hydroxymethylbutenyl diphosphate reductase (HDR), which simultaneously synthesizes IPP and DMAPP in the last step of the pathway. A strong upregulation of HDR gene expression was observed in correlation with carotenoid production during both tomato fruit ripening and Arabidopsis thaliana seedling deetiolation. Constitutive overexpression of the tomato cDNA encoding HDR in Arabidopsis did not increase carotenoid levels in etioplasts. By contrast, light-grown transgenic plants showed higher carotenoid levels and an enhanced seed dormancy phenotype suggestive of increased ABA levels. The analysis of double transgenic Arabidopsis plants overproducing both the enzyme taxadiene synthase (which catalyzes the production of the non-native isoprenoid taxadiene from GGPP) and either HDR or DXS showed a twofold stronger effect of HDR in increasing taxadiene levels. Together, the data support a major role for HDR in controlling the production of MEP-derived precursors for plastid isoprenoid biosynthesis.     

Cloning,expression and characterization of a functional cDNA clone encoding geranylgeranyl diphosphate synthase of Hevea brasiliensis

Geranylgeranyl diphosphate (GGPP) synthase catalyzes the condensation of isopentenyl diphosphate (IPP) with allylic diphosphates to give (all-E)-GGPP. GGPP is one of the key precursors in the biosynthesis of biologically significant isoprenoid compounds. In order to examine possible participation of the GGPP synthase in the enzymatic prenyl chain elongation in natural rubber biosynthesis, we cloned, overexpressed and characterized the cDNA clone encoding GGPP synthase from cDNA libraries of leaf and latex of Hevea brasiliensis. The amino acid sequence of the clone contains all conserved regions of trans-prenyl chain elongating enzymes. This cDNA was expressed in Escherichia coli cells as Trx-His-tagged fusion protein, which showed a distinct GGPP synthase activity. The apparent K(m) values for isopentenyl-, farnesyl-, geranyl- and dimethylallyl diphosphates of the GGPP synthase purified with Ni(2+)-affinity column were 24.1, 6.8, 2.3, and 11.5 microM, respectively. The enzyme shows optimum activity at approximately 40 degrees C and pH 8.5. The mRNA expression of the GGPP synthase was detected in all tissues examined, showing higher in flower and leaf than petiole and latex, where a large quantity of natural rubber is produced. On the other hand, expression levels of the Hevea farnesyl diphosphate synthase were significant in latex as well as in flower.