Biosynthetic pathway of terpenoid indole alkaloids in Catharanthus roseus

Abstract

Catharanthus roseus is one of the most extensively investigated medicinal plants, which can produce more than 130 alkaloids, including the powerful antitumor drugs vinblastine and vincristine. Here we review the recent advances in the biosynthetic pathway of terpenoid indole alkaloids (TIAs) in C. roseus, and the identification and characterization of the corresponding enzymes involved in this pathway. Strictosidine is the central intermediate in the biosynthesis of different TIAs, which is formed by the condensation of secologanin and tryptamine. Secologanin is derived from terpenoid (isoprenoid) biosynthetic pathway, while tryptamine is derived from indole biosynthetic pathway. Then various specific end products are produced by different routes during downstream process. Although many genes and corresponding enzymes have been characterized in this pathway, our knowledge on the whole TIA biosynthetic pathway still remains largely unknown up to date. Full elucidation of TIA biosynthetic pathway is an important prerequisite to understand the regulation of the TIA biosynthesis in the medicinal plant and to produce valuable TIAs by synthetic biological technology.

Keywords

Catharanthus roseus / terpenoidindole alkaloids / biosynthetic pathway / vinblastine / vincristine

Introduction

Alkaloids are a group of nitrogen-containing natural products that are found mainly in plants. Approximately 12 000 plant alkaloids have been discovered, many of which are pharmacologically active and traditionally used as antitussives, purgatives, sedatives and anticancer drugs [1]. Over 20% of plant species produce alkaloids, which are mostly derived from the amino acids, including Phe, Tyr, Trp, Lys and Orn. Among all of the plant alkaloids, terpenoid indole alkaloids (TIAs) are an important subgroup containing more than 3000 structurally diverse molecules mainly found in eight plant families, among which, plants from the Loganiaceae, the Apocynaceae, and the Rubiaceae are the best sources [2]. There are three main structural types according to the arrangement of atoms in the terpenoid portion, the Corynanthe type, such as ajmalicine and akuammicine, the Aspidosperma type, such as tabersonine, and the Iboga type, exemplified by catharanthine [3].

Catharanthus roseus [L.] G. Don (Madagascar periwinkle) is a perennial herb of the Apocynaceae and considered as one of the most extensively investigated medicinal plants and one of the most popular ornamentals. More than 130 terpenoidindole alkaloids (TIAs) are characterized from C. roseus, which represent one of the largest and most diverse groups of alkaloids in this plant. C. roseus is the sole resource of vinblastine and vincristine, which are two of the biggest concerns of TIAs because of their powerful anticancer activities [4]. In addition, some other valuable TIAs from C. roseus have also been developed as leading pharmaceuticals. For example, ajmalicine and serpentine are widely used to treat circulatory disorders [5]. Therefore, C. roseus is a useful model system for the study of TIA biosynthesis [2]. The biosynthesis of TIAs in C. roseus is a complex process with more than 50 biosynthetic events that is composed of the involved genes, enzymes, regulators, and intra-/intercellular transporters [6]. The strictosidine is a common precursor for all the TIAs, which can be converted into a series of monoterpenoid indole alkaloids with varied structures and biological functions. The strictosidine is assembled from the two intermediates, tryptamine and secologanin: the former is synthesized through indole pathway; while the latter is derived from terpenoid biosynthetic pathway (Fig. 1).

Formation of IPP

Isopentenyl pyrophosphate (IPP) is an important intermediate in the biosynthesis of terpenes and terpenoids, which can be produced via two different pathways, the mevalonic acid (MVA) pathway and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Incorporation experiments with [1-¹³C] glucose in C. roseus cell cultures indicated that both the MVA and the MEP pathway are involved in the IPP biosynthesis [7]. The MVA pathway that participates in TIAs production mainly be considered on two levels: (1) it serves as a minor source of precursors for iridoid biosynthesis; (2) it regulates gene expression through protein prenylation [8].

The MEP pathway is composed of seven steps (Fig. 2B), of which six have been characterized in C. roseus. In the MEP pathway, the first step involves a condensation of pyruvate with D-glyceraldehyde-3-phosphate to yield 1-deoxy-D-xylulose-5-phosphate (DXP), catalyzed by the enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXS) [9]. In the second step, DXP is converted into 2-C-methyl-D-erythritol 4-phosphate (MEP) by the enzyme 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR). The third step is catalyzed by 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (CMS), which converts MEP into 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDP-ME). Then CDP-ME is phosphorylated on its 2-hydroxy group by a CDP-ME kinase (CMK). The fifth enzyme 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MECS) converts the 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate into 2-C-methyl-D-erythritol 2,4-cyclo diphosphate (MECDP). Then MECDP is converted into 1-hydroxy-2-methyl-2-butenyl 4-diphosphate (HMBPP) by HMBPP synthase (HDS) in the sixth step. The final step is catalyzed by HMBPP reductase, which converts HMBPP into IPP or dimethylallyl diphosphate (DMAPP) in a 5:1 mixture [10].

The MVA pathway mainly leads to the formation of triterpenes and sesquiterpenes [11,12]. The first step of this pathway is catalyzed by acetoacetyl-CoA thiolase (AACT) [13] (Fig. 2A), which couples two molecules of acetyl-CoA into a molecule of acetoacetyl-CoA. Then acetoacetyl-CoA couples with a third molecule of acetyl-CoA to form 3-hydroxy-3-methyglutaryl-CoA (HMG-CoA) by the catalysis of hydroxymethyglutaryl-CoA synthase (HMGS) [13]. The third step is catalyzed by HMG-CoA reductase (HMGR) [14] to form mevalonate (MVA). Then MVA is phosphorylated by mevalonate kinase (MVK) [15,16] and mevalonate 5-phosphate (MVAP) kinase (PMK) [17] to form mevalonate 5-diphosphate (MVAPP) in the next two steps. Finally, MVAPP is decarboxylated and dehydrated by the ATP-dependent mevalonate 5-diphosphate decarboxylase (MVD) to IPP. Then IPP is isomerized to DMAPP by the catalysis of IPP isomerase (IDI) [18].

From IPP to secologanin

The condensation of one DMAPP with one IPP is catalyzed by geranyl diphosphate synthase (GPPS) in a head to tail fashion to generate geranyl diphosphate (GPP), which is the precursor for the biosynthesis of secologanin [,7]. Secologanin is synthesized through the iridoid pathway comprising nine steps (Fig. 3). In C. roseus, all enzymes in this pathway have been characterized, except for 10-hydroxygeraniol oxidoreductase (10-HGO), whose complete CDS is available in GenBank (Accession number AY352047.1) (Table 1).

In the first step of iridoid pathway, GPP is converted into geraniol by the enzyme geraniol synthase (GES). Simkin et al. (2013) [25] cloned the gene encoding this enzyme in C. roseus. Hydroxylation of geraniol leads to the formation of 10-hydroxylgeraniol, catalyzed by the geraniol 10-hydroxylase (G10H, CYP76B6), which is thought to play a key regulatory role in TIAs biosynthesis. The corresponding cDNA of G10H was cloned and characterized by Collu et al. in 2001 [26]. Cytochrome P450 reductase (CPR) [24], which transfers electrons from NADPH to G10H, is essential for the CYP450 catalyzing reaction. In the third step, 10-hydroxylgeraniol is further oxidized to the dialdehyde, 10-oxogeranial, by 10-HGO. The fourth step is catalyzed by an NADPH-dependent 10-oxogeranial cyclase named iridoid synthase (IRS) [27], which converts 10-oxogeranial to iridodial. Then the cyclized product is further oxidized to 7-deoxyloganetic acid. The enzyme responsible for the oxidation steps is 7-deoxyloganetic acid synthase (7DLS, CYP76A26), which have been functionally characterized by Salim et al. (2014) [28]. The sixth step is catalyzed by 7-deoxyloganetic acid glucosyltransferase (DLGT) [29] to produce 7-deoxyloganic acids. Further hydroxylation of 7-deoxyloganic acid to form loganic acid is catalyzed by 7-deoxyloganic acid 7-hydroxylase (DL7H, CYP72A224) [30]. Then loganic acid is converted into loganin by the catalysis of loganic acid O-methyltransferase (LAMT) [31]. The last step of iridoid pathway is catalyzed by another CYP450 enzyme, secologanin synthase (SLS, CYP72A1) [33,41], to yield secologanin.

Formation of tryptamine

Tryptamine is derived from a single enzymatic conversion of the amino acid L-tryptophan by tryptophan decarboxylase (TDC). The cDNA encoding TDC has been cloned from C. roseus by De Luca et al. (1989) [34]. Tryptophan is synthesized through a biosynthetic pathway consisting of six enzymatically-controlled steps. The first step is formation of anthranilate from chorismate, which is catalyzed by anthranilate synthase (AS). AS and TDC are thought to be the key regulated enzymes in the production of tryptamine [42,43].

Biosynthesis of strictosidine

Strictosidine is the central intermediate in the biosynthesis of many alkaloids in C. roseus, which is derived from the condensation of secologanin and tryptamine by strictosidine synthase (STR) (Fig. 4). A partial cDNA encoding STR protein was isolated from C. roseus by McKnight et al. (1990) [35], and the complete genomic sequence of STR gene was cloned by Pasquali et al. (1992) [44]. The condensation step of secologanin and tryptamine is a committed step in the biosynthesis of TIAs. Some studies revealed that the expression of the STR gene is downregulated by auxin [44] and upregulated by methyl jasmonic acid (MeJA) [45] and fungal elicitation.

The vindoline pathway

Strictosidine is catalyzed by strictosidine β-D-glucosidase (SGD) [36] to form 4,21-dehydrogeissoschizine, which is then catalyzed by many other enzymes in different branches to form diverse TIAs. In one branch, 4,21-dehydrogeissoschizine is converted to form an important metabolite catharanthine. The information on catharanthine biosynthetic pathway is very limited. Another branch leads to the formation of vindoline, which is one precursor of the bisindole alkaloids, including vinblastine and vincristine. The vindoline pathway (Fig. 5) is composed of six steps, in which the tabersonine 16-hydroxylase (T16H) [37,46,47], desacetoxyvindoline 4-hydroxylase (D4H) [48] and deacetylvindoline-4-O-acetyltransferase (DAT) [39,49] are thought to be the mainly regulatory targets of transcription factors.

Biosynthesis of vinblastine and vincristine

The bisindole alkaloids vinblastine and vincristine are derived from the condension of the monomeric alkaloids catharanthine and vindoline. The enzyme catalyzing the dimerization reaction of catharanthine and vindoline is a vacuolar class III peroxidase (CrPrx1) [40], whose full-length gene has been cloned and characterized by Costa et al. CrPrx1 is a single-copy gene with a two introns. The product α-3′,4′-anhydrovinblastinecatalysed by CrPrx1 is the common precursor of all dimeric alkaloids, which can be future converted into vinblastine and vincristine through several steps (Fig. 5).

Conclusions and outlook

In this review, we have summarized the biosynthetic pathway of TIAs in C. roseus and characterization of the related genes encoding the enzymes involved in this pathway. After the past several years’ unremitting efforts, many genes have been cloned and characterized. But the entire biosynthetic pathway of TIAs biosynthesis contains much more steps, which is still unknown and need further elucidated. Recently, the iridoid pathway from GPP to secologanin has been fully elucidated, which means all the sequence data of genes involved in this pathway have been deposited in the GenBank database. This makes it possible to produce secologanin by metabolic engineering or synthetic biology strategy. Synthetic biology is a leading edge technology, which combine biology and engineering strategies to design and construct new biological parts, devices, and systems, or re-design natural biological systems for useful purposes. Many significant plant secondary metabolites such as artemisinic acid (precursor of the anti-malaria drug artemisinin) [50-54], taxol (an expensive anti-cancer drug) [55,56] and ferruginol (precursor of tanshinones) [57-59] have been produced successfully by reconstructing their biosynthetic pathway in Escherichia coli or Saccharomyces cerevisiae. Full dissection of the entire TIA biosynthetic pathway is the prerequisite to produce them by synthetic biology technology. Application of some new technology, such as high-throughput sequencing, virus-induced gene silencing (VIGS), will greatly accelerate the discovery of novel genes in this pathway.

Compliance with ethics guidelines

Xiaoxuan Zhu, Xinyi Zeng, Chao Sun, and Shilin Chen declare that they have no conflict of interest. This manuscript is a review article and does not involve a research protocol requiring approval by the relevant institutional review board or ethics committee.

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