Triterpenoids including steroids are a highly diverse group of natural products widely distributed in plants (Vincken et al., 2007). Plants often accumulate these compounds in their glycosylated form – saponin. Saponins comprise hydrophobic triterpenoid aglycones called sapogenin and one or more hydrophilic sugar moieties.

Biologically, plant saponins are considered defensive compounds against pathogenic microbes and herbivores (Osbourn, 1996; Kuzina et al., 2009; Szakiel et al., 2011). These saponins also have beneficial properties for humans. For example, Panax and Glycyrrhiza plants are well-known traditional herbal medicines containing saponins, ginsenosides, and glycyrrhizin, respectively, with various pharmacological effects (Shibata, 2000, 2001). Saponins have a variety of other applications as well. They show foaming ability when mixed with water, as indicated by the word sapo, meaning soap in Latin. In fact, Saponaria officinalis (common soapwort) and Quillaja saponaria (soapbark) have been used as soap. The saponins of Q. saponaria are also used as emulsifiers in cosmetics and foods. Furthermore, glycyrrhizin is used as a natural sweetener, with 150 times the sweetness of sugar.

In this article, we summarize the genes involved in triterpenoid biosynthesis identified to date and the recent advances in the bioengineering production of useful plant terpenoids; finally, we provide a perspective on the bioengineering of plant triterpenoids.

Terpenoids are built up from C5 units, isopentenyl diphosphate (IPP). IPP is supplied from the cytosolic mevalonic acid (MVA) pathway and the plastidal methylerythritol phosphate (MEP) pathway. Triterpenoids and sesquiterpenoids are biosynthesized via the MVA pathway, whereas monoterpenoids, diterpenoid, and tetraterpenoids are biosynthesized via the MEP pathway. The first diversifying step in triterpenoid biosynthesis is the cyclization of 2,3-oxidosqualene catalyzed by oxidosqualene cyclase (OSC; Abe et al., 1993; Figure 1). In general, animals and fungi have only one OSC, lanosterol synthase (LAS), for sterol biosynthesis. However, higher plants have several OSCs not only for sterol biosynthesis, such as cycloartenol synthase (CAS) and LAS (Ohyama et al., 2009), but also for triterpenoid biosynthesis. The molecular diversity of OSCs enables more than 100 skeletal variations of triterpenoids in plants (Xu et al., 2004). Until now, some dozens of OSC genes from not only model plants but also crops and medicinal plants have been cloned and functionally characterized (reviewed in Kushiro and Ebizuka, 2010). For example, the Arabidopsis thaliana genome has 13 OSC genes, and the functional identification of these genes has been completed, at least, by in vitro experiments. Most of the OSCs from eudicots are phylogenetically classified into some groups, and the reaction products differ from group to group. The site-directed mutagenesis and homology modeling of plant OSCs have been carried out to investigate the reaction mechanisms regarding their product variety (reviewed in Kushiro and Ebizuka, 2010). Of OSCs from various organisms, the structure of human LAS protein was elucidated (Thoma et al., 2004).

After an OSC constructs the basic triterpenoid skeleton, the skeleton is modified to a hydrophobic aglycone called sapogenin. The first modification is oxidation catalyzed by cytochrome P450 monooxygenase (P450), and this step enables further modifications such O-glycosylation. P450 is highly diverse and catalyzes several kinds of chemical reactions committed to the secondary metabolism (Kahn and Durst, 2000).

Glycosylation is essential for saponin biosynthesis. Glycosylation increases the water solubility and changes the biological activity of triterpenoid. Uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs) recognize a wide range of natural products as acceptor molecules.

P450 species and UGTs belong to multigene families and are the key factors for explosive diversification of other natural products in plants. In the case of reported P450 species in saponin biosynthesis, those CYP families vary respecting not only the carbon skeletons of the triterpenoid substrates but also the target positions of the reactions. The diversity of these enzymes makes identification of the genes for saponin biosynthesis difficult. The genes involved in triterpenoid biosynthesis identified in plants to date are presented as follows.

Although almost all production of glycyrrhizin depends on the collection of wild licorice, its harvesting is restricted to prevent exhaustion and desertification in the main producing country, China. Similarly, ginseng requires 4–5 years of careful cultivation and prevention of injury by continuous cropping. Such problems occur not only for saponins but also for other natural plant products. To ensure their stable supply, environment-friendly, and lower-cost alternatives such as biotechnological production are necessary. In the following, we describe the recent advances in the biotechnological production of useful terpenoids.

In plants, overexpression of 3-hydroxy-3-methylgulutaryl-CoA reductase (HMGR), FPP synthase (FPS), and squalene synthase in the isoprenoid pathway has been attempted to increase triterpenoid productivity (Chappell et al., 1995; Schaller et al., 1995; Harker et al., 2003; Lee et al., 2004; Seo et al., 2005; Hey et al., 2006; Muñoz-Bertomeu et al., 2007; Lu et al., 2008; Kim et al., 2010). Despite the improved productivities per unit weight, the transgenic plants sometimes showed growth inhibition (Masferrer et al., 2002; Manzano et al., 2004; Shim et al., 2010), probably caused by metabolic imbalances. In fact, co-overexpression of FPS and the HMGR catalytic domain alleviated the growth inhibition caused by the individual overexpression of FPS or the HMGR catalytic domain (Manzano et al., 2004). Further elucidation of isoprenoid biosynthetic mechanisms is required to improve triterpenoid productivity in plants.

Furthermore, future challenging targets to elucidate for bioengineering of plant triterpenoids should be the regulatory mechanisms of the biosynthetic gene expressions and the accumulation mechanisms of triterpenoids. Saponins are frequently accumulated in specific tissues and organs. Glycyrrhizin and ginsenosides are accumulated in xylems of roots of licorice and ginseng, respectively (Shan et al., 2001; Fukuda et al., 2006). Genes for saponin biosynthesis also express in specific tissues and organs. In avena, Sad genes are expressed in the root epidermal cells accumulating avenacin A-1 (Haralampidis et al., 2001; Qi et al., 2006; Mylona et al., 2008; Mugford et al., 2009). In addition, metabolomic and transcriptomic analyses showed good correlations between expressions of the biosynthetic genes and the accumulations (Matsuda et al., 2010). In fact, recent successes for identification of saponin biosynthetic genes are based on such correlation analysis as described above. Those observations indicate that saponin productions are regulated most likely at the transcription level and thus implying the existence of specific transcription factor(s) for saponin biosynthesis. The engineering of transcription factor is a promising way to modify the biosynthetic pathway in addition to an introduction of multiple biosynthetic enzyme genes (Borevitz et al., 2000; Hirai et al., 2007; Gonzalez et al., 2008). The discovery of novel structural genes involved in the pathway can be achieved by an analysis of overexpressing lines of a transcription factor (Tohge et al., 2005; Luo et al., 2007). Furthermore, the control by the transcription factor would be a useful reference to improve the productivity by the optimization of the expression levels of multiple pathway genes introduced in a heterologous host.

At the subcellular level, saponins are accumulated in vacuoles (Kesselmeier and Urban, 1983; Mylona et al., 2008). However OSCs, P450s and some UGTs for saponin biosynthesis are known as microsomal enzymes (Hayashi et al., 1996; Kurosawa et al., 2002). These facts suggest the presence of a vacuolar transporter of saponin. The transporter is also a target of engineering to improve the accumulation of the target compounds. So far, as a plant terpenoid transporter, an ATP-binding cassette transporter, NpPDR1, involved in secretion of an antifungal diterpenoid, sclareol, in tobacco plant was reported (Jasiński et al., 2001). A homolog of human sterol carrier protein-2 in A. thaliana was identified as a lipid transfer protein (Edqvist et al., 2004). There is, however, no report to identify a triterpenoid transporter in a plant to date. To narrow down the transcription factor and transporter candidates in plant secondary metabolism, omics analyses are also useful strategies (Goossens et al., 2003; Hirai et al., 2007; Morita et al., 2009; Sawada et al., 2009).

Noteworthily, almost all OSCs, catalyzing the first triterpenoid diversification step, cloned from plants have been heterologously expressed in yeast for functional identification (reviewed in Kushiro and Ebizuka, 2010), because OSCs are membrane binding-type proteins and require eukaryotic developed intracellular membrane systems for their heterologous expression. Furthermore, yeast has a sterol biosynthetic pathway for producing the membrane constituent, and this pathway could be converted to a useful triterpenoid-producing pathway.

Previously, we developed a recombinant yeast system for the mechanistic investigation of P450 species in glycyrrhizin biosynthesis (Seki et al., 2008). To supply β-amyrin as the CYP88D6 substrate endogenously, a bAS was constitutively expressed. After the accumulation of β-amyrin in the yeast, CYP88D6 was co-expressed with a CPR as the redox partner. The final yields of 11-oxo-β-amyrin and 11α-hydroxy-β-amyrin at 2 days of culture after the induction were approximately 1.6 and 0.2 mg l⁻¹, respectively. Although we employed Lotus bAS and CPR at that time, G. uralensis bAS and CPR would be more suitable to improve the production.

Genetic engineering in yeast sterol biosynthesis to enhance the availability of β-amyrin may also improve the production of 11-oxo-β-amyrin. Kirby et al. (2008) isolated a bAS from A. annua and achieved 6 mg l⁻¹ of β-amyrin production in yeast expressing an N-terminal-truncated HMGR and restricting the expression of a native OSC, LAS. A large amount of squalene accumulated in yeast means that the yeast can produce even more β-amyrin. In addition to the efforts to improve the common isoprenoid pathway, as already described, enhancement of the catalytic properties of enzymes in triterpenoid biosynthesis should be effective. In Solanaceae, CYP71Ds are involved in sesquiterpenoid phytoalexin biosynthesis. Protein engineering of CYP71Ds based on sequence alignment analysis with phylogenetically related P450 species and homology modeling successfully enhanced the catalytic efficiencies of the enzymes (Takahashi et al., 2007). In Glycyrrhiza, not only glycyrrhizin-producing species but also non-producing species can produce other oleanane-type triterpenoid saponins (Hayashi et al., 2000). The saponin diversity in Glycyrrhiza spp. could be derived from the variation in homologous P450 species and UGTs in saponin biosynthesis. Evaluation of the differences in these enzymes would be useful to improve their activities.

Although the number of identified genes has increased in the last decade, yet there is no saponin biosynthetic pathway of which all genes encoding the proteins involved in the biosynthetic steps have been identified. In fact only one CYP88D6 has been identified in glycyrrhizin biosynthetic pathway that requires two P450 species and two UGTs. The recent transcriptomic and metabolomic approaches have accelerated the elucidation of plant secondary metabolisms (Ziegler et al., 2006; Hirai et al., 2007; Yonekura-Sakakibara et al., 2007, 2008; Liscombe et al., 2009; Okazaki et al., 2009; Matsuda et al., 2010; Saito and Matsuda, 2010), and some saponin biosynthetic genes were identified by such strategies (Achnine et al., 2005; Seki et al., 2008; Naoumkina et al., 2010). Introduction of the current advanced DNA sequence technology in the omics strategies should enhance gene discovery (Li et al., 2010; Sun et al., 2010). In addition to the efforts to discover the proteins for the lacking biosynthetic steps, understanding the regulatory mechanisms of the expression of biosynthetic genes and the accumulation mechanisms of triterpenoids in plant and microbial hosts should enable further promising application for the production of useful triterpenoids.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.