Nature’s assembly line: biosynthesis of simple phenylpropanoids and polyketides

Authors


*(fax +1 314 587 1551; e-mail oyu@danforthcenter.org).

Summary

Plants produce large amounts of phenylpropanoids, both in terms of molecular diversity and absolute quantity of these compounds. The phenylpropanoids, and the related plant polyketides, have multiple biological functions. They serve to attract pollinators, support secondary cell-wall growth, provide protection against various plant diseases, and interact with beneficial soil microbes. Their basic chemical properties also make them useful in the biofuel and biomaterial industries. Phenylpropanoid metabolism begins with the amino acid phenylalanine, which feeds into various biosynthetic pathways that generate a wide range of structurally related polyphenolic compounds. This review focuses on four sub-groups of these polyphenolic compounds – polyketides, stilbenes, isoflavones and catechins. We discuss the biosynthesis of these molecules, their physiological role in plants, and their striking pharmacological and physiological effects on humans. This review also highlights metabolic engineering efforts aimed at increasing or decreasing the amounts of each class of compound in various model plants and crops.

Introduction

The phenylpropanoid pathway produces the majority of phenolic compounds found in nature. These molecules have profound effects on plant growth and development. Plants dedicate a significant amount of energy and invest large carbon resources produced during photosynthesis to make phenylpropanoids. The phenylpropanoids represent the largest pool of secondary metabolites, comprising nearly 20% of total carbon in the terrestrial biosphere (Peters, 2007), with plants synthesizing approximately 10 gigatonnes (10 × 109 tonnes of carbon) of these molecules each year. There are more than 7000 documented phenylpropanoid compounds in plants (Wink, 2003), and they play varied and important roles in many aspects of plant physiology. Other articles in this issue cover some of these biological functions, including the importance of lignin polymers as major cell-wall components (Li et al., 2008), anthocyanins as floral pigments that attract pollinators (Tanaka et al., 2008), flavor and scent compounds derived from phenylpropanoids (Schwab et al., 2008), and the production of valuable bioplastic materials using phenylpropanoid metabolites (van Beilen and Poirier, 2008).

The phenylpropanoids make up the majority of ‘polyphenols’ consumed in our diet. Because they interact with many cellular processes, these molecules directly affect human health (Korkina, 2007). Most phenylpropanoids can function as anti-oxidants due to multiple hydroxyl groups and unsaturated double bonds that can react with radicals and oxidative ions in cells. Some phenylpropanoids act as phyto-estrogens (e.g. isoflavones and coumestrols) or chemo-preventive anti-cancer agents (e.g. resveratrol), or regulate the development of fat cells (e.g. catechins). A subset of the phenylpropanoids, known as polyketides, is an important source of pharmaceuticals due to their diverse chemical structures. In plants, some polyketides serve as intermediates in phenylpropanoid metabolism, while others are molecular end products with interesting biological functions. The benzene or phenol ring structure in the phenylpropanoids and polyketides allows these compounds to cross cellular membranes, and exert their biological activities. For the same reason, many modern medicines are derivatives of plant phenolic compounds. For example, one of the first pharmaceutical drugs, acetylsalicylic acid (aspirin), is a phenylpropanoid. Use of acetylsalicylic acid as a therapeutic agent dates back to the ancient Sumerian and Egyptian cultures, and its precursor was originally isolated from the bark of willow trees more than 100 years ago (MacLagan, 1876). The varied chemical structures of the thousands of identified and unidentified phenylpropanoids from plants are a treasure trove of future medicines awaiting discovery. This review focuses on the biosynthesis and biological functions of four sub-groups of phenylpropanoids (polyketides, stilbenes, isoflavones and catechins).

Start of the phenylpropanoid pathway

Synthesized by the shikimate pathway, the amino acid phenylalanine is the primary starting molecule of this pathway in plants (Figure 1). Phenylalanine ammonia lyase (PAL), the first enzyme of the phenylpropanoid pathway, is ubiquitous in higher plants and catalyzes the deamination of phenylalanine into trans-cinnamic acid (MacDonald and D’Cunha, 2007). Oxidation of this aromatic acid by cinnamate 4-hydroxylase (C4H), a cytochrome P450 enzyme, yields p-coumaric acid (or 4-coumaric acid) (Anterola and Lewis, 2002). In certain monocots, PAL accepts tyrosine as a substrate to directly produce p-coumaric acid, bypassing the requirement for C4H (Rosler et al., 1997). Next, 4-coumaroyl CoA ligase (4CL) attaches a CoA molecule to p-coumaric acid, generating 4-coumaroyl CoA, which provides an active intermediate in multiple branches of the general phenylpropanoid pathway (Dixon et al., 1996). The three enzymes of the pathway (PAL, C4H and 4CL) are highly conserved among plant species because they are important for normal growth and development. Lignin production consumes the majority of p-coumaroyl CoA; however, most plant species divert a portion of this intermediate toward flavonoid biosynthesis. The first enzyme of the flavonoid biosynthesis pathway, chalcone synthase (CHS), is a member of a large family of related polyketide synthases, which synthesize an array of chemicals in plants and bacteria.

Figure 1.

 Start of the phenylpropanoid pathway: phenylalanine to p-coumaroyl CoA.
Conversion of phenylalanine to p-coumaroyl CoA requires the action of three enzymes – phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H) and 4-coumaroyl CoA ligase (4CL). In some monocots, a PAL with tyrosine ammonia lyase (TAL) activity can bypass PAL and C4H by directly forming p-coumaric acid from tyrosine (turquoise box).

The archetypal plant polyketide synthase: chalcone synthase

Polyketides are a diverse collection of natural products found in plants and bacteria. Although the structural diversity of these molecules provides a rich source of pharmaceutically valuable (antibiotics and anti-cancer drugs) and biologically active (pigments and allelochemicals) compounds, their synthesis shares a common chemical logic centered on formation of a polyketone intermediate. Assembly of a generic polyketide involves three distinct phases: (i) loading of a starter molecule, (ii) addition of multiple chemical building blocks to extend the polyketide chain, and (iii) release of the finished molecule, typically through a cyclization reaction (Figure 2).

Figure 2.

 Overview of polyketide assembly in chalcone synthase (CHS).
A p-coumaroyl CoA ‘starter molecule’ is loaded onto a cysteine in the enzyme active site (yellow box). Next, the first of three malonyl CoA molecules binds at the active site and is decarboxylated to form a reactive acetyl CoA intermediate (blue box). Extension of the polyketide chain in the active site by one acetate unit (red) occurs. The process is repeated twice (blue boxes). Hydrolysis of the growing chain can yield polyketide lactones (orange box). The final tetraketide product can cyclize through a Claisen condensation to yield the chalcone product (red box). In stilbene synthase (STS), an aldol condensation results in a stilbene product (turquoise box).

In plants, the archetypal enzyme of polyketide synthesis is chalcone synthase (CHS). This enzyme connects phenylpropanoid and flavonoid metabolism by orchestrating a series of chemical reactions to assemble naringenin chalcone from one p-coumaroyl CoA and three malonyl CoA molecules. As noted above, metabolism of phenylalanine yields p-coumaroyl CoA, and malonyl CoA is provided from fatty acid biosynthesis. A wealth of structural and mechanistic studies on CHS have revealed the molecular details of this process (Ferrer et al., 1999; Jez and Noel, 2000; Jez et al., 2000c, 2001b).

The first step of polyketide synthesis involves the binding or loading of a starter molecule to the enzyme (i.e. p-coumaroyl CoA for CHS) (Figure 2, yellow box). The CoA group found in each of the substrates for CHS provides a common recognition feature analogous to a hand delivering building blocks to the enzyme active site. Once bound, an active-site cysteine reacts with the starter molecule, releasing CoA, and leaving the p-coumaroyl group attached to the enzyme by a thioester bond.

Next, extension of the polyketide begins with binding of the first malonyl CoA to CHS. Within the CHS active site, a catalytic histidine and asparagine dyad catalyzes decarboxylation of malonyl CoA to a reactive acetyl CoA anion. The reactive intermediate acts as a nucleophile to attack the thioester bond of the enzyme-bound p-coumaroyl group. This reaction extends the polyketide chain by an acetate unit, with the diketide (acetate-coumaroyl) reattaching to the active-site cysteine and releasing CoA (Figure 2, blue boxes). The entire process is repeated two more times, generating triketide (acetate-acetate-coumaroyl) and tetraketide (acetate-acetate-acetate-coumaroyl) chains. The physical size of the CHS active site limits the number of additions possible (Jez et al., 2001a).

In the CHS reaction sequence, cyclization of the tetraketide chain by a Claisen condensation yields naringenin chalcone (Figure 2, red box). The starter molecule provides one aromatic ring in the final product, and cyclization of the malonyl-derived polyketide backbone generates the second aromatic ring. Alternatively, the same tetraketide intermediate can undergo an aldol condensation to yield the stilbene resveratrol, as occurs with stilbene synthase (STS) (Austin et al., 2004; Shomura et al., 2005) (Figure 2, light blue box). In addition, hydrolysis of the polyketide chain from the enzyme active site during the extension reactions can lead to triketide and tetraketide ‘derailment’ products, with formation of a lactone ring producing bis-noryangonin and coumaroyl-triacetic acid lactone, respectively (Figure 2, orange box).

Themes and variations in plant polyketide synthesis

Nature capitalizes on the process of polyketide assembly because it affords multiple options for generating chemical diversity. Each chemical step (selection of starter or extender molecule, the number of extension reactions, and the type of cyclization reaction) is versatile and adaptable, with minor changes resulting in new compounds, many of which function as phytoalexins or are of pharmaceutical interest.

Within the family of plant enzymes related to CHS, changes in starter molecule selectivity yield a range of new compounds containing a chalcone-like ring (Austin and Noel, 2003). The acridone (Lukacin et al., 2005; Wanibuchi et al., 2007), benzophenone (Liu et al., 2003), biphenyl (Liu et al., 2004) and phloroisovalerophenone/phloroisobutyrophenone synthases (Klingauf et al., 2005; Paniego et al., 1999) prefer starter molecules other than p-coumaroyl CoA, but catalyze identical extension and cyclization reactions to those catalyzed by CHS (Figure 3a). Recent studies of novel plant polyketide synthases, such as the chromone (Abe et al., 2005b; Morita et al., 2007), aloesone (Abe et al., 2004a, 2006a) and octaketide (Abe et al., 2005a) synthases, reveal that these enzymes can use acetyl CoA as a starter molecule and then perform four to seven extension steps with malonyl CoA molecules to yield pentaketide, heptaketide and octaketide intermediates that cyclize through a Claisen condensation (Figure 3a). Similarly, STS (Austin et al., 2004; Shomura et al., 2005) and stilbenecarboxylate synthase (Eckermann et al., 2003) use other aromatic CoA starter molecules to begin the sequence of reactions, leading to different stilbene molecules (Figure 3b).

Figure 3.

 Chemical diversity of plant polyketides.
Molecules with chalcone (red box), stilbene (pale blue box), lactone (orange box) and linear (white box) cyclization patterns are shown. Starter molecules are colored black and acetate-derived portions are colored in red. The methylmalonyl CoA derived portion of the C-methylated chalcone is colored light purple.

Once considered as side-products resulting from derailment of the growing polyketide chain, many of the lactone-based molecules synthesized by plant polyketide synthases have biological functions. For example, 2-pyrone synthase condenses three acetate units together to form a lactone (Figure 3c) that is later glycosylated into the anti-pathogen molecule parasorboside, a bitterness compound that prevents insect feeding (Eckermann et al., 1998; Jez et al., 2000a). Likewise, the diketide-based bis-noryangonin molecule produced in kava (Piper methysticum) is an effective anti-anxiety compound (Dewick, 2002). Studies on plant polyketide synthases thought to function in the synthesis of certain natural products, such as hydrangic acid in Hydrangea macrophylla and plumbagin in Plumbago indica, suggest that accessory proteins may play a role in guiding assembly of the final molecule, as the coumaroyl triacetic acid lactone and dihydroxymethylphenyl methylpyrone compounds are probably premature hydrolysis products (Akiyama et al., 1999; Springob et al., 2007).

Finally, the simplest variations are in the number of extension reactions and type of molecule used (Figure 3d). Benzalacetone synthase (Abe et al., 2001) catalyzes the single addition of an acetate unit to a p-coumaroyl group. In synthesis of C-methylated chalcones in pine, a CHS-like enzyme accepts methylmalonyl CoA instead of malonyl CoA for the final extension reaction (Schröder et al., 1998) but it is unclear how this enzyme selects methylmalonyl CoA versus malonyl CoA for this reaction.

Engineered plant polyketide synthesis

Exploitation of the chemical versatility of plant polyketide synthesis mimics naturally occurring differences in starter molecule selectivity, extension of the polyketide chain, and variation in cyclization patterns. The assembly-line process of polyketide production allows the application of organic synthesis and/or protein engineering strategies for the generation of novel molecular scaffolds (Figure 4).

Figure 4.

 Engineering plant polyketide synthesis.
Representative molecules resulting from various approaches to engineering chemical diversity in plant polyketide synthases are shown. Portions of molecules derived from the various starter molecules, malonyl CoA and methylmalonyl CoA are colored black, red and light purple, respectively.

The plant polyketide synthase, like most enzymes, displays broad substrate specificity; however, the availability of substrates in vivo dramatically limits the possible ‘natural’ reaction products. Using alternative or non-physiological substrates is the most straightforward approach to generating new polyketides in vitro. For example, both CHS and STS accept CoA esters of long-chain fatty acids up to 14 carbons in length to generate triketide and tetraketide lactones (Abe et al., 2004b). Similarly, synthetic N-acetylcysteamine thioester substrates can replace CoA-linked substrates, albeit with reduced catalytic efficiency (Oguro et al., 2004). In addition, simultaneous variation of both starter and extender molecules leads to production of unnatural aromatic polyketides (Figure 4a) (Abe et al., 2002, 2003).

Detailed three-dimensional information on the plant polyketide synthases and knowledge of their chemical mechanism is guiding efforts aimed at engineering these enzymes for the generation of new molecules (Penning and Jez, 2001). Initial efforts focused on examining how active site variation among enzymes that make the various molecules leads to product specificity. The interconversion of CHS and 2-pyrone synthase showed how three mutations of amino acids completely switch starter molecule specificity and change the cyclization pattern (Jez et al., 2000a). Such approaches also elegantly demonstrated how subtle active site variations govern the choice of Claisen condensation versus aldol condensation during cyclization of the tetraketide intermediate by CHS and STS (Austin et al., 2004).

Changing the size of the polyketide synthase active site leads directly to modification of the reaction sequence. Reducing the volume of the active site in CHS decreases the number of extension reactions and alters the cyclization pattern of the polyketide product (Jez et al., 2001a). Likewise, other substitutions of active site residues that increase the size of the active site allow production of larger polyketides (Figure 4b). Modification of the octaketide-producing polyketide synthase from Aloe arborescens leads to a variety of products including the SEK4/4b octaketides, which were previously shown to be produced by certain bacteria polyketide synthases (Abe et al., 2005a, 2006b). Similarly, three substitutions in chromone synthase, which makes a pentaketide, triple the volume of the active site and result in synthesis of the nonaketide naphthopyrone from nine malonyl CoA molecules (Abe et al., 2007).

Combining protein engineering and providing alternative substrates opens up additional possibilities (Figure 4c). A single point mutation of a phenylalanine to a serine in CHS allows the enzyme to accept an alternative starter molecule (N-methylanthranoyl CoA) to produce a novel polyketide (Jez et al., 2002a). Continued exploration of the interplay between protein structure and polyketide assembly promises to yield new strategies for the biosynthesis of novel molecules.

Following assembly of the polyketide scaffold in plants, a range of additional modifications can tailor the biological activity of the molecule, including additional ring closure reactions (Jez and Noel, 2002; Jez et al., 2000b, 2002b), modifications of the reaction intermediate (Bomati et al., 2005; Oguro et al., 2004), methylation (Bringmann et al., 2007; Dayan et al., 2003; Schröder et al., 2004; Stevens and Page, 2004), prenylation (Klingauf et al., 2005; Kuzuyama et al., 2005; Stevens and Page, 2004) and glycosylation (Das and Rosazza, 2006; Springob et al., 2003). Considerable progress in understanding these reactions suggests the promise of future work aiming to incorporate them into the production and engineering of plant natural products, although how these processes occur in phenylpropanoid and plant polyketide synthesis is beyond the scope of this review.

Stilbenes: synthesis, biological functions, red wine and longer life

As mentioned above, STS is able to synthesize resveratrol (3,5,4′-trihydroxy-trans-stilbene) by condensing one molecule of p-coumaroyl CoA with three molecules of malonyl CoA. Resveratrol is a unique stilbene produced by a limited number of unrelated species, including grapevine (Vitis sp.), peanuts (Arachis hypogaea) and a few varieties of berries (Asensi et al., 2002). There is reasonable evidence to suggest that STS arose independently multiple times from CHS over the course of plant evolution (Tropf et al., 1994). The compound was originally discovered in Japanese knot weed (Polygnum capsidatum), which is a traditional medicinal plant for treating inflammation (Nonomura et al., 1963); however, due to high levels of resveratrol in red wines, research on resveratrol has focused on the health benefits associated with wine consumption. In addition to their importance for plants as a protective compound, or phytoalexin, the biological properties of resveratrol (and related stilbenes) may also be beneficial to human health.

Plants produce resveratrol and related stilbenes as phytoalexins. For example, UV irradiation and fungal attack in grapevine drastically induce resveratrol biosynthesis in leaves and fruit skins, increasing their anti-microbial protection (Jeandet et al., 2002). Early engineering efforts aimed to introduce synthesis of this molecule into plants lacking STS. Defense-induced expression of the grape STS gene in tobacco resulted in the diversion of substrates from endogenous CHS to produce up to 300 μg g−1 fresh weight of resveratrol in tobacco (Hain et al., 1993). The transgenic plants also displayed increased resistance to the tobacco fungal pathogen Botrytis cinerea, indicating that heterologous expression of STS may be toxic to native pathogens that could not detoxify the new phytoalexin.

Transgenic approaches have also introduced stilbene biosynthesis into tomato, lettuce and several other plants, and production of resveratrol led to increased disease resistance in almost all cases (Jeandet et al., 2002); however, the mechanism of toxicity to fungal pathogens remains unknown. Interestingly, conjugation of most of the produced resveratrol with glucose resulted in sequestration of the glycosylated stilbene piceid in vacuoles. One major advantage of engineered phytoalexin accumulation is the prolonged shelf lives of the fruit and seed after harvest. Hairy root cultures aimed at producing large quantities of resveratrol have been generated (Medina-Bolivar et al., 2007). A recent report showed that introduction of the peanut STS gene into hop (Humulus lupulus) and subsequent production of resveratrol improves the nutritional value of beer and enhances disease resistance in hops (Schwekendiek et al., 2007). These studies may have future commercial application.

Originally, the importance of resveratrol and stilbenes to human health was identified as a result of the high levels of these compounds in red wine and the ‘French paradox’. Although the typical French diet is high in saturated fats, the incidence of coronary heart disease is relatively low, and is often attributed to consumption of red wine (and stilbenes). Because stilbenes are anti-oxidants, they may contribute to prevention of heart disease. In addition to acting as an anti-oxidant, resveratrol appears to affect multiple physiological and cellular processes in humans. Clinical experiments indicate that resveratrol consumption reduces serum cholesterol levels, inhibits lipid peroxidation, suppresses platelet aggregation, and exhibits chemopreventive activity towards various cancers (Baur and Sinclair, 2006; Shankar et al., 2007). In both animal models and human studies, resveratrol inhibits carcinogenesis through various mechanisms in various types of tumor (Baur and Sinclair, 2006).

Resveratrol also exhibits drastic effects on life span in animals (Cohen et al., 2004; Howitz et al., 2003; Wood et al., 2004). Although the mechanism of longevity remains unclear, recent experiments show that resveratrol specifically activates sirtuin-like protein deacetylases (Wood et al., 2004). Found in many eukaryotic cells, sirtuins are redox-sensing enzymes that monitor cellular responses to environmental stresses. Conditions that induce oxidative stress, such as calorie starvation, activate sirtuins. Consequently, general metabolic activity slows, apoptosis (programmed cell death) is delayed, and longevity is increased. Following treatment with resveratrol, roundworms (Caenorhabditis elegans), fruit flies (Drosophila melanogaster) and fish (Nothobranchius furzeri) displayed increased life spans without affecting their fertility (Valenzano et al., 2006; Wood et al., 2004). Even in mammals, high-dosage resveratrol feeding significantly extends the life span of obese mice, possibly by countering the negative effects of a high-fat diet (Baur et al., 2006). These health effects are the driving force for future metabolic engineering efforts in plants, microbes and even mammals themselves (Halls and Yu, 2008; Zhang et al., 2006).

Isoflavones: synthesis, function, and soy

Legumes, like soybean, produce isoflavones, which are a specialized group of phenylpropanoids. Unlike the common flavonoid compounds, which have a 2-phenyl-benzopyrone core structure, isoflavones, such as daidzein and genistein, are 3-phenyl-benzopyrone compounds. As with stilbenes, pathogen infection induces the synthesis of isoflavones as phytoalexins.

Biochemically, the synthesis of isoflavones is an offshoot of the flavonoid biosynthesis pathway (Figure 5). Chalcone isomerase (CHI) catalyzes a ring-closure reaction of chalcone to yield the flavonoid naringenin. The entry-point enzyme of the isoflavone pathway is isoflavone synthase (IFS), which is a cytochrome P450 mono-oxygenase (Jung et al., 2000; Steele et al., 1999). IFS uses naringenin as a substrate in a ring-migration reaction. The enzyme hydroxylates the C2 carbon of the substrate, and aryl migration results in movement of the phenyl ring from C2 to C3, yielding 2,5,7,4′-tetrahydroxy-isoflavanone as an unstable reaction intermediate (Sawada et al., 2002). Isoflavanone dehydratase (IDH) converts the intermediate into the isoflavonoid genistein (Akashi et al., 2005). A parallel pathway uses an alternative IFS substrate to produce 5-deoxyisoflavones. This route begins with a legume-specific enzyme, i.e. chalcone reductase (CHR). CHR works in concert with CHS to remove one hydroxyl group from the tetraketide intermediate of CHS, leading to 5′-deoxychalcone (or isoliquiritigenin). The CHI in legumes, which has broader substrate specificity than that of non-legume plants, catalyzes the cyclization of 5′-deoxychalcone to liquiritigenin (Ralston et al., 2005), from which IFS and IDH generate daidzein (Figure 5).

Figure 5.

 Isoflavonoid biosynthesis.
The flavonoid biosynthetic enzymes provide naringenin (or liquiritigenin) for isoflavonoid production (green). An enzyme-catalyzed aryl migration reaction generates the core isoflavonoid structure (orange). Additional modifications, indicated in blue, lead to a variety of isoflavonoid compounds in various plants (yellow).

Following generation of the core isoflavonoid scaffold by IFS, additional metabolism of these molecules can result in compounds with potent anti-microbial activities. For example, biologically active molecules result from methylation and dehydration of the 2-hydroxyisoflavanone intermediate (to form formononetin in alfalfa), glycosylation (to form puerarin in kudzu), hydroxylation and methylation (to form glycitein in soybean) or prenylation (to form wighteone in lupin) (Figure 5, yellow box) (Dixon et al., 1998). Subsequent reactions convert isoflavones into pterocarpans or coumestrols, which are potent anti-fungal compounds. Additionally, because isoflavonoids and flavonoids are the chemical signals that legumes send to the rhizosphere to attract symbiotic rhizobia, these modifications play important roles in determining host specificity during nodulation (Spaink, 1994).

Consumption of foods with high isoflavonoid levels, such as soybean, may have health benefits. The health-promoting effects of isoflavones may result from the weak estrogenic activity they display. Because isoflavonoids are chemically similar to human steroids, these molecules can mimic steroids in the body. For example, isoflavones bind to certain estrogen receptors (Messina and Loprinzi, 2001). In animal models, isoflavones enhance calcium uptake and reduce the occurrence of osteoporosis (Albertazzi, 2002). Moreover, isoflavones may provide an alternative to hormone replacement therapy in clinical treatments of osteoporosis and other post-menopausal symptoms (Vatanparast and Chilibeck, 2007). Likewise, the estrogenic effects of isoflavones show promise in many animal models as chemo-preventive agents for hormone-related cancers of the breast, prostate and colon (Martin et al., 2007).

With limited consumption of isoflavone-rich foods in a typical Western diet, increasing isoflavone content in soybean and other foods offers an interesting approach to obtain the same health benefits as an Oriental diet. Several attempts have aimed to increase isoflavone levels in soybean and introduce isoflavone biosynthesis in non-legume crops. Over-expression of IFS in Arabidopsis, tobacco and maize leads to genistein accumulation in these non-legume plants (Liu et al., 2002; Yu et al., 2000); however, the levels of isoflavones are generally low compared to those in soybean. Moreover, introduction of putative rate-limiting enzymes in the pathway (i.e. PAL, CHS or IFS) has a limited effect on isoflavonoid yields in soybean (Lozovaya et al., 2007). To overcome these limitations, researchers have adopted alternative strategies. One approach involves physically linking proteins in the pathway. Expression of an IFS–CHI fusion protein increased production of isoflavone in Arabidopsis, suggesting that tethering these sequential enzymes in proximity to each other improves pathway flux (Tian and Dixon, 2006). Likewise, over-expression of a synthetic maize transcription factor that activates the expression of multiple genes encoding flavonoid biosynthetic enzymes in soybean seeds resulted in an altered isoflavone profile in transgenic soybean (Yu et al., 2003). In these plants, activation of the flavonoid biosynthetic enzymes that consume naringenin and a lack of activation of downstream isoflavone-metabolizing enzymes led to decreased genistein and increased daidzein levels. Therefore, co-expression of the transcription factor with a silencing construct targeting flavanone 3β-hydroxylase (F3H) resulted in increased total isoflavone content (Yu et al., 2003). F3H is the major flavonoid enzyme that competes with IFS for the common substrate naringenin. Silencing of F3H reduced flavonoid biosynthesis and increased isoflavone accumulations.

In some foods, such as soy-based infant formula, consumers do not welcome phytoestrogens, even though these molecules have no documented adverse effects (Turck, 2007). Various strategies show promise to reduce isoflavonoid content for certain food uses. Disruption of IFS gene expression in soybean hairy root composite plants using RNA-mediated interference significantly reduces daidzein and genistein levels (Subramanian et al., 2005). Similarly, examination of TILLING populations with mutated IFS genes in Medicago truncatula indicates that some legumes can survive without isoflavones; however, the isoflavone-null soybean roots showed increased susceptibility to fungal infections (Graham et al., 2007). This result highlights the important role that isoflavonoids play in maintaining plant health. Interestingly, soybeans lacking isoflavones did not form nodules when inoculated with Bradyrhizobium japonicum, which is one of the nitrogen-fixing symbionts of soybean (Subramanian et al., 2006). Genetic and molecular analysis revealed a previously unknown function of isoflavones as internal inducers of production of bacterial nodulation factor inside of roots. This function is essential for successful nodulation (Subramanian et al., 2007). When rice plants expressing IFS were tested with the symbiotic bacterium B. japonicum, enhanced nodulation factor gene expression was observed (Sreevidya et al., 2006), suggesting that introduction of isoflavone biosynthesis into monocot crops may have significant benefits beyond increasing their nutritional value.

Catechins: synthesis, function, and green tea

Found mainly in fruit skins and seed coats, proanthocyanidins, or condensed tannins, protect plants against pathogens and herbivores as a result of their toxicity and interactions with digestive enzymes of the herbivores (Dixon et al., 2005). Ingestion of these molecules has diverse effects on animals (Cos et al., 2004). In ruminants, proanthocyanidins reduce pasture bloat. Interestingly, proanthocyanidins from blueberry, much like resveratrol, increase the lifespan of C. elegans (Wilson et al., 2006). Negative effects include reducing micronutrient uptake due to the chelating effect of proanthocyanidins on metal ions in animal feeds. The proanthocyanidins are polymers of simple phenylpropanoids known as flavan-3-ols. The monomer components of proanthocyanidines, such as leucoanthocyanidin, (+)-catechin and (−)-epicatechin have diverse biological effects.

To synthesize these monomeric components of proanthocyanidins (Figure 6a), F3H first oxidizes the flavanones generated by CHS and CHI (Marles et al., 2003; Xie and Dixon, 2005). F3H adds a hydroxyl group to the C3 position of the flavanones (hence ‘3-ol’). Next, dihydroflavonol reductase converts the resulting dihydroflavonol into a leucoanthocyanidin. Finally, leucoanthocyanidin reductase transforms this molecule into the final catechin (flavan-3-ol) product (Marles et al., 2003; Tanner et al., 2003; Wilmouth et al., 2002). Alternatively, anthocyanin synthase and anthocyanin reductase convert leucoanthocyanidins into (−)-epicatechin, the epimer of (+)-catechin (Figure 6b) (Pang et al., 2007; Wellmann et al., 2006; Xie et al., 2003). Finally, regio-specific polymerization of leucoanthocyanidin, (+)-catechin, and (−)-epicatechin yields proanthocyanidins.

Figure 6.

 Proanthocyanidin biosynthesis.
(a) The flavonoid biosynthetic enzymes provide naringenin for catetchin production (green). A series of enzyme-catalyzed reactions (purple) leads to the generation of catechin and epicatechins. The stereochemistry of the hydroxyl group in red is determined by the preceding enzymatic reactions.
(b) Chemical structures of catechin epimers (i.e. (+)-catechin and (−)-epicatechin) and catechin derivatives (EGC and EGCG).

As with the proanthocyanidins, the catechin monomers display complex effects on human physiology associated with consumption of green tea, cranberry juice, chocolate and grape seed extracts (Cos et al., 2004). All of these foods contain high levels of catechins. For example, green tea contains many epicatechin derivatives, including epigallocatechin (EGC) and epigallocatechin gallate (EGCG) (Figure 6b). These compounds inhibit cancer cell growth and reduce serum cholesterol levels (Stuart et al., 2006). Catechins and epicatechins in cranberry juice and chocolate have anti-oxidant activities, induce apoptosis in cancer cells, inhibit bacterial growth in urinary tract infections, and reduce atherosclerosis (Aruoma et al., 2006). Lastly, grape seed extracts rich in proanthocyanidins are effective anti-oxidants and may promote cardiac recovery after ischemia (Hwang et al., 2004).

To manipulate proanthocyanidin production in plants, researchers have targeted key enzymes and/or specific transcription factors in the pathway. For example, constitutive expression of the M. truncatula anthocyanidin reductase gene in tobacco, which does not normally accumulate proanthocyanidins, resulted in proanthocyanidin accumulation in the outer layers of flower petals with the compounds localized to vacuoles (Xie et al., 2006). Even when this gene was co-transformed with a transcription factor (PAP1) that specifically activates expression of anthocyanin pathway genes, the distribution of catechins and proanthocyanidins in flowers remained largely unchanged (Xie et al., 2006). This result suggests that unknown factors other than substrate availability limit proanthocyanidin production in heterologous systems.

Conclusion

The molecular foundations of phenylpropanoid synthesis in plants are well understood, as this pathway is arguably the best-studied plant secondary metabolic pathway. Researchers have described thousands of phenylpropanoids and polyketides in plants. Nevertheless, challenges remain in understanding how to control phenylpropanoid synthesis in plants or in microbial production systems, which are emerging as important for bioproduction of these molecules, and in the discovery of new chemical variations and metabolic pathways in this family of plant natural products. For example, the catabolic pathways of these compounds are largely unexplored. Similarly, the polymerization of proanthocyanidins is a long-standing mystery. Despite progress on understanding the structures and reactions of many enzymes in these pathways much work remains to be done. Elucidation of the molecular details of other enzymes, such as the cytochrome P450 mono-oxygenases, and the interactions between proteins in these pathways promise additional insights into the biological chemistry of this system. Finally, although the chemical diversity of phenylpropanoids, including polyketides, stilbenes, isoflavones and catechins, leads directly to varied biological functions in nature and in the human body, many of the effects of these molecules are incompletely understood.

Ancillary