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Metabolic engineering of natural products in plants; tools of the trade and challenges for the future
Available online at www.sciencedirect.com
Metabolic engineering of natural products in plants; tools of the trade and challenges for the future Shuiqin Wu and Joe Chappell Plant natural products play essential roles in plant survivability and many of them are used as nutrients, colorants, flavors, fragrances, and medicines. Genetic engineering of plants for natural products can help alleviate the demands for limited natural resources. Successes in enhancing production capacities have included manipulating blocks of genes coding for segments of pathways, over-expression of putative ratelimiting steps in pathways, expression of transcription factors regulating the entire metabolic pathways, and the construction of novel branch pathways capable of diverting carbon to the biosynthesis of unique metabolites in unexpected intracellular compartments. Further enhancements are likely if more efficient pathways can be constructed, providing for the efficient channeling of intermediates to final products, and if the means for sequestering natural products in planta can be accomplished. Addresses Department of Plant and Soil Sciences, University of Kentucky, 1405 Veterans Drive, Lexington, KY 40546, USA Corresponding author: Chappell, Joe ([email protected])
Current Opinion in Biotechnology 2008, 19:145–152 This review comes from a themed issue on Plant Biotechnology Edited by Joe Chappell and Erich Grotewold Available online 28th March 2008 0958-1669/$ – see front matter # 2008 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2008.02.007
The value of plant natural products There are approximately 300 000 documented species of higher plants on the planet [1] with more than 200 000 individual chemical entities, compounds referred to here as natural products (NPs), isolated and identified from these species till date [2] (Figure 1). Plant NPs can be further divided into primary and secondary metabolites. The primary metabolites, the constituents essential for all living cell types (plants, animal, and microbial), include such metabolites as sugars, fatty acids, amino, and nucleic acids, as well as the chemicals considered ubiquitous to all plants for growth and development, like growth regulators, cell wall components, and metabolites unique to photosynthetic organelles like chloroplasts. Secondary NPs are structurally and chemically much more diverse than the primary NPs, outnumber the primary metabwww.sciencedirect.com
olites by orders of magnitude, and are the primary focus of this review. An equally diverse array of secondary NPs are synthesized by microbial sources [3], but are not further considered here in order to focus our attention to the chemical diversity available from plants, one source that arguably has not been fully utilized for drug development. The vast number of secondary NPs is thought to be a consequence of ecological forces over evolutionary time. For instance, plant defense responses to predators, parasites, and diseases have been associated with the biosynthesis and accumulation of unique compounds representative of particular chemical families, and typically associated with a particular plant family. For example, capsidiol is an antimicrobial sesquiterpene biosynthesized by tobacco cells and tissues responding to microbial challenge [4]. Sesquiterpenes are but one particular class of compounds within the much larger isoprenoid or terpene family of compounds, and different plants within the Solanaceae produce unique and distinct sesquiterpenes [5]. By contrast, plants within the legume family defend themselves by producing flavonoid-type compounds [6], a very different class of compounds to terpenes. Plants in the Brassicaceae appear to have evolved quite a different defense strategy from either Leguminous or Solanacous species, and accumulate glucosinolates constitutively [7]. Secondary NPs have also been associated with interspecies (niche) competition, as well as improving reproductive success by serving as color and volatile cues for pollinators [8]. Given the biological activities and importance of secondary NPs to plants, it is not surprising that these compounds have come to play a crucial role for mankind in foods, food ingredients, and medicines [9]. Just over the past quarter of a century, nearly two-thirds of all newly approved drugs (between 1981 and 2002) were derived or inspired by NPs from natural sources [10,11]. Equally important, over two-thirds of the world population still relies on medicinal plants for their primary pharmaceutical care [12]. The richness of NPs lies within the structural complexity associated with these compounds. Feher and Schmidt [13] illustrated this in a widely cited article, which compared space-filling features of synthetic molecules to those of NPs and approved drugs. Their analysis concluded that drug molecules generally occupied significantly greater volumes than was evident in compounds arising from combinatorial synthetic efforts. By contrast, Current Opinion in Biotechnology 2008, 19:145–152
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Figure 1
An illustration of the plant-derived natural products discussed in this article. Representatives of the fatty acid (orange), cyanoglycoside (purple), phenylpropanoid (green), alkaloid (red) and isoprenoid (blue) families of compounds were chosen to emphasize the structural diversity and spatial complexity of natural products in general, and because many of these are highly desired compounds for their agricultural, medical and industrial applications.
NPs on average occupied similar spatial volumes as approved drugs. Feher and Schmidt [13] correlated the spatial complexity to the degree of chirality evident in the respective molecules: combinatorial synthetic molecules having on average less than one chiral center per molecule while NPs and approved drugs have multiple chiral centers.
The challenges of NPs from natural sources Expanded and extended use of plant NPs are limited largely by their availability. Taxol, the wonder anticancer Current Opinion in Biotechnology 2008, 19:145–152
drug originally extracted from the bark of yew (Taxus) trees accumulates to less than 0.5 g per tree [14]. Complicating matters further, NPs tend to accumulate in plants over long growth periods, often as mixtures of 10s to 100s of different compounds, and the quality of the extractable NPs often varies in relationship to geographical locale and climatic conditions. Sandalwood, for instance, is a highly valued parasitic tree grown in the tropical regions of Asia because it accumulates oils that can be processed into elegant fragrances. However, it needs to grow more than eight years before its wood can be extracted for the commercially valuable essential oils [15]. Unfortunately, the high value of sandalwood oil has resulted in over-harvesting of these trees, endangering the species in some regions, and forcing governments to impose regulations and control over the propagation and harvesting of these trees. If plant NPs are so desirable, but their utility is limited by the factors noted above, what are the alternatives for their production? Synthetic and semi-synthetic approaches are certainly means offering immediate opportunities to meet this challenge, but may not best serve our long-term advantage. The biological activities and specificities of plant NPs are derived largely from the structural complexity of these molecules, characteristics corresponding to the regio-specific modifications and stereo-chemical centers associated with these compounds. Synthetic chemists have developed facile means for inserting such chemical richness into molecules, but often with relatively low yields and resulting in racemic mixtures at each step in multistep syntheses. Having to couple together multiple synthetic steps of such complexity exacerbate the efficiency issues as illustrated by the final yield of 0.002% for the complete synthesis of Taxol [16]. The synthetic approaches are also often dependent on petroleum-based solvents and reactants for the actual reactions and for the purification of intermediates and reaction products, all at a time when reducing our dependence on petrochemicals is necessary and concerns about the hazards associated with the use and recycling of such solvents are worldwide priorities. Capturing the genetic blueprints for the biosynthesis of plant NPs and inserting these into fermentable organisms like yeast and bacteria represent a viable alternative to meet the production limitations [17,18–20,21]. Like synthetic chemistry, fermentation technology is a very mature technology, which has been widely adopted for commercial scale production wherever possible. And many features of fermentation address concerns about harvesting NPs from natural sources. For instance, fermentations are based on the biosynthesis of compounds by enzyme systems, typically yielding single, enantiomeric distinct compounds. Fermentations are performed in highly controllable conditions and in principle are scalable from very small quantities to very large. The www.sciencedirect.com
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downside of fermentation technologies is the reliance on large, costly infrastructures and their demands for energy. The energy demands include those for facilities in operations as well as the feedstocks (carbohydrate, nitrogen sources) used for growing the fermentation organisms. Plant metabolic engineering offers some unique opportunities that can complement, extend, and supplant the synthetic and fermentation options. Engineering plants with unique biosynthetic abilities (for intermediate or final products) requires many of the same tools as for engineering fermentation hosts — genetic blueprints for the actual biosynthetic genes, means for controlling the expression of these gene cassettes during the life-cycle of the plant, and development of downstream processing capacities in order to extract the desired NPs. These are cross-platform benefits. Unique windows include a better understanding of plant metabolism in general and the identification of limiting factors and genetic loci that can be utilized in more traditional breeding/genetic efforts for plant improvement. Arguably, the greatest benefits lie in the potential development of renewable and sustainable production platforms. Engineering the biosynthesis of NPs, including high-volume, high-demand commodity type compounds, into fast growing, high yielding annual plants draws on the natural capacity of plants to use sunlight and CO2 as their primary energy and feedstock sources. Sunlight represents one of the more desirable clean energies known to mankind and utilizing CO2 fixation to drive the biosynthesis of NPs rather than synthetic reactions dependent on petroleum-based chemicals certainly offers an opportunity to help mitigate a greenhouse gas contributing to global warming. It is these facets of plant metabolic engineering we hope to highlight in this article.
Successes till date Plant NPs can be classified into large groups based on their biosynthetic origins: glucosinolates and cyanoglucosides that share common biosynthetic origins from several amino acids and have common features like amine and glucose substituents; phenylpropanoids including flavonoids and polyketides are derived from phenylalanine and tyrosine; isoprenoids (also referred to as terpenoids) arise from the sequential condensation of acetyl-CoA units into a C5 building block, and alkaloids originate from any number of precursors possessing a heterocyclic nitrogen [22] (Figure 1). In the following section, we outline the salient features of these pathways as a preface to discussing some of the recent successes in altering these metabolic pathways in plants. These sections then provide the context for subsequent sections identifying some of the opportunities and challenges for the future engineering of these pathways. Engineering fatty acid metabolism
Although fatty acids are not typically considered as secondary metabolites, fatty acid derivatives can serve the www.sciencedirect.com
same defense functions as other secondary compounds in plants [23] and many have been identified for their industrial utilities [24]. Alteration of the fatty acid composition and profile in triglycerides was one of the first examples of a directed change in plant metabolism, wherein a thioesterase gene from the California Bay tree was introduced into Arabidopsis thaliana under the control of a seed-specific promoter diverted a significant portion of medium-chain fatty acids (laurate), rather than longchain fatty acids, from acyl-ACP into the triacylglycerol fraction accumulating in seeds [25]. Additional progress in altering the profile of triacyl lipids in developing oil seeds for fatty acids of importance to humans was recently reported by Wu et al. [26]. Using a combination of up to nine genes catalyzing a variety of condensation and desaturation reactions, these investigators were able to increase the levels of the pharmaceutically important long chain polyunsaturated fatty acids archidonic acid (AA) and eicosapentaenoic acid (EPA) from approximately 1% to a maximum of 25% of the total triacyl glycerol lipid levels. Neither of these studies, nor many others [24,27], reported significant enhancement in the overall fatty acid levels in seeds, just a change in the fatty acid profiles, which has led to speculation about the bottleneck in this metabolism and the identification of diacyl glycerol transferase as limiting biosynthesis [28]. Engineering phenylpropanoid metabolism
Plants produce a variety of compounds by the condensation of malonyl-CoA units with a so-called starter molecule typically derived from phenylalanine, in reactions that share similarity to those for fatty acid biosynthesis. However, the phenylpropanoid derivatives have one or more aromatic ring(s) in contrast to the long, un-branched aliphatic chains terminated by a carboxylic acid for fatty acids. The biosynthesis of these phenylpropanoid derivatives also occurs in the cytoplasm, but the precursors are derived from metabolism in other organelles including the chloroplast and mitochondria. Phenylalanine hence serves as an important precursor to polyketide biosynthesis as well as other chemical constituents like monolignols (components of cell wall lignans), and the highly colored anthocyanins and procyanidins pigments. Engineering of phenylpropanoid metabolism has been explored for well over 20 years because of its potential in agricultural and pharmaceutical applications. Even so, recent efforts highlight important progress in the development of alternative engineering strategies for phenylpropanoid metabolism in plants. Many different groups have tried to increase the levels of chalcones and stilbenes in plants because of their putative health benefits and their potential roles in fighting microbial diseases in plants [6]. In many cases, over-expression of a polyketide synthase gene from a distantly related plant species under the transcriptional control of a strong, constitutive promoter did in fact yield transgenic plants accumulating Current Opinion in Biotechnology 2008, 19:145–152
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detectable but very low levels of new phenylpropanoids. More recently, Xie et al. [29] (2006) reported that the over-expression of an Arabidopsis anthocyanidin reductase gene plus a Myb transcription factor previously associated with anthocyanin accumulation also in Arabidopsis, were sufficient to enhance the accumulation of anthocyanins and their diversion to condensed and oligomerized proanthocyanidins in vegetative tissues of tobacco and the model legume Medicago truncatula. The level of proanthocyanidins accumulating were particularly important with regards to their potential for controlling pasture bloat, a serious and potentially lethal condition in grazing cattle associated with excess methane production. Engineering alkaloid metabolism
Plant-derived alkaloids are a complicated group of compounds whose backbone scaffolds arise from a variety of biosynthetic origins and are named according to the heterocyclic nitrogen sources (Figure 1). For instance, pyridine alkaloids arise from nicotinic acid while indole alkaloids originate from tryptophan. Alkaloid biosynthesis is also complicated by the involvement of multiple cellular compartments and tissue-specific transformations (see Facchini review in this issue of COB for details). Nonetheless, significant progress in engineering alkaloids metabolism has been reported, which also highlights the need for advanced engineering designs. The genes responsible for caffeine biosynthesis have been identified and have been used to both upregulate and suppress caffeine metabolism. Ogita et al. [30,31] for instance used an RNAi strategy to silence the CaMXMT methyltransferase catalyzing the conversion of 7-methylxanthine to theobromine mRNA in two species of Coffea. These authors observed not only a suppression of the corresponding CaMXMT mRNA and enzyme activity but also a suppression of two other methyltransferases, CaXMT and CaDXMT, both necessary for caffeine biosynthesis also. Suppression of all three methyltransferases was not surprising because these genes share extensive sequence identity with one another. By contrast, Kim et al. [32] reported over-expression of these three Coffea methyltransferase genes in transgenic tobacco and observed 0.2–5 mg of caffeine/g fresh weight of transgenic leaf material. Wild-type (control) tobacco does not synthesize caffeine and therefore no caffeine accumulated in un-transformed leaves. Consistent with caffeine’s putative role as a defense compound in coffee plants, the transgenic tobaccos also exhibited a greatly enhanced resistance to cutworm, Spodoptera litura, herbivory. In one of the first examples of engineering plant metabolism via the manipulation of a transcriptional factor, van der Frits and Memelink [33] first isolated the ORCA3 Current Opinion in Biotechnology 2008, 19:145–152
transcription factor based on its ability to bind to ciselements found in the promoters of several terpene indole alkaloid (TIA) biosynthetic genes. Subsequent overexpression of the ORCA3 gene in Catharanthus roseus cell cultures was sufficient to elevate the level of several crucial TIA intermediates, tryptophan and tryptamine, as well as the overall accumulation of terpene indole alkaloids when the cultures were supplemented with downstream terpene biosynthetic precursors whose biosynthesis was independent of ORCA3. Engineering isoprenoid metabolism
Isoprenoid biosynthesis is an equally complicated biochemical labyrinth as that for phenylpropanoids or alkaloids. This occurs partly because of the involvement of two pathways that operate simultaneously in each cell. The mevalonate pathway (MEV) operates in the cytoplasm relying on acetyl-CoA units for the biosynthesis of isopentenyl diphoshpate, IPP, the universal 5-carbon building block for all isoprenoids. The methyl erythritol phosphate (MEP) pathway also yields the 5-carbon building block IPP but is localized to the plastid compartment and is initiated from pyruvate and 3-phosphoglycerate derived from fixed CO2 and the operation of the Calvin cycle. Both pathways lead to the formation of IPP and its allylic isomer dimethylallyl pyrophosphate (DMPP), the basic building blocks giving rise to all kinds of linear and cyclic compounds consisting of five carbons up to those consisting of 1000s of carbons as found in polymers like rubber [34]. Like many other secondary pathways, isoprenoids are decorated with many substituents added to the core scaffolds by coordinately regulated enzymes localized to the cytoplasm (Figures 1 and 2). Isoprenoids are the largest group of plant natural product based on the absolute number of compounds identified and the prospects for new derivatives to be found or biosynthesized (Table 1). They are broadly used in medicines, flavors, and fragrances [35]. Like many of the transgenic efforts noted above, manipulation of isoprenoid metabolism in plants has been tried for many years by the insertion of single gene constructs with the encoded gene products targeted to their respective cytoplasmic or plastidic compartments [36–39]. And while there has been some success in terms of transgenic plants producing a specific terpene end-product, the success has been tempered by the relatively modest levels of the terpene accumulating. Plant cells have a very complicated intracellular organization with metabolite flow between compartments highly regulated and orchestrated depending on the biosynthetic needs of the plants (Figure 2). This is the case for isoprenoid biosynthesis as well as several other NP pathways in plants. Moreover, the typical strategies for manipulating the isoprenoid pathways have been to over-express a suspect-limited enzyme targeted to their native intracelluwww.sciencedirect.com
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Figure 2
A schematic representation for several natural product biosynthetic pathways in plants to illustrate how coordinate interactions between intracellular compartments are important for the biosynthesis of these diverse and complex molecules. TCA, tricarboxylic acid cycle; IPP, isopentenyl diphosphate; Phe, phenylalanine; G3P, glyceraldehyde 3-phosphate.
lar location, either the cytoplasm or the chloroplast. And as already noted, such efforts have resulted in the biosynthesis of novel end products, but not at very high levels.
when targeted to the native locale. In particular, the upstream 5-carbon IPP produced in the chloroplast was diverted to 15-carbon FPP biosynthesis by a plastidtargeted FPP synthase. The newly created pool of FPP was then channeled to sesquiterpene production using a plastid-targeted sesquiterpene synthase.
Recently our laboratory has demonstrated an alternative system to generate transgenic plants yielding high levels of isoprenoids by the construction of novel branch pathways in expected and unexpected intracellular locales [40]. Sesquiterpenes (C15 terpenes) are typically biosynthesized in the cytosolic compartment via mevalonate pathway. When a novel sesquiterpene biosynthetic pathway was redirected from its natural cytosolic location to the chloroplast compartment, it dramatically increased terpene accumulation 100–10 000 times greater than
The success of Wu et al. [40] should be assessed relative to the work of others. In particular, Kappers et al. [41] overexpressed a strawberry mono-/sesqui-terpene synthase FaNES1 targeted to the mitochondria in Arabidopsis and observed the generation of small amounts of 4,8-dimethyl-1,3(E), 7-nonatriene, a nerolidol (C15) derivative. Although only minute amounts of this com-
Table 1 Structural diversity is greater amongst the secondary natural products (glucosinolates, phenylpropanoids, alkaloids and isoprenoids) rather than the primary products (lipids, carbohydrates, amino acids), and based on chemical rationalizations and predictions, there are many more secondary NPs waiting to be discovered (derived from [22,49]) Chemical class
# of compounds identified
Estimate of % discovered
Example uses
Lipids Carbohydrates Amino acids
100s 100s 100s
>80 >80 >90
Food additives, manufacturing Food processing, industrial Food and health supplements, industrial manufacturing
Glucosinolates, cyanoglucosides Phenylpropanoids Alkaloids Isoprenoids
100s 8000 12 000 25 000
>80 20–40 50–60 1–10
Food additives, medicinals Food and nutrient supplements, pharmaceuticals Pharmaceuticals Food manufacturing, flavor and fragrances, pharmaceuticals, manufacturing
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pound were produced, it was sufficient to attract carnivorous predator mites and demonstrated that a limited amount of FPP was probably biosynthesized in the mitochondria.
Lessons learned and challenges for the future Targeting of enzymes to non-native cellular compartments is obviously one means for sequestering and facilitating the biosynthesis of unique NPs. It is of course predicated on the availability of a suitable substrate pool, or at least the ability to build this capacity into the transgenic line, as described by Wu et al. [40]. However, what makes for the success of this strategy, in general, is perhaps the absence of the normal regulatory mechanisms that control the flux of carbon down any particular biosynthetic pathway and the absence of competition for pathway intermediates. Hence, if the strategy of Wu et al. [40] is to be generalized, then one would expect that redirecting other branch pathways, a branch of the shikimate pathway, for example, from the chloroplast, to the cytoplasm would have a similar impact on enhancing the accumulation of down-stream products. Many investigators have suggested that metabolic pathways may be organized into complexes or metabolons that more efficient channel substrate and intermediates down catalytic cascades to final products. For instance, phenylalanine ammonia lyase serves as the first enzyme committing phenylalanine to flavonoid/lignin metabolism and has been found to associate with 4-coumarate CoA ligase and cinnamate 4-hydroxylase in such a way as to limit the release of intermediates to the bulk solvent and has been characterized by direct physical interactions [42,43]. The work of Tattersall et al. [44] also suggested that Arabidopsis engineered for dhurrin, a cyanogenic glycoside, biosynthesis occurred via formation of a metabolon. In this case, when two multifunctional cytochrome P450s (CYP79A1 and CYP71E1), and one UDP-glucosyltransferase (UGT85B1) from sorghum were co-expressed in Arabidopsis, endogenously produced tyrosine was diverted to dhurrin biosynthesis without the accumulation of the cyanogenic biosynthetic intermediates. The lack of intermediate accumulation suggests limited escape of intermediates once they start down the dhurrin biosynthetic pathway or metabolon, which would have been likely if the three enzymes operated independently and in isolation from one another. The notion of metabolons is appealing to genetic engineers because it argues that more predictable outcomes of manipulating a pathway might be possible if the mechanisms responsible for metabolon assembly were known. Although there has been some success in physically linking successive enzymes into more efficient metabolic chains in vitro [45], Wu et al. [40] did not report much success with this type of approach. Perhaps it is not too surprising that these chimeric/hybrid proteins are not Current Opinion in Biotechnology 2008, 19:145–152
physiologically relevant. These artificial hybrid enzymes may fold and interact with other proteins in unusual ways, inadvertently distorting bona fide protein–protein interactions necessary for metabolon formation. Perhaps there are still other missing protein factors like Erg28 in yeast [46,47]. Erg28 is a small, apparently noncatalytic protein which serves to facilitate the assembly of enzymes for efficient sterol biosynthesis in yeast and man [48].
Conclusions and perspectives Plants have evolved a delicate regulatory system to coordinate metabolic activities. As a general view, genes are spatially and temporally expressed in precise patterns. At the cellular level, enzymes are localized to different but specific compartments. In some cases, multiple enzymes will gather together to form a complex in order to improve catalytic efficiency by minimizing intermediate diffusion and preventing intermediates from being lost to competing pathways and futile cycles. For the next generation of genetic engineers dedicated to improving Natural Product biosynthesis, being able to predictably generate sustainable production platforms for renewable resources in plants means taking full advantage of these rules (i.e. understanding the fundamentals).
Acknowledgements Work from the authors’ laboratory discussed here has been supported by grants from NSF and Firmenich, SA. We thank Drs Jeanne Rasbery and Walter Suza for their critical comments on this manuscript.
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40. Wu SQ, Schalk M, Clark A, Miles RB, Coates R, Chappell J: Redirection of cytosolic or plastidic isoprenoid precursors elevates terpene production in plants. Nat Biotechnol 2006, 24:1441-1447. The authors demonstrated an alternative means for achieving high yields of terpene production by redirecting a novel sesquiterpene biosynthetic pathway from its natural cytosolic location to the chloroplast compartment. Terpene accumulation was 100s–10 000s times higher than previous attempts. 41. Kappers IF, Aharoni A, van Herpen TWJM, Luckerhoff LLP, Dicke M, Bouwmeester HJ: Genetic engineering of terpenoid metabolism attracts, bodyguards to Arabidopsis. Science 2005, 309:2070-2072. 42. Jorgensen K, Rasmussen AV, Morant M, Nielsen AH, Bjarnholt N, Zagrobelny M, Bak S, Moller BL: Metabolon formation and metabolic channeling in the biosynthesis of plant natural products. Curr Opin Plant Biol 2005, 8:280-291. 43. Winkel BSJ: Metabolic channeling in plants. Annu Rev Plant Biol 2004, 55:85-107. 44. Tattersall DB, Bak S, Jones PR, Olsen CE, Nielsen JK, Hansen ML, Hoj PB, Moller BL: Resistance to an herbivore through engineered cyanogenic glucoside synthesis. Science 2001, 293:1826-1828. The entire dhurrin biosynthetic branch pathway consisting of two cytochrome P450s CYP71E1 and CYP79A1, and one glycosyltransferase HMNGT were transferred from their native host plant (sorghum) to Arabidopsis. The lack of intermediate accumulation suggested the
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formation of a metabolon for the efficient shuttling of metabolites for dhurrin biosynthesis. 45. Brodelius M, Lundgren A, Mercke P, Brodelius PE: Fusion of farnesyldiphosphate synthase and epi-aristolochene synthase, a sesquiterpene cyclase involved in capsidiol biosynthesis in Nicotiana tabacum. Eur J Biochem 2002, 269:3570-3577. 46. Mo CQ, Bard M: Erg28p is a key protein in the yeast sterol biosynthetic enzyme complex. J Lipid Res 2005, 46:1991-1998. Erg28p is an endoplasmic reticulum transmembrane protein and does not appear to have any enzymatic function in yeast. The authors demonstrated that Erg28p broadly interacts with many enzymes in the ergosterol biosynthetic pathway, and that it probably acts as a scaffold to tether those proteins together in forming a large, multienzyme complex. 47. Mo C, Valachovic M, Randall SK, Nickels JT, Bard M: Proteinprotein interactions among C-4 demethylation enzymes involved in yeast sterol biosynthesis. Proc Natl Acad Sci U S Am 2002, 99:9739-9744. 48. Gachotte D, Eckstein J, Barbuch R, Hughes T, Roberts C, Bard M: A novel gene conserved from yeast to humans is involved in sterol biosynthesis. J Lipid Res 2001, 42:150-154. 49. Buckingham Je: Dictionary of Natural Products on CD-ROM. London: Chapman & Hall; 1998:6.1.
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Metabolic engineering of natural products in plants; tools of the trade and challenges for the future Shuiqin Wu and Joe Chappell Plant natural products play essential roles in plant survivability and many of them are used as nutrients, colorants, flavors, fragrances, and medicines. Genetic engineering of plants for natural products can help alleviate the demands for limited natural resources. Successes in enhancing production capacities have included manipulating blocks of genes coding for segments of pathways, over-expression of putative ratelimiting steps in pathways, expression of transcription factors regulating the entire metabolic pathways, and the construction of novel branch pathways capable of diverting carbon to the biosynthesis of unique metabolites in unexpected intracellular compartments. Further enhancements are likely if more efficient pathways can be constructed, providing for the efficient channeling of intermediates to final products, and if the means for sequestering natural products in planta can be accomplished. Addresses Department of Plant and Soil Sciences, University of Kentucky, 1405 Veterans Drive, Lexington, KY 40546, USA Corresponding author: Chappell, Joe ([email protected])
Current Opinion in Biotechnology 2008, 19:145–152 This review comes from a themed issue on Plant Biotechnology Edited by Joe Chappell and Erich Grotewold Available online 28th March 2008 0958-1669/$ – see front matter # 2008 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2008.02.007
The value of plant natural products There are approximately 300 000 documented species of higher plants on the planet [1] with more than 200 000 individual chemical entities, compounds referred to here as natural products (NPs), isolated and identified from these species till date [2] (Figure 1). Plant NPs can be further divided into primary and secondary metabolites. The primary metabolites, the constituents essential for all living cell types (plants, animal, and microbial), include such metabolites as sugars, fatty acids, amino, and nucleic acids, as well as the chemicals considered ubiquitous to all plants for growth and development, like growth regulators, cell wall components, and metabolites unique to photosynthetic organelles like chloroplasts. Secondary NPs are structurally and chemically much more diverse than the primary NPs, outnumber the primary metabwww.sciencedirect.com
olites by orders of magnitude, and are the primary focus of this review. An equally diverse array of secondary NPs are synthesized by microbial sources [3], but are not further considered here in order to focus our attention to the chemical diversity available from plants, one source that arguably has not been fully utilized for drug development. The vast number of secondary NPs is thought to be a consequence of ecological forces over evolutionary time. For instance, plant defense responses to predators, parasites, and diseases have been associated with the biosynthesis and accumulation of unique compounds representative of particular chemical families, and typically associated with a particular plant family. For example, capsidiol is an antimicrobial sesquiterpene biosynthesized by tobacco cells and tissues responding to microbial challenge [4]. Sesquiterpenes are but one particular class of compounds within the much larger isoprenoid or terpene family of compounds, and different plants within the Solanaceae produce unique and distinct sesquiterpenes [5]. By contrast, plants within the legume family defend themselves by producing flavonoid-type compounds [6], a very different class of compounds to terpenes. Plants in the Brassicaceae appear to have evolved quite a different defense strategy from either Leguminous or Solanacous species, and accumulate glucosinolates constitutively [7]. Secondary NPs have also been associated with interspecies (niche) competition, as well as improving reproductive success by serving as color and volatile cues for pollinators [8]. Given the biological activities and importance of secondary NPs to plants, it is not surprising that these compounds have come to play a crucial role for mankind in foods, food ingredients, and medicines [9]. Just over the past quarter of a century, nearly two-thirds of all newly approved drugs (between 1981 and 2002) were derived or inspired by NPs from natural sources [10,11]. Equally important, over two-thirds of the world population still relies on medicinal plants for their primary pharmaceutical care [12]. The richness of NPs lies within the structural complexity associated with these compounds. Feher and Schmidt [13] illustrated this in a widely cited article, which compared space-filling features of synthetic molecules to those of NPs and approved drugs. Their analysis concluded that drug molecules generally occupied significantly greater volumes than was evident in compounds arising from combinatorial synthetic efforts. By contrast, Current Opinion in Biotechnology 2008, 19:145–152
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Figure 1
An illustration of the plant-derived natural products discussed in this article. Representatives of the fatty acid (orange), cyanoglycoside (purple), phenylpropanoid (green), alkaloid (red) and isoprenoid (blue) families of compounds were chosen to emphasize the structural diversity and spatial complexity of natural products in general, and because many of these are highly desired compounds for their agricultural, medical and industrial applications.
NPs on average occupied similar spatial volumes as approved drugs. Feher and Schmidt [13] correlated the spatial complexity to the degree of chirality evident in the respective molecules: combinatorial synthetic molecules having on average less than one chiral center per molecule while NPs and approved drugs have multiple chiral centers.
The challenges of NPs from natural sources Expanded and extended use of plant NPs are limited largely by their availability. Taxol, the wonder anticancer Current Opinion in Biotechnology 2008, 19:145–152
drug originally extracted from the bark of yew (Taxus) trees accumulates to less than 0.5 g per tree [14]. Complicating matters further, NPs tend to accumulate in plants over long growth periods, often as mixtures of 10s to 100s of different compounds, and the quality of the extractable NPs often varies in relationship to geographical locale and climatic conditions. Sandalwood, for instance, is a highly valued parasitic tree grown in the tropical regions of Asia because it accumulates oils that can be processed into elegant fragrances. However, it needs to grow more than eight years before its wood can be extracted for the commercially valuable essential oils [15]. Unfortunately, the high value of sandalwood oil has resulted in over-harvesting of these trees, endangering the species in some regions, and forcing governments to impose regulations and control over the propagation and harvesting of these trees. If plant NPs are so desirable, but their utility is limited by the factors noted above, what are the alternatives for their production? Synthetic and semi-synthetic approaches are certainly means offering immediate opportunities to meet this challenge, but may not best serve our long-term advantage. The biological activities and specificities of plant NPs are derived largely from the structural complexity of these molecules, characteristics corresponding to the regio-specific modifications and stereo-chemical centers associated with these compounds. Synthetic chemists have developed facile means for inserting such chemical richness into molecules, but often with relatively low yields and resulting in racemic mixtures at each step in multistep syntheses. Having to couple together multiple synthetic steps of such complexity exacerbate the efficiency issues as illustrated by the final yield of 0.002% for the complete synthesis of Taxol [16]. The synthetic approaches are also often dependent on petroleum-based solvents and reactants for the actual reactions and for the purification of intermediates and reaction products, all at a time when reducing our dependence on petrochemicals is necessary and concerns about the hazards associated with the use and recycling of such solvents are worldwide priorities. Capturing the genetic blueprints for the biosynthesis of plant NPs and inserting these into fermentable organisms like yeast and bacteria represent a viable alternative to meet the production limitations [17,18–20,21]. Like synthetic chemistry, fermentation technology is a very mature technology, which has been widely adopted for commercial scale production wherever possible. And many features of fermentation address concerns about harvesting NPs from natural sources. For instance, fermentations are based on the biosynthesis of compounds by enzyme systems, typically yielding single, enantiomeric distinct compounds. Fermentations are performed in highly controllable conditions and in principle are scalable from very small quantities to very large. The www.sciencedirect.com
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downside of fermentation technologies is the reliance on large, costly infrastructures and their demands for energy. The energy demands include those for facilities in operations as well as the feedstocks (carbohydrate, nitrogen sources) used for growing the fermentation organisms. Plant metabolic engineering offers some unique opportunities that can complement, extend, and supplant the synthetic and fermentation options. Engineering plants with unique biosynthetic abilities (for intermediate or final products) requires many of the same tools as for engineering fermentation hosts — genetic blueprints for the actual biosynthetic genes, means for controlling the expression of these gene cassettes during the life-cycle of the plant, and development of downstream processing capacities in order to extract the desired NPs. These are cross-platform benefits. Unique windows include a better understanding of plant metabolism in general and the identification of limiting factors and genetic loci that can be utilized in more traditional breeding/genetic efforts for plant improvement. Arguably, the greatest benefits lie in the potential development of renewable and sustainable production platforms. Engineering the biosynthesis of NPs, including high-volume, high-demand commodity type compounds, into fast growing, high yielding annual plants draws on the natural capacity of plants to use sunlight and CO2 as their primary energy and feedstock sources. Sunlight represents one of the more desirable clean energies known to mankind and utilizing CO2 fixation to drive the biosynthesis of NPs rather than synthetic reactions dependent on petroleum-based chemicals certainly offers an opportunity to help mitigate a greenhouse gas contributing to global warming. It is these facets of plant metabolic engineering we hope to highlight in this article.
Successes till date Plant NPs can be classified into large groups based on their biosynthetic origins: glucosinolates and cyanoglucosides that share common biosynthetic origins from several amino acids and have common features like amine and glucose substituents; phenylpropanoids including flavonoids and polyketides are derived from phenylalanine and tyrosine; isoprenoids (also referred to as terpenoids) arise from the sequential condensation of acetyl-CoA units into a C5 building block, and alkaloids originate from any number of precursors possessing a heterocyclic nitrogen [22] (Figure 1). In the following section, we outline the salient features of these pathways as a preface to discussing some of the recent successes in altering these metabolic pathways in plants. These sections then provide the context for subsequent sections identifying some of the opportunities and challenges for the future engineering of these pathways. Engineering fatty acid metabolism
Although fatty acids are not typically considered as secondary metabolites, fatty acid derivatives can serve the www.sciencedirect.com
same defense functions as other secondary compounds in plants [23] and many have been identified for their industrial utilities [24]. Alteration of the fatty acid composition and profile in triglycerides was one of the first examples of a directed change in plant metabolism, wherein a thioesterase gene from the California Bay tree was introduced into Arabidopsis thaliana under the control of a seed-specific promoter diverted a significant portion of medium-chain fatty acids (laurate), rather than longchain fatty acids, from acyl-ACP into the triacylglycerol fraction accumulating in seeds [25]. Additional progress in altering the profile of triacyl lipids in developing oil seeds for fatty acids of importance to humans was recently reported by Wu et al. [26]. Using a combination of up to nine genes catalyzing a variety of condensation and desaturation reactions, these investigators were able to increase the levels of the pharmaceutically important long chain polyunsaturated fatty acids archidonic acid (AA) and eicosapentaenoic acid (EPA) from approximately 1% to a maximum of 25% of the total triacyl glycerol lipid levels. Neither of these studies, nor many others [24,27], reported significant enhancement in the overall fatty acid levels in seeds, just a change in the fatty acid profiles, which has led to speculation about the bottleneck in this metabolism and the identification of diacyl glycerol transferase as limiting biosynthesis [28]. Engineering phenylpropanoid metabolism
Plants produce a variety of compounds by the condensation of malonyl-CoA units with a so-called starter molecule typically derived from phenylalanine, in reactions that share similarity to those for fatty acid biosynthesis. However, the phenylpropanoid derivatives have one or more aromatic ring(s) in contrast to the long, un-branched aliphatic chains terminated by a carboxylic acid for fatty acids. The biosynthesis of these phenylpropanoid derivatives also occurs in the cytoplasm, but the precursors are derived from metabolism in other organelles including the chloroplast and mitochondria. Phenylalanine hence serves as an important precursor to polyketide biosynthesis as well as other chemical constituents like monolignols (components of cell wall lignans), and the highly colored anthocyanins and procyanidins pigments. Engineering of phenylpropanoid metabolism has been explored for well over 20 years because of its potential in agricultural and pharmaceutical applications. Even so, recent efforts highlight important progress in the development of alternative engineering strategies for phenylpropanoid metabolism in plants. Many different groups have tried to increase the levels of chalcones and stilbenes in plants because of their putative health benefits and their potential roles in fighting microbial diseases in plants [6]. In many cases, over-expression of a polyketide synthase gene from a distantly related plant species under the transcriptional control of a strong, constitutive promoter did in fact yield transgenic plants accumulating Current Opinion in Biotechnology 2008, 19:145–152
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detectable but very low levels of new phenylpropanoids. More recently, Xie et al. [29] (2006) reported that the over-expression of an Arabidopsis anthocyanidin reductase gene plus a Myb transcription factor previously associated with anthocyanin accumulation also in Arabidopsis, were sufficient to enhance the accumulation of anthocyanins and their diversion to condensed and oligomerized proanthocyanidins in vegetative tissues of tobacco and the model legume Medicago truncatula. The level of proanthocyanidins accumulating were particularly important with regards to their potential for controlling pasture bloat, a serious and potentially lethal condition in grazing cattle associated with excess methane production. Engineering alkaloid metabolism
Plant-derived alkaloids are a complicated group of compounds whose backbone scaffolds arise from a variety of biosynthetic origins and are named according to the heterocyclic nitrogen sources (Figure 1). For instance, pyridine alkaloids arise from nicotinic acid while indole alkaloids originate from tryptophan. Alkaloid biosynthesis is also complicated by the involvement of multiple cellular compartments and tissue-specific transformations (see Facchini review in this issue of COB for details). Nonetheless, significant progress in engineering alkaloids metabolism has been reported, which also highlights the need for advanced engineering designs. The genes responsible for caffeine biosynthesis have been identified and have been used to both upregulate and suppress caffeine metabolism. Ogita et al. [30,31] for instance used an RNAi strategy to silence the CaMXMT methyltransferase catalyzing the conversion of 7-methylxanthine to theobromine mRNA in two species of Coffea. These authors observed not only a suppression of the corresponding CaMXMT mRNA and enzyme activity but also a suppression of two other methyltransferases, CaXMT and CaDXMT, both necessary for caffeine biosynthesis also. Suppression of all three methyltransferases was not surprising because these genes share extensive sequence identity with one another. By contrast, Kim et al. [32] reported over-expression of these three Coffea methyltransferase genes in transgenic tobacco and observed 0.2–5 mg of caffeine/g fresh weight of transgenic leaf material. Wild-type (control) tobacco does not synthesize caffeine and therefore no caffeine accumulated in un-transformed leaves. Consistent with caffeine’s putative role as a defense compound in coffee plants, the transgenic tobaccos also exhibited a greatly enhanced resistance to cutworm, Spodoptera litura, herbivory. In one of the first examples of engineering plant metabolism via the manipulation of a transcriptional factor, van der Frits and Memelink [33] first isolated the ORCA3 Current Opinion in Biotechnology 2008, 19:145–152
transcription factor based on its ability to bind to ciselements found in the promoters of several terpene indole alkaloid (TIA) biosynthetic genes. Subsequent overexpression of the ORCA3 gene in Catharanthus roseus cell cultures was sufficient to elevate the level of several crucial TIA intermediates, tryptophan and tryptamine, as well as the overall accumulation of terpene indole alkaloids when the cultures were supplemented with downstream terpene biosynthetic precursors whose biosynthesis was independent of ORCA3. Engineering isoprenoid metabolism
Isoprenoid biosynthesis is an equally complicated biochemical labyrinth as that for phenylpropanoids or alkaloids. This occurs partly because of the involvement of two pathways that operate simultaneously in each cell. The mevalonate pathway (MEV) operates in the cytoplasm relying on acetyl-CoA units for the biosynthesis of isopentenyl diphoshpate, IPP, the universal 5-carbon building block for all isoprenoids. The methyl erythritol phosphate (MEP) pathway also yields the 5-carbon building block IPP but is localized to the plastid compartment and is initiated from pyruvate and 3-phosphoglycerate derived from fixed CO2 and the operation of the Calvin cycle. Both pathways lead to the formation of IPP and its allylic isomer dimethylallyl pyrophosphate (DMPP), the basic building blocks giving rise to all kinds of linear and cyclic compounds consisting of five carbons up to those consisting of 1000s of carbons as found in polymers like rubber [34]. Like many other secondary pathways, isoprenoids are decorated with many substituents added to the core scaffolds by coordinately regulated enzymes localized to the cytoplasm (Figures 1 and 2). Isoprenoids are the largest group of plant natural product based on the absolute number of compounds identified and the prospects for new derivatives to be found or biosynthesized (Table 1). They are broadly used in medicines, flavors, and fragrances [35]. Like many of the transgenic efforts noted above, manipulation of isoprenoid metabolism in plants has been tried for many years by the insertion of single gene constructs with the encoded gene products targeted to their respective cytoplasmic or plastidic compartments [36–39]. And while there has been some success in terms of transgenic plants producing a specific terpene end-product, the success has been tempered by the relatively modest levels of the terpene accumulating. Plant cells have a very complicated intracellular organization with metabolite flow between compartments highly regulated and orchestrated depending on the biosynthetic needs of the plants (Figure 2). This is the case for isoprenoid biosynthesis as well as several other NP pathways in plants. Moreover, the typical strategies for manipulating the isoprenoid pathways have been to over-express a suspect-limited enzyme targeted to their native intracelluwww.sciencedirect.com
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Figure 2
A schematic representation for several natural product biosynthetic pathways in plants to illustrate how coordinate interactions between intracellular compartments are important for the biosynthesis of these diverse and complex molecules. TCA, tricarboxylic acid cycle; IPP, isopentenyl diphosphate; Phe, phenylalanine; G3P, glyceraldehyde 3-phosphate.
lar location, either the cytoplasm or the chloroplast. And as already noted, such efforts have resulted in the biosynthesis of novel end products, but not at very high levels.
when targeted to the native locale. In particular, the upstream 5-carbon IPP produced in the chloroplast was diverted to 15-carbon FPP biosynthesis by a plastidtargeted FPP synthase. The newly created pool of FPP was then channeled to sesquiterpene production using a plastid-targeted sesquiterpene synthase.
Recently our laboratory has demonstrated an alternative system to generate transgenic plants yielding high levels of isoprenoids by the construction of novel branch pathways in expected and unexpected intracellular locales [40]. Sesquiterpenes (C15 terpenes) are typically biosynthesized in the cytosolic compartment via mevalonate pathway. When a novel sesquiterpene biosynthetic pathway was redirected from its natural cytosolic location to the chloroplast compartment, it dramatically increased terpene accumulation 100–10 000 times greater than
The success of Wu et al. [40] should be assessed relative to the work of others. In particular, Kappers et al. [41] overexpressed a strawberry mono-/sesqui-terpene synthase FaNES1 targeted to the mitochondria in Arabidopsis and observed the generation of small amounts of 4,8-dimethyl-1,3(E), 7-nonatriene, a nerolidol (C15) derivative. Although only minute amounts of this com-
Table 1 Structural diversity is greater amongst the secondary natural products (glucosinolates, phenylpropanoids, alkaloids and isoprenoids) rather than the primary products (lipids, carbohydrates, amino acids), and based on chemical rationalizations and predictions, there are many more secondary NPs waiting to be discovered (derived from [22,49]) Chemical class
# of compounds identified
Estimate of % discovered
Example uses
Lipids Carbohydrates Amino acids
100s 100s 100s
>80 >80 >90
Food additives, manufacturing Food processing, industrial Food and health supplements, industrial manufacturing
Glucosinolates, cyanoglucosides Phenylpropanoids Alkaloids Isoprenoids
100s 8000 12 000 25 000
>80 20–40 50–60 1–10
Food additives, medicinals Food and nutrient supplements, pharmaceuticals Pharmaceuticals Food manufacturing, flavor and fragrances, pharmaceuticals, manufacturing
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pound were produced, it was sufficient to attract carnivorous predator mites and demonstrated that a limited amount of FPP was probably biosynthesized in the mitochondria.
Lessons learned and challenges for the future Targeting of enzymes to non-native cellular compartments is obviously one means for sequestering and facilitating the biosynthesis of unique NPs. It is of course predicated on the availability of a suitable substrate pool, or at least the ability to build this capacity into the transgenic line, as described by Wu et al. [40]. However, what makes for the success of this strategy, in general, is perhaps the absence of the normal regulatory mechanisms that control the flux of carbon down any particular biosynthetic pathway and the absence of competition for pathway intermediates. Hence, if the strategy of Wu et al. [40] is to be generalized, then one would expect that redirecting other branch pathways, a branch of the shikimate pathway, for example, from the chloroplast, to the cytoplasm would have a similar impact on enhancing the accumulation of down-stream products. Many investigators have suggested that metabolic pathways may be organized into complexes or metabolons that more efficient channel substrate and intermediates down catalytic cascades to final products. For instance, phenylalanine ammonia lyase serves as the first enzyme committing phenylalanine to flavonoid/lignin metabolism and has been found to associate with 4-coumarate CoA ligase and cinnamate 4-hydroxylase in such a way as to limit the release of intermediates to the bulk solvent and has been characterized by direct physical interactions [42,43]. The work of Tattersall et al. [44] also suggested that Arabidopsis engineered for dhurrin, a cyanogenic glycoside, biosynthesis occurred via formation of a metabolon. In this case, when two multifunctional cytochrome P450s (CYP79A1 and CYP71E1), and one UDP-glucosyltransferase (UGT85B1) from sorghum were co-expressed in Arabidopsis, endogenously produced tyrosine was diverted to dhurrin biosynthesis without the accumulation of the cyanogenic biosynthetic intermediates. The lack of intermediate accumulation suggests limited escape of intermediates once they start down the dhurrin biosynthetic pathway or metabolon, which would have been likely if the three enzymes operated independently and in isolation from one another. The notion of metabolons is appealing to genetic engineers because it argues that more predictable outcomes of manipulating a pathway might be possible if the mechanisms responsible for metabolon assembly were known. Although there has been some success in physically linking successive enzymes into more efficient metabolic chains in vitro [45], Wu et al. [40] did not report much success with this type of approach. Perhaps it is not too surprising that these chimeric/hybrid proteins are not Current Opinion in Biotechnology 2008, 19:145–152
physiologically relevant. These artificial hybrid enzymes may fold and interact with other proteins in unusual ways, inadvertently distorting bona fide protein–protein interactions necessary for metabolon formation. Perhaps there are still other missing protein factors like Erg28 in yeast [46,47]. Erg28 is a small, apparently noncatalytic protein which serves to facilitate the assembly of enzymes for efficient sterol biosynthesis in yeast and man [48].
Conclusions and perspectives Plants have evolved a delicate regulatory system to coordinate metabolic activities. As a general view, genes are spatially and temporally expressed in precise patterns. At the cellular level, enzymes are localized to different but specific compartments. In some cases, multiple enzymes will gather together to form a complex in order to improve catalytic efficiency by minimizing intermediate diffusion and preventing intermediates from being lost to competing pathways and futile cycles. For the next generation of genetic engineers dedicated to improving Natural Product biosynthesis, being able to predictably generate sustainable production platforms for renewable resources in plants means taking full advantage of these rules (i.e. understanding the fundamentals).
Acknowledgements Work from the authors’ laboratory discussed here has been supported by grants from NSF and Firmenich, SA. We thank Drs Jeanne Rasbery and Walter Suza for their critical comments on this manuscript.
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