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Plant production systems for bioactive small molecules
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Plant production systems for bioactive small molecules Eng-Kiat Lim and Dianna Bowles Bioactive small molecules are important dietary components of food, as well as being widely used in diverse industrial sectors, from flavours, fragrances and sweeteners through to natural pesticides and pharmaceuticals. Plants already manufacture many of these bioactives, but often in yields that are not commercially competitive. There are a variety of new pathway engineering, cell culture and molecular breeding strategies in use and in development to improve yield and the robust supply of bioactives in planta. In the future, biorefining applications are likely to play a significant role in providing chemical intermediates for bioactive production from biomass feedstocks. Address Centre for Novel Agricultural Products, Department of Biology, University of York, York YO10 5DD, UK Corresponding author: Bowles, Dianna ([email protected])
Current Opinion in Biotechnology 2012, 23:271–277 This review comes from a themed issue on Plant biotechnology Edited by Dianna Bowles and Stephen Long Available online 3rd January 2012 0958-1669/$ – see front matter # 2011 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2011.12.008
Introduction Plants have evolved an exquisite sensitivity to changes in their environments, responding rapidly and adapting to maximise opportunities and minimise risks. One consequence of this ability is the extraordinary molecular diversity that exists today in land-based, fresh water and marine plants. The range of small molecule metabolites is vast and reflects functionalities related to biotic and abiotic stress responses, developmental and metabolic plasticity as well as interactions with other beneficial and harmful organisms in the ecosystem. The utility of small molecules, many made by the pathways of plant secondary metabolism, has long been recognised in many industrial sectors. Recent reviews include those functionalities related to health [1], nutrition [2], fragrances and flavours [3], sweeteners [4], natural pesticides and insect repellents [5,6] (Table 1). Often the molecular nature of the bioactive in each application has been identified and has provided the starting point for designing new strategies to increase www.sciencedirect.com
the productivity of the plant system. Undoubtedly as the activity screens widen and the assay technologies improve, the number of small molecules with interesting and economically relevant properties will evermore increase. Whilst plants produce valuable small molecules, the yield may not be high and the plant production system has rarely been optimised into a robust agricultural or horticultural crop. Many of the potentially useful land-based species continue to be cultivated in extensive low-input systems in developing countries by small-scale farmers and these uncertainties can lead to insecure supply chains. The cultivation of aquatic species currently focuses on microalgae, but technologies require considerable improvements in efficiency. There are the added post-harvest issues of extraction and the barriers preventing a robust availability of pure products for the downstream commercial manufacturers. These difficulties have led to an increased interest in synthetic biology and microbial fermentation, specifically when the metabolic steps and gene products involved in the synthesis of a high value small molecule are known. Nevertheless, with the advances in molecular breeding and gene technologies, the full benefits of plant-based synthesis are starting to be realised either as an alternative or as a complement to industrial fermentation. First, current research strategies are reviewed to assess the range of new biotechnologies in development for increasing yields of bioactive small molecules and increasing the quality of the products made by the plant. The application of these technologies to specific examples is then summarised. The reader is also referred to a number of relevant reviews in plant natural products [1–7] and plant secondary metabolism [8,9].
Strategies for increasing productivity and quality Pathway engineering
In principle, engineering the metabolic pathways affecting the yield of specific bioactives is the most direct route to altering plant productivity. In practice, this can be difficult to achieve given the plasticity of plant metabolism and the lack of detailed understanding both about individual steps in the biosynthesis and the direct and indirect impacts of altering flux through the pathways. However, in recent years there have been successful strategies in which key biosynthetic genes from native or non-native sources have been overexpressed, genes encoding enzymes that divert precursors from the desired Current Opinion in Biotechnology 2012, 23:271–277
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Table 1 Examples of bioactive small molecules referenced in the review. Small molecules
Reference
Artemisinin, quinine, morphine, taxol Vitamins, carotenoids, flavonols, catechins, anthocyanins, genistein, daidzein, resveratrol, lycopene Vanillin, menthol, eugenol, limonene, 2-phenylethel alcohol, linalool, benzyl acetate, ionones, anethole, cinnamaldehyde Rebaudioside A, stevioside D-Limonene, pyrethrins, rotenone, azadirachtin, sabadilla p-Menthane-3,8-diol, citronellal, limonene, geraniol, isopulegol, d-pinene, 1,8-cineole, a-pinene, eugenol, carvacrol
[1] [2] [3]
Functionality Health Nutrition Fragrances/flavours Sweeteners Pesticides Insect repellents
pathways have been knocked out, and protein engineering has been combined with the transgenic approach to express enzymes or regulator proteins with altered activities. For example, levels of bioactives such as beneficial fatty acids [10], stilbene and flavonoid anti-oxidants [11,12], anticholinergic alkaloids [13], and glucosinolates for use as chemoprotective agents [14], have all been enhanced via overexpression of the relevant plant biosynthetic genes. Genes from non-plant sources have also been used in plant metabolite engineering. For example, the content of carotenoids in potato tubers have been increased through overexpression of multiple bacterial genes [15]. A more striking example has been demonstrated by Naqvi et al. [16] in which a transgenic corn containing high levels of multivitamins was generated through introducing biosynthetic genes from three distinct metabolic pathways into the plant (Figure 1). Changing the expression of transcription factors in transgenic plants, or the use of RNA silencing has also proved to be successful strategies. Recent examples include the altered expression of Myb transcription factors such as PAP1 and LAP1 to activate the anthocyanin pathway and generate transgenic plants with high anthocyanin and phenolic contents [17,18]. Also RNAi has been used to knock out metabolic pathways reprogramming metabolic flux from synthesis of the flavone apigenin to the isoflavone genistein [19]. Another interesting application of RNAi in metabolic engineering is to knock out the native pathway and then use the transgenic cells as biotransformation factories; Runguphan et al. [20] have used this approach successfully to synthesise non-natural alkaloids. End-product toxicity and feedback inhibition may restrict the over-production of bioactives in plants due to the insolubility and toxicity of many small molecules. To avoid these problems, plants have developed specialised structures for storage. Some lipophilic molecules are synthesised and accumulated in oil ducts and trichomes, whereas many others are modified with sugars to increase their water solubility and are maintained in the vacuole, an intracellular compartment with massive storage Current Opinion in Biotechnology 2012, 23:271–277
[4] [5] [6]
capacity. Sugar modifications of small molecules are carried out by glycosyltransferases [21]. Overexpression of glycosyltransferases can lead to the high production of specific small molecule glycosides, as demonstrated in the study of phenylpropanoid homeostasis [22]. Thus, coexpression of genes encoding biosynthetic enzymes with those genes encoding glycosyltransferases can be an important alternative means to increase the yield of bioactives in plants. A major issue in the application of transgenic approaches to pathway engineering is the time and cost required to generate stably transformed plants, with no certainty that the new lines will be useful for commercial production of the bioactive. Any strategy to pre-evaluate the transgenic design is therefore highly desirable. In this context, transient gene expression in Nicotiana benthamiana by infiltration of recombinant agrobacteria has increasingly been used as a rapid assessment tool [23]. Recent examples include the optimisation of metabolic engineering for fatty acids [24,25] and glucosinolates [26]. These strategies, combined with recent advances in gene discovery [27,28–30,31], protein engineering of key biosynthetic enzymes [32,33,34], and the development of in silico mathematical modelling to guide experimental design [35], all point to the probability that pathway engineering will be used to increase the yields of many more diverse bioactives over the coming years. Culture technologies
The use of plant tissue culture systems such as suspension cells and hairy root cultures have several advantages over cultivated plants for the production of bioactives. First, genetic modification in a contained system can readily be applied without the regulatory barriers associated with field grown crops. Second, a cell/tissue culture system can be scaled up in bioreactors with controllable production rates. Also, given the relative simplicity of the culture systems, harvest and extraction processes generally incur relatively low costs. Taxol, the anti-cancer drug, is perhaps the most well-known bioactive that has been successfully produced via tissue culture technology. www.sciencedirect.com
Bioactive small molecule production in plants Lim and Bowles 273
Figure 1
Dehydroascorbate β-CAROTENE
Monodehydroascorbate
Lycopene
L-ASCORBATE
Oryza sativa dehydroascorbate reductase
Pantoea ananatis carotene desaturase ζ-Carotene
L-Galactose-1,4-lactone
Pantoea ananatis carotene desaturase Phytoene Zea mays phytoene synthase 1
L-Galactose
Geranylgeranylpyrophosphate × 2
L-Galactose-1-phosphate
Geranylpyrophosphate
GDP-L-galactose
Mevalonic acid
GDP-D-mannose
Acetyl-CoA
D-Mannose-1-phosphate
TETRAHYDROFOLATE-Glun Tetrahydrofolate
GTP
Dihydrofolate
Dihydroneopterin triphosphate
Chorismate
E. coli GTP cyclohydrolase I
Aminodeoxy-chorismate Dihydropteroate
Dihydroneopterin phosphate
p-Aminobenzoate
Plastid
Hydroxymethyldihydropterin pyrophosphate
Dihydroneopterin
Hydroxymethyldihydropterin
Hydroxymethyldihydropterin
Mitochondrion Current Opinion in Biotechnology
A transgenic corn plant was engineered using multiple constructs to affect three distinct metabolic pathways to generate high contents of b-carotene (precursor of vitamin A), ascorbic acid (vitamin C) and folic acid (vitamin B9) [16]. The enzyme reactions affected through engineering are colourcoded, together with the final products that have increased levels.
There are a number of strategies in common use for enhancing yields of bioactives in cell/tissue culture systems. These can involve optimisation of growth conditions to allow scale-up of the biomass for production, use of abiotic and biotic elicitors, and coculture with other organisms. For example, the biomass of a hairy root culture system of Artemisia annua has been scaled up to 20 L in a mist reactor [36]. Application of an electrical stimulus has been used as an effective abiotic elicitor to stimulate the production of phenolics in tissue cultures [37], and a range of elicitors such as oligosaccharides and polysaccharides, jasmonic acid and sodium nitroprusside have been shown to promote levels of many different bioactives in culture www.sciencedirect.com
systems including artemisinin, isoflavones, stilbenes, diterpenes and alkaloids [38–42]. Co-culture with other organisms, such as fungi, has also been used to enhance the production of podophyllotoxin and taxol, two anticancer bioactives, in Linum album and Taxus chinensis suspension cells, respectively [43,44]. In earlier years, the major challenge facing the use of plant cell/tissue cultures for bioactive production was the relatively low productivity of the culture systems available. However, this limitation can now be overcome by modern bioreactor technologies, such as those that enable the cultures to be scaled up in 20 000 L bioreactors [45]. Combining these new production systems Current Opinion in Biotechnology 2012, 23:271–277
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with the continuing advances in gene discovery and pathway engineering, it is highly probable that cell culture technologies will increasingly be used in the future to bring many more plant bioactives to commercialisation.
Fast-track breeding
Plant breeding is the traditional approach to improve crop quality. In recent years, this approach has also been applied to medicinal plants for selecting cultivars with higher content of bioactives. For rapid and effective selection of desirable cultivars, ‘fast-track breeding’ benefits from knowledge of the genetic maps of the plants, relevant quantitative trait loci (QTL) and biomarkers associated with the synthesis and yield of target bioactives. Since the complete genome of Arabidopsis thaliana was published in 2000, there have been many additional plant genomes sequenced, including those of rice, grapevine, corn, apple, potato and soybean. These have contributed substantial information to underpin yield improvement of bioactives including isoflavonoids, flavonoids, terpenes and many other bioactive natural products, such as reviewed in [8]. The yield of bioactives in field crops will always reflect not only both the content of the bioactives, but also importantly, the biomass of the plant. Extractability of the improved varieties is also an important parameter, with lack of contaminating co-products determining utility for the commercial end-users. Detailed genetic information of several medicinal plants, such as Artemisia annua [46], Catharanthus roseus [47], Taxus cuspidate [48], Allium sativum [49], Epimedium sagittatum [50], Papaver somniferum [51], have been reported recently. These databases represent powerful tools to support the molecular breeding of bioactives in plants, as well as providing a foundation for synthetic biology and industrial fermentation routes. In this context, artemisinin, made by the Chinese medicinal plant, Artemisia annua, underpins the production of artemisinin combination therapies (ACTs). These are essential anti-malarials, recommended by WHO. Cost of supply of artemisinin, together with the capacity and robustness of its production are significant factors for manufacturers of the pharmaceuticals, particularly given the estimated rise in global demand of ACTs over the coming years. Increased knowledge of the genes encoding the enzymes involved in synthesis of artemisinic acid and regulation of the pathway [52,53–55], together with the first genetic map of A. annua and identification of relevant QTL [46], have all contributed to new strategies for enhanced production of the bioactive. Both the cost of product to manufacturers and ease of its availability are likely to dictate future market-led decisions to increase supply of artemisinin through higher crop yields Current Opinion in Biotechnology 2012, 23:271–277
in the field, versus the use of microbial-based industrial fermentation. Improved sequencing technologies have facilitated rapid developments in forward genetic research. It is clear that many more non-GM molecular breeding platforms will be established in the next years to generate high-yielding varieties. The use of GM can offer an alternative route but whilst the technologies may be available, the cost and time of bringing the new product through regulatory hurdles to the market often precludes its selection as a commercialisation route.
Feedstocks for modification and synthesis Plant biomass is now acknowledged as an alternative to the use of fossil reserves for fuel production. In parallel, the use of plant-based feedstocks for chemical production has also gained interest and biorefinery applications have become many and varied. A zero-waste biorefinery capable of using inputs from a diverse range of crop/ forestry feedstocks presents an immense opportunity to maximise the value of biomass and produce multiple chemical intermediates. In turn, high value bioactives can either be extracted and further modified post-extraction, or synthesised de novo from those intermediates. Feedstocks from plant biomass include carbohydrates, lignin and lipids and their potential for bioactive production has been reviewed, such as in [56]. For example, carbohydrates and lignin can lead to chemical intermediates such as levulinic acid, vanillin and many other aromatic molecules for the synthesis of pharmaceuticals [56]. Lipids can be used to produce beneficial polyunsaturated fatty acids such as omega-3 [57]. In addition to the principal feedstocks, a variety of small molecules, whilst representing only a small proportion of the biomass, have also attracted considerable interest in biorefining, due to their high value either as chemical intermediates or bioactives. For example, shikimic acid, a key intermediate for the antiviral drug Tamiflu, is extracted from the tree species, Liquidambar styraciflua, before further conversion of the biomass to biofuels [58]. In the coming years, combining designer feedstocks with new biotechnologies and chemical/biochemical possessing will undoubtedly widen the utility of biorefineries to produce specific bioactives in high yields.
Conclusion The decline of fossil reserves and the consequent increase in costs of energy and petrochemicals derived from those reserves are leading to recognition that a global bio-based economy needs to be developed. In future years, the products of agriculture and forestry must support industrial sectors beyond the provision of food, animal feed and energy. High value bioactive chemicals will increasingly be derived directly or indirectly from www.sciencedirect.com
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plants. In addition to the highlighted research in land plants, microalgal biotechnology has recently emerged as a new platform for bioactive production [59]. The biotechnologies to increase productivity are already available. As assays are developed to discover new bioactivities of plant natural products, increasing applications will be coming to market.
Acknowledgement The Leverhulme Trust is thanked for a senior research fellowship to DJB to study the bioactivity of plant natural products.
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57. Octave S, Thomas D: Biorefinery: toward an industrial metabolism. Biochimie 2009, 91:659-664. 58. Martin E, Duke J, Pelkki M, Clausen EC, Carrier DJ: Sweetgum (Liquidambar styraciflua L.): extraction of shikimic acid coupled to dilute acid pretreatment. Appl Biochem Biotechnol 2010, 162:1660-1668.
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59. Harun R, Singh M, Forde GM, Danquah MK: Bioprocess engineering of microalgae to produce a variety of consumer products. Renew Sust Energy Rev 2010, 14:1037-1047. This review discusses recent developments in microalgae biotechnology and their potential for production of both bioactive chemicals and bioenergy.
Current Opinion in Biotechnology 2012, 23:271–277
Plant production systems for bioactive small molecules Eng-Kiat Lim and Dianna Bowles Bioactive small molecules are important dietary components of food, as well as being widely used in diverse industrial sectors, from flavours, fragrances and sweeteners through to natural pesticides and pharmaceuticals. Plants already manufacture many of these bioactives, but often in yields that are not commercially competitive. There are a variety of new pathway engineering, cell culture and molecular breeding strategies in use and in development to improve yield and the robust supply of bioactives in planta. In the future, biorefining applications are likely to play a significant role in providing chemical intermediates for bioactive production from biomass feedstocks. Address Centre for Novel Agricultural Products, Department of Biology, University of York, York YO10 5DD, UK Corresponding author: Bowles, Dianna ([email protected])
Current Opinion in Biotechnology 2012, 23:271–277 This review comes from a themed issue on Plant biotechnology Edited by Dianna Bowles and Stephen Long Available online 3rd January 2012 0958-1669/$ – see front matter # 2011 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2011.12.008
Introduction Plants have evolved an exquisite sensitivity to changes in their environments, responding rapidly and adapting to maximise opportunities and minimise risks. One consequence of this ability is the extraordinary molecular diversity that exists today in land-based, fresh water and marine plants. The range of small molecule metabolites is vast and reflects functionalities related to biotic and abiotic stress responses, developmental and metabolic plasticity as well as interactions with other beneficial and harmful organisms in the ecosystem. The utility of small molecules, many made by the pathways of plant secondary metabolism, has long been recognised in many industrial sectors. Recent reviews include those functionalities related to health [1], nutrition [2], fragrances and flavours [3], sweeteners [4], natural pesticides and insect repellents [5,6] (Table 1). Often the molecular nature of the bioactive in each application has been identified and has provided the starting point for designing new strategies to increase www.sciencedirect.com
the productivity of the plant system. Undoubtedly as the activity screens widen and the assay technologies improve, the number of small molecules with interesting and economically relevant properties will evermore increase. Whilst plants produce valuable small molecules, the yield may not be high and the plant production system has rarely been optimised into a robust agricultural or horticultural crop. Many of the potentially useful land-based species continue to be cultivated in extensive low-input systems in developing countries by small-scale farmers and these uncertainties can lead to insecure supply chains. The cultivation of aquatic species currently focuses on microalgae, but technologies require considerable improvements in efficiency. There are the added post-harvest issues of extraction and the barriers preventing a robust availability of pure products for the downstream commercial manufacturers. These difficulties have led to an increased interest in synthetic biology and microbial fermentation, specifically when the metabolic steps and gene products involved in the synthesis of a high value small molecule are known. Nevertheless, with the advances in molecular breeding and gene technologies, the full benefits of plant-based synthesis are starting to be realised either as an alternative or as a complement to industrial fermentation. First, current research strategies are reviewed to assess the range of new biotechnologies in development for increasing yields of bioactive small molecules and increasing the quality of the products made by the plant. The application of these technologies to specific examples is then summarised. The reader is also referred to a number of relevant reviews in plant natural products [1–7] and plant secondary metabolism [8,9].
Strategies for increasing productivity and quality Pathway engineering
In principle, engineering the metabolic pathways affecting the yield of specific bioactives is the most direct route to altering plant productivity. In practice, this can be difficult to achieve given the plasticity of plant metabolism and the lack of detailed understanding both about individual steps in the biosynthesis and the direct and indirect impacts of altering flux through the pathways. However, in recent years there have been successful strategies in which key biosynthetic genes from native or non-native sources have been overexpressed, genes encoding enzymes that divert precursors from the desired Current Opinion in Biotechnology 2012, 23:271–277
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Table 1 Examples of bioactive small molecules referenced in the review. Small molecules
Reference
Artemisinin, quinine, morphine, taxol Vitamins, carotenoids, flavonols, catechins, anthocyanins, genistein, daidzein, resveratrol, lycopene Vanillin, menthol, eugenol, limonene, 2-phenylethel alcohol, linalool, benzyl acetate, ionones, anethole, cinnamaldehyde Rebaudioside A, stevioside D-Limonene, pyrethrins, rotenone, azadirachtin, sabadilla p-Menthane-3,8-diol, citronellal, limonene, geraniol, isopulegol, d-pinene, 1,8-cineole, a-pinene, eugenol, carvacrol
[1] [2] [3]
Functionality Health Nutrition Fragrances/flavours Sweeteners Pesticides Insect repellents
pathways have been knocked out, and protein engineering has been combined with the transgenic approach to express enzymes or regulator proteins with altered activities. For example, levels of bioactives such as beneficial fatty acids [10], stilbene and flavonoid anti-oxidants [11,12], anticholinergic alkaloids [13], and glucosinolates for use as chemoprotective agents [14], have all been enhanced via overexpression of the relevant plant biosynthetic genes. Genes from non-plant sources have also been used in plant metabolite engineering. For example, the content of carotenoids in potato tubers have been increased through overexpression of multiple bacterial genes [15]. A more striking example has been demonstrated by Naqvi et al. [16] in which a transgenic corn containing high levels of multivitamins was generated through introducing biosynthetic genes from three distinct metabolic pathways into the plant (Figure 1). Changing the expression of transcription factors in transgenic plants, or the use of RNA silencing has also proved to be successful strategies. Recent examples include the altered expression of Myb transcription factors such as PAP1 and LAP1 to activate the anthocyanin pathway and generate transgenic plants with high anthocyanin and phenolic contents [17,18]. Also RNAi has been used to knock out metabolic pathways reprogramming metabolic flux from synthesis of the flavone apigenin to the isoflavone genistein [19]. Another interesting application of RNAi in metabolic engineering is to knock out the native pathway and then use the transgenic cells as biotransformation factories; Runguphan et al. [20] have used this approach successfully to synthesise non-natural alkaloids. End-product toxicity and feedback inhibition may restrict the over-production of bioactives in plants due to the insolubility and toxicity of many small molecules. To avoid these problems, plants have developed specialised structures for storage. Some lipophilic molecules are synthesised and accumulated in oil ducts and trichomes, whereas many others are modified with sugars to increase their water solubility and are maintained in the vacuole, an intracellular compartment with massive storage Current Opinion in Biotechnology 2012, 23:271–277
[4] [5] [6]
capacity. Sugar modifications of small molecules are carried out by glycosyltransferases [21]. Overexpression of glycosyltransferases can lead to the high production of specific small molecule glycosides, as demonstrated in the study of phenylpropanoid homeostasis [22]. Thus, coexpression of genes encoding biosynthetic enzymes with those genes encoding glycosyltransferases can be an important alternative means to increase the yield of bioactives in plants. A major issue in the application of transgenic approaches to pathway engineering is the time and cost required to generate stably transformed plants, with no certainty that the new lines will be useful for commercial production of the bioactive. Any strategy to pre-evaluate the transgenic design is therefore highly desirable. In this context, transient gene expression in Nicotiana benthamiana by infiltration of recombinant agrobacteria has increasingly been used as a rapid assessment tool [23]. Recent examples include the optimisation of metabolic engineering for fatty acids [24,25] and glucosinolates [26]. These strategies, combined with recent advances in gene discovery [27,28–30,31], protein engineering of key biosynthetic enzymes [32,33,34], and the development of in silico mathematical modelling to guide experimental design [35], all point to the probability that pathway engineering will be used to increase the yields of many more diverse bioactives over the coming years. Culture technologies
The use of plant tissue culture systems such as suspension cells and hairy root cultures have several advantages over cultivated plants for the production of bioactives. First, genetic modification in a contained system can readily be applied without the regulatory barriers associated with field grown crops. Second, a cell/tissue culture system can be scaled up in bioreactors with controllable production rates. Also, given the relative simplicity of the culture systems, harvest and extraction processes generally incur relatively low costs. Taxol, the anti-cancer drug, is perhaps the most well-known bioactive that has been successfully produced via tissue culture technology. www.sciencedirect.com
Bioactive small molecule production in plants Lim and Bowles 273
Figure 1
Dehydroascorbate β-CAROTENE
Monodehydroascorbate
Lycopene
L-ASCORBATE
Oryza sativa dehydroascorbate reductase
Pantoea ananatis carotene desaturase ζ-Carotene
L-Galactose-1,4-lactone
Pantoea ananatis carotene desaturase Phytoene Zea mays phytoene synthase 1
L-Galactose
Geranylgeranylpyrophosphate × 2
L-Galactose-1-phosphate
Geranylpyrophosphate
GDP-L-galactose
Mevalonic acid
GDP-D-mannose
Acetyl-CoA
D-Mannose-1-phosphate
TETRAHYDROFOLATE-Glun Tetrahydrofolate
GTP
Dihydrofolate
Dihydroneopterin triphosphate
Chorismate
E. coli GTP cyclohydrolase I
Aminodeoxy-chorismate Dihydropteroate
Dihydroneopterin phosphate
p-Aminobenzoate
Plastid
Hydroxymethyldihydropterin pyrophosphate
Dihydroneopterin
Hydroxymethyldihydropterin
Hydroxymethyldihydropterin
Mitochondrion Current Opinion in Biotechnology
A transgenic corn plant was engineered using multiple constructs to affect three distinct metabolic pathways to generate high contents of b-carotene (precursor of vitamin A), ascorbic acid (vitamin C) and folic acid (vitamin B9) [16]. The enzyme reactions affected through engineering are colourcoded, together with the final products that have increased levels.
There are a number of strategies in common use for enhancing yields of bioactives in cell/tissue culture systems. These can involve optimisation of growth conditions to allow scale-up of the biomass for production, use of abiotic and biotic elicitors, and coculture with other organisms. For example, the biomass of a hairy root culture system of Artemisia annua has been scaled up to 20 L in a mist reactor [36]. Application of an electrical stimulus has been used as an effective abiotic elicitor to stimulate the production of phenolics in tissue cultures [37], and a range of elicitors such as oligosaccharides and polysaccharides, jasmonic acid and sodium nitroprusside have been shown to promote levels of many different bioactives in culture www.sciencedirect.com
systems including artemisinin, isoflavones, stilbenes, diterpenes and alkaloids [38–42]. Co-culture with other organisms, such as fungi, has also been used to enhance the production of podophyllotoxin and taxol, two anticancer bioactives, in Linum album and Taxus chinensis suspension cells, respectively [43,44]. In earlier years, the major challenge facing the use of plant cell/tissue cultures for bioactive production was the relatively low productivity of the culture systems available. However, this limitation can now be overcome by modern bioreactor technologies, such as those that enable the cultures to be scaled up in 20 000 L bioreactors [45]. Combining these new production systems Current Opinion in Biotechnology 2012, 23:271–277
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with the continuing advances in gene discovery and pathway engineering, it is highly probable that cell culture technologies will increasingly be used in the future to bring many more plant bioactives to commercialisation.
Fast-track breeding
Plant breeding is the traditional approach to improve crop quality. In recent years, this approach has also been applied to medicinal plants for selecting cultivars with higher content of bioactives. For rapid and effective selection of desirable cultivars, ‘fast-track breeding’ benefits from knowledge of the genetic maps of the plants, relevant quantitative trait loci (QTL) and biomarkers associated with the synthesis and yield of target bioactives. Since the complete genome of Arabidopsis thaliana was published in 2000, there have been many additional plant genomes sequenced, including those of rice, grapevine, corn, apple, potato and soybean. These have contributed substantial information to underpin yield improvement of bioactives including isoflavonoids, flavonoids, terpenes and many other bioactive natural products, such as reviewed in [8]. The yield of bioactives in field crops will always reflect not only both the content of the bioactives, but also importantly, the biomass of the plant. Extractability of the improved varieties is also an important parameter, with lack of contaminating co-products determining utility for the commercial end-users. Detailed genetic information of several medicinal plants, such as Artemisia annua [46], Catharanthus roseus [47], Taxus cuspidate [48], Allium sativum [49], Epimedium sagittatum [50], Papaver somniferum [51], have been reported recently. These databases represent powerful tools to support the molecular breeding of bioactives in plants, as well as providing a foundation for synthetic biology and industrial fermentation routes. In this context, artemisinin, made by the Chinese medicinal plant, Artemisia annua, underpins the production of artemisinin combination therapies (ACTs). These are essential anti-malarials, recommended by WHO. Cost of supply of artemisinin, together with the capacity and robustness of its production are significant factors for manufacturers of the pharmaceuticals, particularly given the estimated rise in global demand of ACTs over the coming years. Increased knowledge of the genes encoding the enzymes involved in synthesis of artemisinic acid and regulation of the pathway [52,53–55], together with the first genetic map of A. annua and identification of relevant QTL [46], have all contributed to new strategies for enhanced production of the bioactive. Both the cost of product to manufacturers and ease of its availability are likely to dictate future market-led decisions to increase supply of artemisinin through higher crop yields Current Opinion in Biotechnology 2012, 23:271–277
in the field, versus the use of microbial-based industrial fermentation. Improved sequencing technologies have facilitated rapid developments in forward genetic research. It is clear that many more non-GM molecular breeding platforms will be established in the next years to generate high-yielding varieties. The use of GM can offer an alternative route but whilst the technologies may be available, the cost and time of bringing the new product through regulatory hurdles to the market often precludes its selection as a commercialisation route.
Feedstocks for modification and synthesis Plant biomass is now acknowledged as an alternative to the use of fossil reserves for fuel production. In parallel, the use of plant-based feedstocks for chemical production has also gained interest and biorefinery applications have become many and varied. A zero-waste biorefinery capable of using inputs from a diverse range of crop/ forestry feedstocks presents an immense opportunity to maximise the value of biomass and produce multiple chemical intermediates. In turn, high value bioactives can either be extracted and further modified post-extraction, or synthesised de novo from those intermediates. Feedstocks from plant biomass include carbohydrates, lignin and lipids and their potential for bioactive production has been reviewed, such as in [56]. For example, carbohydrates and lignin can lead to chemical intermediates such as levulinic acid, vanillin and many other aromatic molecules for the synthesis of pharmaceuticals [56]. Lipids can be used to produce beneficial polyunsaturated fatty acids such as omega-3 [57]. In addition to the principal feedstocks, a variety of small molecules, whilst representing only a small proportion of the biomass, have also attracted considerable interest in biorefining, due to their high value either as chemical intermediates or bioactives. For example, shikimic acid, a key intermediate for the antiviral drug Tamiflu, is extracted from the tree species, Liquidambar styraciflua, before further conversion of the biomass to biofuels [58]. In the coming years, combining designer feedstocks with new biotechnologies and chemical/biochemical possessing will undoubtedly widen the utility of biorefineries to produce specific bioactives in high yields.
Conclusion The decline of fossil reserves and the consequent increase in costs of energy and petrochemicals derived from those reserves are leading to recognition that a global bio-based economy needs to be developed. In future years, the products of agriculture and forestry must support industrial sectors beyond the provision of food, animal feed and energy. High value bioactive chemicals will increasingly be derived directly or indirectly from www.sciencedirect.com
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plants. In addition to the highlighted research in land plants, microalgal biotechnology has recently emerged as a new platform for bioactive production [59]. The biotechnologies to increase productivity are already available. As assays are developed to discover new bioactivities of plant natural products, increasing applications will be coming to market.
Acknowledgement The Leverhulme Trust is thanked for a senior research fellowship to DJB to study the bioactivity of plant natural products.
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