The effect of bacterial isochorismate synthase on the Brassica rapa metabolome
Simoh, S.
Citation
Simoh, S. (2008, June 11). The effect of bacterial isochorismate synthase on the Brassica rapa
metabolome. Retrieved from https://hdl.handle.net/1887/12944Version: Corrected Publisher’s Version
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Chapter 8
Future perspectives in engineering plant secondary metabolism
Sanimah Simoh, Huub JM Linthorst and Robert Verpoorte
Our life depends on plants. Plants are not only the primary sources of food and nutrition for human and animal populations but they also have been widely used for centuries to prevent and cure diseases. In modern days, the perception that plants are
‘safe and natural’ results in the growing interest of the public to use plant-based products in day to day life. For most of the population in developing countries such as Africa and Asia, plant-based medicines are used as primary health care because they are easily accessible and affordable. Nowadays, about 25 percent of drugs used worldwide are plant-derived pharmaceuticals (Raskin et al., 2002). The World Health Organization (WHO) estimates that the current market sales of phytomedicinal products is around US $ 60 billion annually (http://www.who.int/
mediacentre/factsheets/fs134/) and the demand is likely to increase more than US $5 trillion in 2050 (Kala et al., 2006). Usually all the economically important compounds are the products of plant secondary metabolism including terpenoids, alkaloids, polyketides, phenylpropanoids and flavonoids. Among the important drugs currently used are paclitaxel from Taxus spp., vincristine and vinblastin from Catharantus roseus, digoxin from Digitalis spp, morphine and codeine from Papaver somniferum and quinine from Cinchona spp.
Even though the role of plants to produce secondary metabolites beneficial to economy has long been known, most of them are poorly explored. In the tropical rainforest, it is estimated than only 1 percent of the species has been studied by scientists for their bioactive compounds and use (http://www.rain- tree.com/facts.html). In Peninsular Malaysia for example, from 10,000 species of higher plants and 2000 species of lower plants available, 16% are known to have
medicinal properties (Saha et al., 2004) and approximately only a hundred have been fully investigated for their potential (http://www.arbec.com.my/ biotech/html). Many of the valuable plants rich in secondary metabolites are endangered species and produce extremely low concentrations of the desired compounds. For instance, it is claimed that over-harvesting the Pacific yew (Taxus brevifolia) for its anticancer compound, could make the plant becoming extinct. This is because of the very slow growth of the plants and low yield of the compound in the bark (40-100 mg/kg) (Cragg et al., 1993). Currently, it is estimated that around 4000-10,000 valuable medicinal plants are categorized as extinct species (Edward, 2004) and this number is expected to increase in the near future. In the case of tropical rainforest, the valuable plants are also subject to over-exploiting by indigenous peoples, deforestation for commercial logging and land clearing for urban developments. The minimal enforcement of regulation and law to protect biodiversity in developing countries adds to this problem.
Biotechnological approaches such as plant cell culture technology which is often considered as an alternative to ensure the continuous supply of these plants/compounds play a main role to overcome the abovementioned problems.
However, the main limitation one faces many times with this technology is the low accumulation of the desired secondary metabolites. Moreover, downstream processing is necessary to obtain the desired product. So far, only few commercial products from plant cell cultures are available on the market. Metabolic engineering (ME) which is established nearly 15 years ago (Tyo et al., 2006) offers a promising solution to enhance the level of the compounds which are present in trace amounts in plant cell/organ cultures or intact plants. ME deals with manipulating multiple components in networks, pathways or whole organisms (Bulter, 2003). Thus, the application of this technology for manipulating certain pathways in plants for production of economically important secondary metabolites has gained much interest in recent years. Some recent developments in this field are reviewed by Oksman-Caldentey and Inzé (2004), Davies (2007) and Verpoorte (2007). ME becomes more applicable by taking advantage of the advancement in gene transfer technology. For example the biosynthetic capability of one species can be expanded by introducing the gene for enzymes from other sources in order to produce certain compounds that normally do not exist in that species (Nessler, 1994). This technology can also be implemented to improve certain traits in agricultural crops. This includes development of a plant
resistant to pathogen attack by expression of certain compounds, to reduce the level of undesired metabolites in crops that might be detrimental to humans and animals as well as to increase desired metabolites in the intact plants (Gómez-Galera et al., 2007). Advancement of ME currently reached the stage that multi point engineering is used instead of single point as multi-step manipulation may help in controlling the metabolic flux in a predictable manner (Capell and Christou, 2004).
The basic important step for ME is to develop and establish the transformation system of the plant of interest. This includes the availability of the cloned gene and the gene delivery method which allows the efficient transfer of the gene of interest to the plant cell as well as the expression system for up or down regulation of the gene in the targeted pathways (Gómez-Galera et al., 2007). The establishment of the regeneration system of the plant of interest is even more crucial especially if one deals with recalcitrant species. With the exception of the model plant, Arabidopsis thaliana, most of the plant transformation systems are using explants as starting materials which means tissue culture systems have to be dealt with. Thus, longer time is needed to obtain the transformed plants. Even though published protocols are expected to be applicable to similar plants, one still need to establish a reliable laboratory protocol.
The stability of the transformed plant is another factor that needs to be considered.
Often, the transformed plants are unable to produce fertile seeds which are needed for the subsequent generation. The regeneration factor again plays a main role if cross pollination has to be carried out instead of self-fertilization for the plants to set seeds.
This means that the proportion of transformed plants must be high enough in order to get stable transformation for the next generation since only few transformed plants behave in the expected way, as variation in gene expression between individual transformants occurs (De Block, 1993).
In the past years, an increased number of plant biosynthetic genes and enzymes have been cloned and identified, enhancing the current state of knowledge towards plant biosynthetic pathways leading to production of secondary metabolites. Major biosynthetic steps of flavonoids, phenolic derivatives, terpenes and alkaloids have been unraveled and many of the enzymes and genes related to the compounds have been characterized and identified as well (Petersen, 2007). The refinement of plant transformation and gene expression systems is also a major advantage. All these achievements apparently help ME to have more impact on manipulating plant pathways for efficient production of desired metabolites. Moreover, the emerging new
functional genomic tools i.e. transcriptomics, proteomics and metabolomics as well as computational biology shed new light on this field. The role of metabolomics is obvious. As the production of plant secondary metabolites is tightly controlled by complex regulatory networks and one pathway may link to several pathways of primary and secondary metabolism, manipulation of a target pathway may affect other pathways which leads to the production of unintended or unexpected metabolites.
Furthermore, plant secondary metabolism pathways are normally induced in response to biotic or abiotic stress and one pathway may produce more than one metabolite (Fischbach and Clardy, 2007), thus manipulation of a pathway for a desired metabolite might increase or decrease the concentration of other metabolites in the same pathway. Metabolomics is able to diagnose these alterations. To date, transcriptomic and metabolomic data are integrated in order to get insight in the effect of manipulating plant pathways in the whole plant metabolism. Few attempts to link between the transcriptome and metabolome level in plants have been made for examples for A. thaliana (Dubouzet et al., 2007; Tian et al., 2007) and rice (Hirai et al., 2004). The application of these high throughput methods will accelerate the research on ME as the assessment of the changes as a result of manipulating the pathways can be done in holistic way at the level of genome, transcriptome and metabolome. The development of bioinformatics plays a role in facilitating research related to ME.
In developed nations the goals of ME research is more towards generating value- added traits in existing plants especially to obtain the new crops that have high consumers appeal such as to increase flavonoids and carotenoids in tomato and potato (Davies, 2007). In the case of “Golden Rice’, it could help by preventing Vitamin A deficiency in developing countries where rice is consumed as a major staple food.
Developing countries, where biodiversity resources are well known, are in good position to exploit this opportunity. The aim of ME related research might be relevant to increase the production of useful metabolites. The success of projects related to this technology needs concerted effort from plant biologists, biochemists and metabolic engineers as well as the public and private sector.
