1.1. Tropane alkaloids: history and relevance
Historically, plants have been the major source of active compounds for the pharmaceutical industry. Today, plant extracts and its active principles represent 25% of the annual commercialized drugs in the United States (Kinghorn and Seo, 1996, Butler 2004, Prakash Rout et al., 2009, Qurishi et al., 2010). Moreover, the World Health Organization has estimated that more than the 80% of the population in developing countries relies for their health care on traditional medicines mostly from plant origin (Canter et al., 2005). The plant active principles are, in general, a product of the plant secondary metabolism; they are low-molecular-weight compounds that in general participate in defence mechanisms against diseases (phytoalexins) or as attractants for pollinator insects (pigments and fragrances) (Canter et al., 2005). The production of each group of secondary metabolites, as counterpart of primary metabolites, is in general restricted to a limited number of species, and they usually are organ-specific and furthermore, tissue-specific.
According to their chemical properties, secondary metabolites could be classified in three main groups: a) terpenes, b) phenols and c) alkaloids. Terpenes and terpenoids are the primary constituents of the essential oils, widely used as natural flavour additives for food and fragrances. Phenols are a class of chemical compounds consisting of a hydroxyl group directly bonded to an aromatic hydrocarbon group that seem to be universally distributed in plants. They are essential for the growth and reproduction of plants, and are produced as a response for defending injured plants against pathogens. Finally, alkaloids are characterized for being alkaline nitrogenated organic compounds, derived from aminoacids, generally insoluble in water and soluble in alcohol, ether, chloroform, etc. Among the alkaloids, those derived from tropane have received particular attention for their properties.
1.2. Tropane alkaloids
Tropane alkaloids are distributed among the plant families
Hyoscyamine and scopolamine are the end products of the tropane alkaloid biosynthetic pathway having both pharmacological applications for its action in the parasympathetic nervous system. Scopolamine, a 6,7-β-epoxide of hyoscyamine, is derived from hyoscyamine by the intermediate form 6β-hydroxyhyoscyamine (Fig. 2). The pharmaceutical industry employs hyosciamine, and its racemic form atropine, to obtain hyoscine N-butyl bromide that is used for its antispasmodic action since 1951. This semisynthetic derivative has pharmacological proprieties similar to those from scopolamine but with less activity and more adverse effects (Tytgat, 2007). Figure 2 compares the industrial process for producing the atropine racemic form of hyosciamine, and the scopolamine biosynthetic pathway in plants.
Scopolamine is one of the earlier alkaloids purified from plant sources, described by Albert Ladenburg in 1880. It is the most valuable of the tropane alkaloids being its worldwide demand 10-fold the demand of the sum of hyosciamine (its precursor) and atropine (the semisynthetic drug) (Moyano et al., 2003). As atropine, scopolamine is an anticholinergic drug, besides it also has anti-muscarinic activity. Anticholinergic drugs are, in general, competitive and reversible inhibitors from acetylcholine with effects at parasympathetic level. Because of that pharmacological activity scopolamine has a number of uses in medicine. Its primary use is for the therapy of nausea (transdermal patchs), motion sickness and intestinal cramping. Also, it is used for ophtalmic purposes (to induce mydriasis and cycloplegia) and as a general depressant in combination with narcotic painkillers. It also has secondary uses are a preanesthetic agent, a drying agent for sinuses, lungs and related areas and to reduce motility and secretions in the gastrointestinal tract (tinctures). Uncommonly, it is also used for some forms of Parkinsonism, combined with opioids (e.g.: with morphine). The production and purification of scopolamine is extremely expensive which have made that several groups have focused in finding scopolamine analogs or synthetic forms.
1.2.2. Biosynthetic pathway, key points: PMT and H6H
As was mentioned above, scopolamine and hyoscyamine are the most relevant tropane alkaloids widely in use. Their metabolic pathway starts in putrescine, a polyamine that is shared by several metabolic pathways (e.g.: pyridinic alkaloids). The role of putrescine methyl transferase (PMT; EC 188.8.131.52) is to sequester putrescine from the polyamine pool, which is the cross-point between the primary and secondary metabolism towards the tropane alkaloid pathway. The enzyme PMT catalyses the reaction from putrescine to N-methyl putrescine with S-adenosyl methionine as methyl donor (Zhang et al., 2004, Stenzel et al., 2006). It was described by Mizusaki et al. (1973) in tobacco for the first time, afterwards it has also been described in
2. Strategies for scopolamine production
The chemical complexity of tropane alkaloids turns almost impossible its substitution by other compounds or the
2.1. Metabolic engineering
The in-depth understanding of biosynthetic pathways, along with the increasing number of cloned genes involved in biosynthesis, enable the exploration of metabolic engineering as a potential effective approach to increase the yield of specific metabolites. It could be achieved by enhancing rate-limiting steps or by blocking competitive pathways. Metabolic engineering is a multidisciplinary powerful tool that uses from bioinformatics, genetics, biochemistry, systems biology, molecular biology, biochemical engineering, etc, for re-direct the metabolic flux generally in order to increase secondary metabolite production yields (e.g.: alkaloids). It also could be useful for a rational and directed modification of metabolic pathways to understand their design and regulation and to study the impact of intermediates and end products in a specific organism.
When the improvement of a productive process is the main goal, the
2.1.1. Hairy root culture initiation and maintenance
Hairy roots are obtained by infection of the plant with different
As we have said before, the synthesis of tropane alkaloids is produced in the pericycle of roots (Hashimoto et al., 1991) being the final products translocated to the aerial part of the plant. It is not surprising then that the establishment of hairy roots was considered as an alternative strategy for scopolamine production (Oksman-Caldentey et al., 1991, Oksman-Caldentey and Arroo, 2000, Palazón et al., 2003 and 2008). It was reported that
Even though hairy roots are scopolamine producers the yields achieved are low, which has fostered the quest of an alternative production strategy. In the 1990’s the first studies about the influence of plant growth regulators on scopolamine yields were performed. The effect of the addition of GA7 on kinetics of growth and alkaloid accumulation in two different
The term elicitor was first introduced to describe the action of biomolecules able to induce phytoalexin production. In general, elicitors induce defence systems and increase the resistance to pathogens in plants. Also, pathogens biomolecules derived from the pathogen cell wall (exogenous elicitors), and compounds released from plants by the action of the pathogen, triggers that defence response (Angelova et al., 2006).
At the end of the 1990’s several groups started the study of the effect of elicitors on secondary metabolism. Hence, it was evident that biotic elicitors (produced by pathogenic microorganisms or released from their cell walls by plant enzymes) were able to induce changes in secondary metabolites patterns. Also, the release of certain compounds, such as cellulose or pectinase and molecules active in the signal transduction pathway (salicylic acid, jasmonic acid), could also promote the plant defence response (Benhamou, 1996; Guo et al., 1997; Lawrence et al., 2000). That response includes the production of phytoalexins (pathogeneses related proteins), protease inhibitors and a variety of other defence compounds (among them alkaloids).
3. Metabolic and genetic engineering
Molecular biology, as a tool for DNA manipulation, could be used for engineering metabolic pathways. Figure 3 shows the usual strategies employed for overproducing a specific metabolite (C ).
The strategies metabolic engineering uses to increase secondary metabolite production are a) the overexpression of secondary metabolite precursors (A); b) the overexpression of genes whose products are rate-limiting (B), c) the creation of new branches in the biosynthetic pathway (E to C), d) the blocking of reactions (C to D) redirecting them to the C pathway, among others. Also, there are other strategies for overproducing C such as a) manipulation of regulatory genes, b) a positive or negative regulation of gene expression, c) selection of mutants with increased expression of metabolites, d) use of specific promoters for tissue/organ specific expression, e) iRNA technology and gene silencing. Each of the strategies above mentioned has unpredictable consequences in the cell homeostasis.
3.1. Overexpression of key enzymes
Overexpression of a key enzyme in the biosynthetic pathway is one of the strategies used to increase secondary metabolite production. In general, the overexpressed enzyme catalyzes a bottleneck step. In the tropane alkaloid pathway the key enzyme is H6H. As was mentioned above, H6H catalyzes two reactions, the hydroxylation at position 6, rendering an intermediate 6-hydroxyhyoscyamine; and the epoxidation that leads to the end product scopolamine. Therefore, overexpression of H6H is an attractive strategy for poor scopolamine producing species with hyoscyamin-rich accumulation.
Another strategy focus on the lack of substrates in the tropane alkaloid pathway. Thus, the carbon flux is redirect from the primary to the secondary metabolism, being PMT the key and pivot enzyme between both metabolisms. There is a strong expression of the
The overexpression of only one enzyme in a complex metabolic net could not be sufficient to increase some secondary metabolite expression. Particularly, there are several works about the overexpression of more than one of the enzymes involved in the alkaloid tropane pathway (Zhang et al., 2004, Liu et al., 2010, Chunxian et al., 2011). However, not significant scopolamine yields were attained. The unsuccessful results could be attributed to the transgenic transformation processes itself. The mechanism of integration of transgenes into plant DNA is poorly understood, the integration of many genes at one or a few loci could not happen by chance. Also, multiple copies of one or more transgenes can result in postranscriptional gene silencing and turn unstable and reduce gene expression (Matzke & Matzke; 1998; Palazón et al., 1998; Bulgakov et al., 2004; Kutty et al., 2011). Evidently,
The genetic transformation with numerous in-tandem genes could be troublesome. For hairy root induction and
are needed. New and simple DNA transfer methods simplify the process as, for example, the Gateway system, based on bacteriophague Lambda site-specific recombination system (Karimi et al., 2002; Attanassov et al., 2009; Dubin et al., 2008, Xu & Quinn, 2008). Figure 4 shows a protocol designed in our lab for the production of a PCR fragment for
4. New approaches
In this chapter, we have reviewed some of the most relevant strategies for improving tropane alkaloid biosynthesis such as the establishment of scopolamine overproducing organ cultures, the elicitation and the genetic transformation with homologous genes. Nevertheless, the knowledge generated and the strategies in use have demonstrated that the tropane alkaloid metabolism is immersed into a complex net of metabolic pathways with a delicate equilibrium quite difficult to be manipulated. Modern system biology tools, like elicitation and overexpression, allow the carbon flux redirection with some limitations. These margins cannot be overcome and decelerate the development of a competitive and sustainable production platform. Those troubles and limitations have fostered new strategies based on functional genomics (Goossens et al., 2002; Goossens & Rischer, 2007, Oksman-Caldentey & Inzé, 2004) such as biotransformation.
Biotransformation is one of those new strategies. The production of scopolamine and other alkaloids was studied in engineered
Another approach was the bioconversion of hyoscyamine to scopolamine using recombinant
In the last two decades plant biotechnology has made considerable advances in the quest of a scopolamine and other tropane alkaloids productive process. Several groups have explored a wide spectrum of strategies that have led to the exhaustive knowledge of the tropane alkaloid pathway, its limiting steps and some of the regulation pathways. It is evident that genetic transformation is a promissory tool for engineering tropane alkaloid biosynthetic metabolism in order to produce high amounts of scopolamine. Combining genetic transformation and metabolic engineering would be a powerful strategy to re-direct the metabolic flux towards that biosynthetic pathway. It became clear, from the results already published, that the overexpression of
This work was supported by Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) and the Agencia Nacional de Producción Científica y Tecnológica (ANPCyT) (PICT2007 0552).