November 20, 2005

The Role of Metabolic Engineering for the Production of Secondary Metabolites of Plants

By Khan, M T H; Ather, A; Gambari, R

Plants are very interesting and useful sources of molecules of great importance for the development of new pharmaceutical products. Metabolic engineering has proven to be a valuable tool for large production of several biomedically as well as industrially relevant compounds from micro-organisms as well from higher plants. Current development of studies employing transgenic and/or recombinant technologies have opened up opportunities of the metabolic engineering/ metabolo-mics of biosynthetic pathways, leading to the growth of manufacturing of high-value secondary metabolites, even at the commercial level.

KEY WORDS: Metabolic engineering - Transcriptome - Secondary metabolite - Agrobacterium - Recombinant DNA technology - Metabolomics - Phytoalexin.

During molecular evolution, plants have developed very efficient approaches to magnetize pollination or seed-dispersing animals (such as birds or insects), for shielding themselves against UV-light and for their defence against herbivores, micro-organisms and other plants. In natural growth conditions, plants compete for space, light, water and nutrients. The major strategies used are based on the production of secondary metabolites, including essential oils and fragrances behaving either as attractant for pollinations or as repellent. The pollination is also favored by colorants, molecules like flavonoids, anthocyanins and carotinoids. Flavonoids are also involved in the absorption of UV-radiation.1

The very high variety of biochemical pathways and production of secondary metabolites is the basis for the well-known feature of plants to serve as a tremendous source for isolation of molecules of great interest for the development of new medicinal agents considered less risky than those of microbial or animal origin.2 However, it should be pointed out that the beneficial medicinal effects of plant material might be also due to the endogenous combination of several secondary products. On the other hand, approaches involving genetic engineering of medicinal plants has been described to increase yield of pharmaceutically important single secondary metabolites.3

Table I presents some of plant-derived pharmaceutical products, including the source plant species, their price in US dollar and their therapeutic indications.4

The plant molecular biology moved since long time ago from studies of single gene functions useful to extrapolate basic principles in a very simplistic manner, to a much more detailed analysis of processes known to be regulated by a multitude of tightly interconnected and even interdependent factors. By definition, the modern plant molecular science, and in particular its applied component plant biotechnology requires a holistic, multidisciplinary and comprehensive approach to achieve its objectives.5

TABLE I.-Pharmaceutical products from natural sources (mainly from, plants) (modified from Rao et al.4).

The enhanced classification of the biomolecular complexes, branching, and the knowledge of the interactive metabolic pathways of many organisms are feasible with advances in genome sequencing, transcriptome and proteome analysis and may provide more suitable starting platforms for engineering of secondary metabolite production. Indeed, knowledge and exploitation of the dynamic nature of gene expression and enzyme regulation may prove to be highly useful in metabolic and combinatorial engineering approaches. In combination with additional techniques for metabolic pathway modification, such as site directed mutagenesis, the goals of improved secondary metabolite production and employment of novel products can be readily achieved.6

Tools for genetic transformations of plants

Genetic transformation of plants is possible using different and well known DNA delivery technologies, the most successful of which are (a) the introduction of genes employing vectors transferred by Agrobacterium and (b) direct delivery strategies.7

Agrobacterium is a plant pathogenic bacterium occurring in the soil that has the ability to transfer part of its DNA into the genome of the plant. Two species of Agrobacterium are of particular importance in plant tissue culture, A. tumefaciens (which causes the formation of an undifferentiated tumor, known as "crown gall tumor") and A. rhizogenes (which induces the formation of fast-growing plagiotropic, highly branched root tissue, known as "hairy root"). Both of these phenotypes are the results of the ability of the plasmid of Agrobacterium to incorporate several of its genes into the genome of the host plant.7

As far as gene transfer approaches for direct uptake of DNA by protoplasts, several methods were developed and reported.7,8 Since DNA is not lipophilic, it is relatively incapable of passing through the plasma membrane without some treatments to increase permeability, such as in the case of the use of polyethylene glycol (PEG). The PEG method for transformation allows the insertion of the DNA in the plastome. In case of plasmid, transformation can be accomplished by a combination of biolistic and PEG transformation technologies.7,8 Furthermore, DNA uptake into protoplasts can also be induced electrically (electroporation) in order to produce temporary pores in the plasma membrane.7

Some other alternative DNA delivery methods have been reviewed by Songstad et al.9 They reviewed about the updates of silicon carbide fibers, electroporation, electrophoresis, microinjection, etc., and associated advantages and disadvantages.9

Why biotechnology?

The biotechnological approaches generally consist of in vitro growth of cells, tissues, organs or the entire organisms and genetic manipulation of them to obtain desired compounds. The development of the micropropagation methods for a number of medicinal plant species has been already reported and needs to be extended.10 Cryopreservation of cells is an area of great interest in the preservation of medicinal plants and it has already been applied in the case of several plant species. The development and adoption of plant cell culture and organ culture methods have lead to the production of plant products on a large scale.4

TABLE II.-Partial list of the most important issues of biotechnology in the production of secondary metabolites from plants (from Rao et al4).

TABLE III.-Examples of the chemical classes and sub-classes of compounds isolated from higher plant cultures in vitro (modified from Stockigt et al.11).

Table II shows different issues related to the biotechnological advances in the usual projects found on plant-derived secondary metabolites.4 Table III presents some of the classes and sub- classes of compounds isolated and reported so far from the higher plant cultures in vitro.11

Genetic modification of plants

The genetic transformation allowed by plant technologies is a versatile platform for cultivar improvement as well as for studying gene function.12

Transgene integration in plants transformed by either Agrobacterium-mediated or direct DNA delivery methods occurs via illegitimate recombination.5 Recent investigations aimed at genetically dissecting transgene integration mechanism(s) have provided new insights into the process and are expected to fast track the elucidation of factors that control stable and predictable transgene expression.5

However, it is surprising that relatively few metabolic pathways have been modulated using recombinant DNA technology. One reason for this is that such pathways must be studied in the context of the whole cell, rather than at the single pathway level, and that even the simplest modifications can have unpredictable consequences throughout the complete system.5

Genetic engineering of secondary metabolic pathways aims to either increase or decrease the quantity of a single compound or group of interconnected compounds. To decrease the production of a certain unwanted (or of a group of) compound(s), several approaches have been reported. An enzymatic step in the pathway can be knocked out, for example, by reducing the level of the corresponding mRNA via antisense, or RNA interference technologies, or by over- expressing an antibody against the enzyme.13 The antisense approach has been successfully used for changing flower colors.13 In this case other approaches include diversion of the flux into a competitive pathway or an increase in the catabolism of the target compound.13 More often, the goal is to increase the production of certain compounds in the normal producing plant species.

Major issues and new trends in metabolic engineering

Genetic modification by plant transformation allows stable alterations of the biochemical processes involved in important traits such as yield, nutritional quality and resistance to diseases. The achievements in modification of secondary metabolism in plants have been reviewed, including as the alternative approaches to alter the secondary metabolism.4.6,7

There are at least 3 distinct uses of the metabolic engineering (metabolomics) in plants:

1. Increase or decrease of the amount of metabolites and production of novel compounds.

2. Improving quality traits of food and fodders or alter traits of horticultural plants.

3. Increasing the resistance of plants against pest and diseases.

1. In the first strategy of metabolomics the aim is to increase the level of certain compounds, like medicinal products, colorants, flavors and fragrances, foreconomically important production. For example, strictosidine synthase (Str) has been over-expressed in Catbamnthus roseus leading to the production of 200 mg/mL terpenoicl indole alkaloids.14 Another interesting example is that of the stable transgenic hairy root clone of Hyoscymus muticus, over- expressing the hyscyamine 6β-hydroxylase; this transformation ultimately leads to the production of 17 mg/mL scopolamine, which was about 100 times more than the wild-type clones.15 In another example, a new pathway was created by introducing a gene encoding a biosynthetic enzyme like tryptophan decarboxylase (Tdc) from Catberanthus roseus, in tobacco resulting in a high production of tryptamine (up to 1 mg/gm fresh weight).9

2. The second approach consists in the development of dietary values of the foodstuff by activating pathways leading to health endorsing molecules. For instance, following genetic engineering approaches the provitamin A (β-carotene) biosynthetic pathway has been activated into rice endosperm, in order to facilitate the production of vitamin A in the rice, reducing a serious health problem due to vitamin A deficiency in many countries using rice as the chief staple-food.7

3. The third issue is the use of metabolomics of secondary metabolism to increase or broaden the spectrum of disease resistance. An example is the introduction of the biosynthesis of novel phytoalexins or structural variants of the naturally occurring phytoalexins, or by modifying expression of transcriptional regulators of phytoalexin pathways. For instances, the introduction of stilbene synthase gene in tobacco and tomato has increased the resistance against Botrytis and Phytophthora infection, respectively. This protection was clue to the production of the phytoalexin resveratrol.7


Plant cell and transgenic hairy root cultures are promising potential alternative sources for the production of high-value secondary metabolites of commercial relevance. Recent developments in transgenic research have opened up the possibility of the metabolic engineering of biosynthetic pathways to produce high- value secondary metabolites.4


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2. Shanks JV, Morgan J. Plant 'hairy root' culaire. Gun- Opin Biotechnol 1999;10:151-5.

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6. Mijts BN, Schmidt-Dannert C. Engineering of secondary metabolite pathways. Curr Opin Biotech 2003;14:597-602.

7. Hallard DAC. Transgenic plant cells for the production of indole alkaloids. Thesis, Leiden University. Leiden, 2000, p. 1-16.

8. Kofer W, Eibl C, Steinmuller K. PEG-mediated plasmid transformation in higher plants. In Vitro Cell Develop Biol Plant 1998;34:303-9.

9. Songstad DD, De Luca V, Brisson N. High levels of tryptamine accumulation in transgenic tobacco expressing tiyptophan decarboxylase. Plan Physiol 1990;94:l410-3.

10. Naik GR. Micropropagation studies in medicinal and aromatic plants. In: Khan IA, Khanun A editors. Role of biotechnology in medicinal and aromatic plants. Hyderabad: Ukaz Publications; 1998. p. 50-6.

11. StockigtJ, Obitz P, Flakenhagen H, Lutterbach R, Enclress R. Natural products and enzymes from plant cell cultures. Plant Cell Tissue Org Cult 1995;43:914-20.

12. Hansen G, Wright MS. Recent advances in the transformation of plants. Trends Plant Sd 1999:4:226-31.

13. Verpoorte R, Memelink J. Engineering secondary metabolite production in plants. Curr Opin Biotechnol 2002;13:181-7.

14. Canel C, Lopes Cardoso MI, Whitmer S. Effects of over- expression of strictosidine synthase and tiyptophan decarboxylase on alkaloid production by cell cultures of Catharanthus roseus. Planta 1998;205:4l4-9.

15. Jouhikainen K, Lindgren L, Jokelainen T. Enhancement of scopolamine production in Hyoscyamus muticus L. hairy root cultures by genetic engineering. Planta 1999;208:545-51.

M. T. H. KHAN,1,2 A. ATHER 2, R. GAMBARI 2,3

1 Pharmacology Research Laboratory

Faculty of Pharmaceutical Sciences

University of Science and Technology, Chittagong, Bangladesh

2 ER-GenTech, Department of Biochemistry

and Molecular Biology

University ofFerrara, Ferrara, Italy

3 Biotechnology Center, University ofFerrara, Ferrara, Italy

M. T. H. Khan is the recipient of a grant from MCBN-UNESCO (grant no. 1056), and fellowships from CIB (Italy) and Associazione Veneta per Ia Lotta alia Talassemia (AVLT, Italy).

Address reprint requests to: M. T. H. Khan, Centre di Biotecnologie, Universit degli Studi di Ferrara, Via Fossato di Mortara 74, Ferrara 44100, Italy.

Copyright Edizioni Minerva Medica Sep 2005