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Introduction

Secondary metabolites can be defined as plant substances which do not involved

directly for the growth and development of the plant but important for the defense system of

the plant. It also acts as attractant to the pollination agents, seed dispersion agents besides

functions in competition interaction (Verpoorte et al. 1999). Plant is the best source for

secondary metabolites. A lot of higher plants have been used as the main producer of natural

products which can be used in many fields such as pharmaceutical, agrochemicals, flavoring,

perfumes, food additives and insecticides (Verpoorte et al. 2002). Since secondary

metabolites can provide a lot of benefit to human, numerous afforts have been done to

enhance the production of the seconday metabolites by reseachers around the world.

There is a lot of methods have been used to increase the pproduction of secondary

metabolites from plant. They include media optimization, selection and screening,

biotransformation and scale up, and also through genetic approaches (Verpoorte et al. 1999).

The efforts that involved in the genetics approaches will be discussed in detail in this writing

but before that, we will take a look briefly into the other methods as mentioned above that

have been extensively used to increase the production of secondary metabolites from plant.

Non-genetic Approaches

Since the last two decades, a lot of methods have been applied to obtain larger amount

of secondary metabolites from plant or plant cells. One of the earliest methods which have

been extensively used is the medium optimization. The application of this method involves

the manipulation of several important parameters such as lighting, temperature, the

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composition of the medium such as types and concentration of suitable plant growth

regulators or elicitors to induce the biosynthesis of plant secondary metabolites (Zhang et al.

2004, Matkowski 2008). The best medium for obtaining high biomass growth with high

production of the required secondary metabolite in high quantity is the main priority in this

method.

In selection and screening method, cell lines that produce high amount of compound

of interest are selected and it is repeated to obtain cell lines producing higher amount of that

compound. This method has been used in berberine production from cell culture ofCoptis

japonica and the production was successfully enhanced through this method (Verpoorte et al.

2002). Biotransformation is a method where specific secondary metabolites are produced by

the action of the enzymes in the plant cells to convert the precursor of that compound which

provided to the cell culture exogenously. This method has been used to increase the

production of vanillic acid from Vanilla planifolia using phenylalanine as the precursor,

modification of monoterpene into monoterpene glycoside in Mentha Canadensis and the

production of active compounds Rosin and Rosavin from alcohol cinnamyl inRhodiola rosea

(Matkowski 2008).

Elicitation also has been widely used to enhance the production of plant secondary

metabolites. Elicitor is a biotic or abiotic molecule that can induce defense mechanisms in

plant especially in the production of phytoalexins, the low molecular weight molecules which

usually produced when the plant is infected by pathogen (Verpoorte et al 1999). One example

of abiotic elicitor is the jasmonic acid. The application of jasmonic acid as elicitor has

successfully increased the production of -tocopherol in sun flower and Arabidopsis thaliana

cell cultures (Matkowski 2008). Example of biotic elicitor can be seen in the application of

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yeast extract as the elicitor in enhancing the production of silymarin in Silybum marianum

(Ibid).

Plant secondary metabolite usually produced in higher amount from differentiated

tissues compared to undifferentiated tissues (Verpoorte 2002). As the consequence, many

researchers prefer shoot and hairy root culture rather than cell culture. For the research

purpose, most researcher produce plant secondary metabolites in small scales. But when the

secondary metabolites are targeted for the commercial purpose, systematic upscale researches

are required and cell suspension culture are usually used combined with other medium

optimization methods. An example of up scaling approach is in the production of shikonin

usingLithospermum erythrorhizon (Matkowski 2008).

Anyway, not all plant secondary metabolites production can be increased by those

methods. Besides the application of the cell culture produces relatively low amount of

secondary metabolites for commercialization, some types of secondary metabolites are not

sensitive to the medium modification, hormone composition or even with the addition of

elicitors (Verpoorte et al. 1999). The application of genetic approaches seem to have

significant potential to solve this problems, thus become complement to the existing

approaches in enhancing the production of plant secondary metabolites. The next section will

focus on the genetic approaches that have been applied to increase many plant secondary

metabolites.

Genetic Approaches

Biosynthetic pathway mapping

Genetic approaches require very deep understanding about the pathways that involved

in the biosynthesis of the targeted secondary metabolites. The first step that needs to be done

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is the mapping of the biosynthetic pathway producing compound of interest (Verpoorte et al.

1999, 2002). There are some techniques that have been made to build the secondary

metabolites biosynthetic pathway map. One of them is by using the intermediate molecules

which labeled with radioactive to estimate the pathway involved in the production of the

targeted molecules. It is done by analyzing the incorporation of the labeled intermediates in

the targeted product (Verpoorte et al. 2002). Other technique is developing mutant

microorganism to identify enzymes and genes involved in the biosynthetic pathway. The

combination of transcriptome analysis, proteome analysis and metabolome analysis also been

applied by looking at the differences between plants that produce the compound and the

plants that do not produce the compound (ibid). Another technique used to contribute in

building the biosynthetic pathway map is by targeting one step in the biosynthetic pathway,

the enzyme that functions in that step then isolated, gene or genes encoding the enzyme was

or were cloned to be analyzed. Through this technique, detail information about the enzymes

involved in the pathway can be obtained.

There are several aspects need to be taken into consideration in building the plant

secondary metabolites biosynthetic pathway map (Verpoorte et al 2000). First, there are

reactions in the biosynthetic pathway that do not require the action of any enzyme. The

conversion of alkaloid isoquinoline neopine into codeinone in morphine biosynthetic pathway

can be an example for this. Second, there are also enzymes that can process two different

reactions such as the conversion of hiosciamine into 6-hydroxihiosciamine then into

scopolamine only through the action of single enzyme which is hiosciamine 6-hydroxilase.

The third aspect is the selectivity and specificity of the enzyme. Enzymes that have high

specificity usually can only act on one specific substrate while enzymes that have low

specificity can act on several different substrates.

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By developing the biosynthetic pathway map, we can target specific stages in the

pathway to be manipulated so that the production of compound of interest can be increased.

Genetic approaches that will be focused on specific pathway also can be done precisely and

the probability of success in efforts to increase the production of the secondary metabolites

will become higher.

DNA microarray

DNA microarray technology has been developed based on the requirement for the

thorough and efficient strategy to measure the expression of all genes in the genome

(Mohammad Yaseen et al. 2009). DNA microarray can be defined as proper arrangement of

thousand of identified nucleotides or genes that printed on impermeable solid support such as

glass, silicon chip or nylon membrane (Lorkowski & Cullen 2003). As an effort to increase

the production of plant secondary metabolites, this technology is applied to study the profiles

of gene expression that related to the important secondary metabolites production in plant of

interest.

The expression of genes in plant is influenced by several factors such as

developmental stages of the plant and biotic or abiotic stresses that the plant perceives

(Verpoorte et al. 2002). To see the changes in genes expression, we can use two population of

plant. The first population represents plant that has developed to specific developmental stage

or has perceived specific stresses. The second population in the other hand represents the

control population. It also can be applied to represent different plant tissues or organs. RNA

is extracted from both populations and cDNA then synthesized from the extracted RNA and

can be used in probe synthesis to generate the DNA microarray (Lorkowski & Cullen 2003).

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Oligonucleotides that specific to the examined genes are printed onto two pieces of solid chip

represent both population of plant.

After the labeled probes from both population hybridized to the printed nucleotides on

the chip and visualized, differences of expression level of the examined genes can be seen

based on the intensity of signal produced from the hybrid between the two microarray chips.

Another method developed to generate the microarray chip only requires single microarray

chip to analyze changes in genes expression (Lorkowski & Cullen 2003). In this method,

different types of labeling molecule used to represent two different populations. Changes in

level of examined genes expression is determined based on the color formed after

hybridization. If the expression level of the genes increased in the tested population compared

to the control population, the color must be from the molecule that used to label the tested

population. In the other hand, if the expression level of the genes decreased in the tested

population compared to the control population, the color must be from the molecule that used

to label the control population and if the expression level of the gene does not change the

color must be from the combination of molecules that used to label the tested population and

the control population.

By comparing the changes in gene expression level with changes in production of

secondary metabolites, genes that involved in biosynthesis of specific types of secondary

metabolites can be identified. This approach has been used to identify genes that involved in

secondary metabolites biosynthesis which induced by UV-B ray in Arabidopsis thaliana. 70

genes have been found which encode proteins related to photosynthesis, pathogenesis,

antioxidant enzymes, enzymes that involved in lignin and isoflavonoid biosynthesis and also

proteins that involved in signal transduction (Brosche et al. 2002). It is proven that DNA

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microarray is very useful in exploring molecular mechanisms and pathway networks in

secondary metabolites biosynthesis. This approach definitely has high potential to be applied

in broader and larger scale of efforts in enhancing secondary metabolites production.

Metabolic engineering

Other genetic approach that widely used to amplify secondary metabolites production

from plant is the genetic engineering. In the scope of secondary metabolites production, it is

also known as metabolic engineering. Metabolic engineering can be defined as optimization

activities of genetic and regulatory process in the cell to enhance the production of some

compound from the cell through the application of recombinant DNA technology (Sharma &

Sharma 2009). This approach has been applied in enhancing secondary metabolites from

plant since 1990s. In most cases, metabolic engineering researches have been done by

focusing on the enzymes that function in rate limiting manner or competitive to the secondary

metabolites biosynthetic pathways (Verpoorte 1999). The information about the biosynthetic

pathways has been obtained by the generation of biosynthetic pathway map.

After identifying the limiting factor in the biosynthetic pathway, modification can be

made by several ways to get the optimum production of targeted compounds such as

introducing genes that isolated from more efficient organism, using promoter that can

increase the expression of the targeted genes and application of antisense technology which

also part of the gene suppression technology which usually used to obtain plant with some

eliminated trait. In enhancing production of secondary metabolite antisense technology is

applied to suppress competitive pathways which may inhibit the biosynthetic pathways of the

targeted compound. Besides increasing the carbon flux for the compound of interest, this

approach can be applied to decrease the production of unwanted compound like what have

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been done to the Nicotiana attenuate in reducing the nicotine content in that plant. Constructs

that expressed the sense and antisense of PMT gene encoding for putrescine

methyltransferase (the main site for nicotine biosynthesis) was introduced into the plant cell

mediated byAgrobacterium it was observed that the corresponding transcript was reduced in

the root (Saedler & Baldwin 2003).

Metabolic engineering also has been applied in introducing and overexpressing genes

encoding a rate limiting enzyme, putrescine N-methyltransferase (PMT) and hyoscyamine 6-

hydroxilase (H6H) for the biosynthesis of scopolamine into the root culture ofHyoscyamus

niger. Transgenic hairy root clones that expressing both pmt and h6h genes were found

producing higher amount of scopolamine compared to the wild type and clones which only

have single gene (pmtorh6h) (Zhang et al. 2003).

Combinatorial approach

The next genetic approach that we will discuss in this writing is the combinatorial

approach. This approach has been used to enhance the production of some secondary

metabolites that naturally produced in really low amount by plant. Combinatorial approach

can be classified as a new approach applied in increasing plant secondary metabolites. It also

can be used to generate novel compound (Julsing et al. 2006). The basic concept of this

approach is the combination of several metabolic pathways from different organisms and

introducing them into the same genetic level in a host organism. As the consequence, the

precursors which required for the synthesis of the compounds of interest are provided by

these pathways with the action of proteins encoded by introduced genes (Ibid). This approach

has been used for the production of some important classes of plant secondary metabolites

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such as alkaloid (vinblastin, vinkristin), terpenoid (antermisinin, paclitaxel, carotenoid), an

also flavonoid.

Microorganism usually used as the host in combinatorial approach since the structure

of the cell in simple compared to plant. In the production of carotenoid for example, Candida

utilis fungi has been manipulated as the host for the production of lycopene, -carotene and

astaxanthin. The production of carotenoid in the host cell requires the biosynthesis of its

intermediate geranylgeranyl diphosphate (GGDP). E. coli produce C15 precursor forGGDP

which is pharnesyl diphosphate (FDP). The extension of this prenyl chain to C20 occurs by

the diphosphate synthase activity. This enzyme encoded by CrtE gene which isolated from

Erwinia sp. The production of GGDP from FDP is catalyzed by the expression of

prenyltransferase. Enzyme GGDP synthase which encoded by gps from Archaeoglobus

fulgidis also expressed inE. coli. Expression of this GGDP synthase can produce GGDP with

more efficient since it acts by catalyzing three chain extension reactions starting from C5

producing C20 products.

Conclusion

Genetic approaches actually contribute a lot in enhancing plant secondary metabolite

production. Besides providing detail information about how the compounds of interest are

produced from the plants, it also offers chances to increase the production of some compound

that were very difficult to get in sufficient amount through the cell culture approaches before.

Novel compounds also can be obtained through the genetic approaches such as metabolic

engineering and combinatorial approach. Even though there is a lot of discoveries have been

made in the biosynthetic pathways and regulatory systems for the production of secondary

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metabolites in plant, there is a lot more need to be discovered and deeply understood and this

is the main challenge that the researchers in these field have to face. Anyway, development

and improvement in the methodologies applied in this approach may become the catalysts to

the progress of efforts made to gain deeper understanding of biosynthetic pathways network

in plant. This understanding can be applied to increase the production of plant secondary

metabolite efficiently for the welfare of human life.

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Mattijs, K. J., Albert, K., Herman, J. W., Wim, J. Q. & Oliver, K. 2006. Combinatorial

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Mohammad Yaseen, K., Saleh, A., Vimal, K. & Shalini, R. 2009. Recent advances in

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Saedler, R. & Baldwin, I. T.2003. Virus-induced gene silencing of jasmonate-induced direct

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