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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.
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
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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 of u
and the production was successfully enhanced through this method (Verpoorte et al.
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 active compounds Rosin and Rosavin from alcohol cinnamyl in
(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 Į-tocopherol in sun flower and
cell cultures (Matkowski 2008). Example of biotic elicitor can be seen in the application of
yeast extract as the elicitor in enhancing the production of silymarin in "
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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
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.
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Denetic 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
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 (). 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
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
can be an example for this. Second, there are also enzymes that can process two different
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
By developing the biosynthetic pathway map, we can target specific stages in the
Denetic 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
A 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
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.
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. R A
is extracted from both populations and c A then synthesized from the extracted R A and
can be used in probe synthesis to generate the A microarray (Lorkowski & Cullen 2003).
r
Oligonucleotides that specific to the examined genes are printed onto two pieces of solid chip
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
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 . 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 A
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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
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
activities of genetic and regulatory process in the cell to enhance the production of some
compound from the cell through the application of recombinant A 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
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
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 in reducing the nicotine content in that plant. Constructs
that expressed the sense and antisense of gene encoding for putrescine
methyltransferase (the main site for nicotine biosynthesis) was introduced into the plant cell
mediated by it was observed that the corresponding transcript was reduced in
Metabolic engineering also has been applied in introducing and overexpressing genes
hydroxilase (H6H) for the biosynthesis of scopolamine into the root culture of
Transgenic hairy root clones that expressing both and genes were found
producing higher amount of scopolamine compared to the wild type and clones which only
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 (). This approach
has been used for the production of some important classes of plant secondary metabolites
0
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, u
fungi has been manipulated as the host for the production of lycopene, ȕ-carotene and
astaxanthin. The production of carotenoid in the host cell requires the biosynthesis of its
intermediate geranylgeranyl diphosphate (DDP). ! produce C15 precursor for DDP
which is pharnesyl diphosphate (FP). The extension of this prenyl chain to C20 occurs by
the diphosphate synthase activity. This enzyme encoded by CrtE gene which isolated from
Erwinia . The production of DDP from FP is catalyzed by the expression of
prenyltransferase. Enzyme DDP synthase which encoded by from
also expressed in ! . Expression of this DDP synthase can produce DDP with
more efficient since it acts by catalyzing three chain extension reactions starting from C5
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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.
ovel 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
c
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Matkowski, A. 2008. Plant in vitro culture for the production of antioxidant ± A review.
£ !" :548-560
Mattijs, K. J., Albert, K., Herman, J. W., Wim, J. Q. & Oliver, K. 2006. Combinatorial
biosynthesis of medicinal plant secondary metabolites. £ ! :265-
279
Mohammad Yaseen, K., Saleh, A., Vimal, K. & Shalini, R. 2009. Recent advances in
medicinal plant biotechnology. # £ 0:9-22
Verpoorte, R., Heijden, R., Hoopen, H. J. D. & Memelink, J.1999. Metabolic engineering of
plant secondary metabolite pathways for the production of fine chemicals. £
:467-479
Verpoorte, R., Heijden, R. & Memelink, J. 2000. Engineering the plant cell factory for
secondary metabolite production. À:323±343
Verpoorte, R., Contin, A. & Memelink, J. 2002. Biotechnology for the production of plant
secondary metabolites. ":13-25
Zhang, W., Franco, C., Curtin, C. & Conn, C. 2004. To Stretch the Boundary of Secondary
Metabolite Production in Plant Cell-Based Bioprocessing: Anthocyanin as a Case Study.
Journal of Biomedicine and Biotechnology :264±271
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