As a most notable member in the stilbenoid family, resveratrol (3,5,4′-trihydroxy-trans-stilbene) has a wide range of biological activities which may have the impact on human health, including antioxidant, anti-inflammatory, cardioprotective, antiviral, anticancer, and antiaging properties as demonstrated in in vitro and animal studies. The concentration of this amino acid is higher in tomato compared with many vegetables such as carrots, onions or pepper. In addition, glutamate provides the characteristic ‘‘umami taste’’ to foods with high free glutamate content such as cheese, tomato and mushrooms, which are major ingredients in cooking.

1. Production and Biosynthesis of resveratrol and L-glutamate
by genetic engineering methods
Yusuf Farrokhzad
yusuf.farokhzad@modares.ac.ir
6/15/2018 1

2. Metabolic engineering is referred to as
the directed improvement of cellular
properties through the modification of
specific biochemical reactions or the
introduction of new ones, with the use of
recombinant DNA technology.
What is metabolic engineering?
Metabolic engineering is generally defined as the
redirection of one or more enzymatic reactions to
improve the production of existing compounds,
produce new compounds, or mediate the
degradation of undesirable compounds. It involves
the redirection of cellular activities by modifying
the enzymes, endo-cellular localization, and
regulatory functions within cells.
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Stilbenoids:
Stilbenoids are plant defense phenolic compounds
that exhibit numerous biological activities with
potential applications in human health.
These compounds are present in non-taxonomically
related plants species, such as grapevine (Vitis vinifera),
peanut (Arachis hypogaea), and blueberry (Vaccinium
spp.).

4.  Stilbenoids also provide protection against
oxidative and other abiotic stresses such as UV
radiation.
 As a most notable member in the stilbenoid
family, resveratrol (3,5,4′-trihydroxy-trans-
stilbene) has a wide range of biological activities
which may have impact on human health,
including antioxidant, anti-inflammatory,
cardioprotective, antiviral, anticancer, and
antiaging properties as demonstrated in in vitro
and animal studies.
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 Role in plant
 Health properties

5. In plant tissues, its production is either constitutive or inducible and
is controlled by the key enzyme, stilbene synthase (STS),
which belongs to a multigene family of the type III group of the
polyketide synthase superfamily.
 Stilbene synthase (STS):
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6.  Pathway biosynthetic of Resevratrol:
STS catalyses the condensation of three
molecules of coumaroyl-CoA to form resveratrol.
The synthesis of resveratrol takes place in a
single enzymatic step with CoA-esters of
cinnamic acid derivatives and three malonyl-CoA
units as starting blocks. STS and chalcone
synthase (CHS) are key enzymes of the flavonoid
biosynthesis pathway.
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7. 1. Hairy Root Cultures of Vitis Species
biosynthetic potential of the parental plant
high growth rate and genetic stability
The first study describing the stilbenoid profile in a Vitis species hairy
root culture has been carried out in muscadine grape (Vitis
rotundifolia.Two hairy root lines, Fry-3A and Nob-2Cot, were produced via
A. rhizogenes-mediated transformation. The accumulation of four
stilbenoids, resveratrol, piceatannol, ε-viniferin, and piceid, in these two
hairy root culture lines was studied upon treatment of cultures with the
stress hormone methyl jasmonate (MeJA).
 Resevratrol Biosynthesis by Hairy Root
Cultures:
The levels of stilbenoids were determined by high-performance liquid
chromatography. Later, the effects of MeJA and hydrogen peroxide on the
accumulation of stilbenoids in cultures of line Fry-3A were further
determined along a time course from 0 to 96 h.
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8. The synthesis of resveratrol is
strongly enhanced by fungal attacks,
UV irradiation and other
environmental stress conditions.
2. Resevratrol Biosynthesis by non-
GM methods:
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9.  Resveratrol Production in Transgenic
Plants:
Promising results have been obtained with STS-
encoding genes in transgenic plants, thus confirming
that plant molecular engineering with resveratrol
may lead to a novel
STS genes have been transferred to a number of
crops either to improve.
The resistance of plant
to stresses(biotic and
abiotic stresses such as
fungal pathogens and
UV radiation).
The nutritional value
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10. 1.Two stilbene synthase genes from Vitis
vinifera, (Vst1 and Stsy) have been the most
common genes used for plant transformation.
 Other STS-encoding genes have also been
used:
2. AhRS gene from Arachis hypogea
3. SbSTS1 gene from Sorghum bicolor
4.STS-encoding gene from Parthenocissus
henryana
To increase the levels of stilbene production,
some investigators have used chimeric genes or
a combination of two STS encoding genes
(Vst1 and Vst2).
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11. To date, the STS-encoding genes for plant
transformation have been expressed under the
control of a limited number of promoters, in
particular the well-characterized constitutive
promoter pCaMV35S, its own stress-responsive
promoter pVst1, the fungus-inducible promoter
pPR10.1 or the tissue-specific promoter p-nap. As
expected, the pCaMV35S promoter triggered
strong and constitutive stilbene accumulation in
most studies, but, as a consequence, in some cases,
caused a drastic depletion of the
endogenous pools of precursors.
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13.  These analyses have shown various stilbene levels and spatiotemporal distributions, leading
to a considerable variability in terms of relative amounts of different forms.
In particular, the glycosylation of polyphenolic compounds occurs frequently in plants not only to protect
cells from their potential toxic effects but also to prevent their oxidation and enzymatic degradation. In
the case of resveratrol, the free compound is first synthesized before being glycosylated by endogenous
glycosyl-transferases.
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Free resveratrol and its glycosylated forms have both been detected in transgenic plants. The stilbene
content also depends strongly upon plant species, probably on account of different endogenous pools of
enzymes or precursors, as well as differences in secondary metabolic pathways.

14. In transgenic tomato, resveratrol synthesis was found to increase the overall antioxidant
properties of the fruit, as well as the ascorbate/glutathione content
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 Biological activity Resevratrol in transgenic plant:

15. As far as concerns the amounts of stilbenes detected in
the various studies, no common trend has been found,
which may possibly be explained by the plant species as
well as the specific genetic constructs used for
transformation. In tomato and apple, the free to
glycosylated resveratrol ratio naturally depends on the
fruit ripening stage.
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These two compounds accumulate differentially in fruit
tissues at the mature stage. These variations may be the
result of different endogenous β-glycosidase expression
patterns. Even more than the plant species considered,
the stilbene levels and forms in transgenic plants depend
not only on tissues or organs used as source material,
but also on the fruit ripening stages.

16. Free amino acid production during tomato fruit ripening:
focus on L-glutamate
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Tomato (Solanum lycopersicum):
Tomato is an important worldwide crop and represents one of
the most highly consumed vegetables in Western countries.
Tomato plays an important role in human diet and provides
health benefits as source of vitamins (A and K), minerals
(phosphorus and potassium) and antioxidants (phenolics,
folate, vitamins C and E, lycopene and b-carotene)
Interest in finding the way to improve the flavor of
tomatoes are increasing, given that consumers are
complaining about poor tomato organoleptic properties.
The ripening process involves drastic changes in fruit
characteristics that improve palatability of fruit. The taste
of tomato is enhanced during this process, which results
from a combination of sugars, organic acids and free
amino acids.

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Free amino acid content of tomato fruit pericarp increases
markedly during ripening transition of tomato fruit, suggesting a
high protein turnover. In particular, free glutamate content of
ripe tomato fruit is much higher in all the cultivated varieties
than in tomato wild species.
The concentration of this amino acid is higher in
tomato compared with many vegetables such as carrots,
onions or pepper. In addition, glutamate provides the
characteristic ‘‘umami taste’’ to foods with high free
glutamate content such as cheese, tomato and
mushrooms, which are major ingredients in cooking.

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Glutamate occupies a in the metabolism of
aminoacids in plants. Biochemical and genetic approaches
had been pursued to investigate the sources of glutamate
accumulation in tomato fruit.
1. glutamate decarboxylase (GDC; EC 4.1.1.15), which
catalyzes the decarboxylation of glutamate to c-aminobutyrate
(GABA)
2. glutamine synthetase (EC 6.3.1.2), involves in the synthesis
of glutamine from glutamate
3. glutamate dehydrogenase (GDH; EC 1.4.1.3) that catalyzes
the amination of 2-oxoglutarate (synthetic reaction) and the
deamination of glutamate (catabolic reaction).
Glutamate metabolizing enzymes in the pericarp of
ripening tomato fruit such as:

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 Transgenic tomato plants with altered levels
of glutamate were produced by introducing a
gene for GDC in the anti-sense orientation,
by overexpressing legdh1, which encodes the
beta subunit of tomato NADH-GDH, and
by expression of a gdhA gene for NADPH-
dependent GDH of Aspergillus nidulans.
 L-Glutamate is the main N-form of tomato
phloem sap therefore, it would also be
supplied to the fruit, although to a lesser
extent due to:
1.low sink activity of ripe fruits.
2.the degradation of endogenous peptides.

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 The observed changes in transcripts and
metabolites during fruit ripening indicate that the
ripening process involves substantial turnover of
existing and newly synthesized proteins. Different
carboxypeptidases had been already identified in
tomato fruit. In addition, two c-glutamyl
transpeptidases which catalyze the glutamate
transfer to either water (hydrolysis) or to an
acceptor amino acid or peptide, were shown to
increase during the tomato ripening transition.
 This fact implicates that glutamate could also be
liberated from c-glutamyl substrates such as
glutathione or other molecules with a c-linked
terminal glutamate residue.

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Among the pathways that could
produce glutamate depicted in Fig,
the activity of 2-oxoglutarate-
dependent L-a-amino acid
transaminases (EC 2.6.1) and
GABA transaminase (GABA-T; EC
2.6.1.19) was previously detected in
ripening tomato fruits.
Proposed pathways of glutamate production in tomato fruits

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