3. Introduction
Metabolic pathway engineering involves the directed modification of the cellular
activities by manipulation of enzymatic, transport and regulatory functions through
introduction, deletion or modification of metabolic pathways using recombinant DNA
technology and other molecular biological tools.
Future prospects for using metabolic pathway engineering to increase crop productivity,
both in terms of quantity and quality.
RNA interference in designing transgenic crops
Durum wheat (cv. Svevo and cv Ofanto):- Amylose content modified by targeted
manipulation of the starch biosynthetic pathway.
Gossypium hirsutum, cv. Coker 312.
δ-cadinene synthase is the key enzyme in gossypol biosynthesis.
Inhibition of δ-cadinene gene disturbed terpenoid biosynthesis to eliminate gossypol
from seed while retaining a full complement of this and related terpenoid in the rest of
plant for maintaining capability against insect and diseases.
4. Following are main objective for metabolic improvement :
1. Metabolic engineering approaches for modification of metabolite biosynthesis.
2. Metabolic engineering approaches for vitamin biosynthesis.
3. Metabolic engineering approaches for mineral biosynthesis.
4. Metabolic engineering approaches for nutraceuticals/functional foods.
5. Metabolic engineering approaches for functional foods to improve gut health.
6. Metabolic engineering approaches for reduction of antinutrients or allergens
7. Non-transgenic methods for metabolic engineering of crops.
5. 1. Metabolic engineering approaches for modification of metabolite
biosynthesis
Two basic approaches to modifying a biosynthetic pathway to increase amounts of
desirable compounds may be identified – manipulation of pathway flux, or
introduction of novel biosynthetic activities from other organisms.
The methods for increasing, preventing or redirecting flux into or within the
pathway include: increasing levels of a rate-limiting biosynthetic enzyme inhibition
of the activity of a gene that competes for a limited substrate supply, and up- or
down-regulation of the pathway using regulatory factors.
For reducing production of undesirable compounds the well-proven approach is to
inhibit gene activity for one of the biosynthetic enzymes.
RNA interference (RNAi) is an effective and reliable approach for preventing
enzyme production, with examples of better performance than using antisense or
sense-inhibition constructs.
Results for the flavonoid pathway demonstrate that all of the above-mentioned
approaches can be applied successfully to modify plant metabolite production.
6. This includes not only the use of transgenes for biosynthetic enzymes but also
extensive examples of the power of transcription factor (TF) transgenes to up-regulate
the pathway.
For example, at least eight biosynthetic genes leading to anthocyanin production were
greatly up-regulated by a single MYB transgene in petunia, and overexpression of an
AtMYB12 transgene in arabidopsis (Arabidopsis thaliana) caused the specific up-
regulation of four biosynthetic genes for flavonol production.
7. 2. Metabolic engineering approaches for vitamin biosynthesis
‘Golden Rice’ cultivars that could make a significant contribution of provitamin A to
the diet of large population groups.
Provitamin A is the term for the plant carotenoid pigments that are converted to
retinol (vitamin A) in animals through their degradation in an active and regulated
process.
All-trans--carotene has the most provitamin A activity, and it is this carotenoid that has
been the prime focus of the molecular breeding programme.
No rice germplasm capable of synthesising carotenoids in the endosperm has been
identified for use in conventional breeding programmes, so GM is the only current
option for producing rice cultivars high in carotenoids.
The direct engineering of retinol biosynthesis into plants has not been targeted, one
reason being that retinol supplementation may cause side-effects at amounts greater
than 5× the RDA, while amounts of provitamin A are safe at least up to 100× the RDA.
Recently developed versions of Golden Rice have up to 37g /g−1 DW total carotenoids
in the endosperm (of which 31 g g−1 Dw is-carotene)
8. Vitamin E is a collective term for a group of lipid-soluble antioxidants of
photosynthetic organisms called tocochromanols, comprised of tocopherols and
tocotrienols.
There are four types of naturally occurring tocopherols and tocotrienols and that
differ in the number and position of methyl groups on the aromatic ring.
The identification of genes for the biosynthetic steps has enabled a range of
successful GM approaches for increasing vitamin E activity in plant sources.
These have included both increasing total tocochromanol amounts and altering the
specific forms produced.
Examples of the latter include increasing amounts of tocotrienols (which are more
commonly produced in monocots) as opposed to tocopherols and promoting
production of tocopherol, which has the highest vitamin E activity in mammals.
9. 3. Metabolic engineering approaches for mineral biosynthesis:-
More than 20 minerals are required as part of the human diet.
Iron, zinc and iodine are the mineral elements most frequently lacking in diets, but
elements such as calcium, magnesium and selenium are also deficient in the diets of
some populations.
GM approaches to increasing the amounts of minerals in food crops have focused
mostly on iron, zinc and selenium to date.
However, other modifications of food functionality, such as introduction of fructans,
may also affect the absorption of minerals from the diet.
some variation in iron amounts in the available rice germplasm, issues with the
bioavailability of iron may be hard to address by non-GM breeding.
Using GM, there has been little progress in engineering increased iron uptake into the
plant as a whole, but there has been good progress in increasing the distribution of the
iron into the edible parts and improving bioavailability factors.
10. Selenium
The amount of selenium in the diet varies greatly with selenium concentration in
soils.
Increasing amounts of selenium in the diet is essential for some populations, while
for others the amounts of selenium in soil contribute to selenium toxicity.
Metabolic engineering approaches have been targeted at increasing selenium uptake
into plants, and storage in non-toxic forms, both for phytoremediation and to supply
adequate dietary selenium.
11. 4. Metabolic engineering approaches for nutraceuticals/functional foods:-
successful engineering for foods with enhanced amounts of nutraceuticals has
been
limited to a relatively small group of compounds, principally flavonoids, fatty acids
and
carotenoids, and for carotenoids this has mostly been focused on provitamin A
production.
One reason for the small group of nutraceutical compounds targeted is that many of
the
suggested beneficial compounds are produced by biosynthetic pathways for which
there
is limited information.
The desired amounts of vitamins such as provitamin A may be well-known, but such
information is lacking for many of the nutraceuticals that have attracted interest
over
the past few years .
The action of isoflavonoids as phytoestrogens has been known for some time, but
12. 5. Metabolic engineering approaches for functional foods to improve gut health
The gastrointestinal tract is a key area for mediating the action of dietary plant
material on human health.
Environmental factors have a particularly large influence on a range of gut disorders,
such as irritable bowel syndrome, and modifying dietary components could help to
prevent or treat of such chronic conditions.
Foods and food products designed to benefit gut health may include addition of micro-
organisms (probiotics), digestion-resistant carbohydrates (as prebiotics and dietary
fibre) or specific bioactive compounds, such as phenolics.
At present, the metabolic engineering of plants for non-nutrient based improvement
of human gut health is at an early stage.
However, there have already been notable achievements in modifying biosynthesis of
carbohydrates that impact on gut health, specifically altering the type of starch
accumulated and modification or introduction of fructan biosynthesis.
Fructans are soluble carbohydrates comprised of fructose polymers, and occur as
carbon storage molecules in plants of several phylogenetically diverse families.
13. Fructans cannot be digested directly by humans, but are fermented by the gut
bacteria.
There is evidence for a range of health benefits from inclusion of fructans in the diet.
It is thought that they have prebiotic qualities, acting as a food supply for, and
encouraging the abundance of, beneficial gut bacteria such as Lactobacilli and
Bifidobacteria species.
There is also evidence, including from extensive animal trials, that fructans have
beneficial effects on mineral absorption, blood lipid composition, and prevention of
colon cancer, through prebiotic and independent mechanisms.
Extracted fructan, often obtained from chicory, is already in use as a functional food
ingredient, as a soluble low-calorie fibre with associated human health benefits.
14. 6. Metabolic engineering approaches for reduction of antinutrients or allergens
Some crop plants may produce undesirable compounds such as antinutrients,
allergens or toxins, and preventing the production of these in the edible parts is a
target for metabolic engineering.
Cassava (Manihot esculenta) is an interesting example, as it is one of the major food
crops in Africa.
Significant problems associated with cassava as a food staple are its low tuber protein
content, post-harvest losses, and a high content of the cyanogenic glucosides
linamarin and lotaustralin.
When tissue is disrupted, the cyanogenic glucosides are converted to compounds that
include the toxin hydrogen cyanide.
Appropriate processing of the crop can reduce amounts of hydrogen cyanide in the
food, but generally also results in loss of proteins, vitamins, and minerals.
15. Although conventional breeding has generated cultivars with reduced generation of
hydrogen cyanide, no cultivars eliminate cyanogenic glucoside production.
Jørgensen et al. used RNAi to prevent production of the cytochrome P450 enzyme that
makes the first committed step in the biosynthesis of linamarin and lotaustralin, and
generated transgenic plants with elimination of cyanogenic glucosides in the leaves
(<1% of non-transgenic amounts) and a 92% reduction of glucoside amounts in
tubers.
Other examples of using GM for reduction of toxins or antinutrients, specifically;
improving iron bioavailability through reducing phytic acid amounts in rice and the
reduction of sinapate ester content in seeds of oil-seed rape (Brassica napus).
Metabolic engineering has also been used to generate lines of coffee (Coffea arabica)
with reduced caffeine production. Proanthocyanidins can be regarded as antinutrients,
particularly against iron uptake, even though they may also have positive bioactive
properties.
16. 7. Non-transgenic methods for metabolic engineering of crops
Gene technology methods that enabled the transfer of traits from existing germplasm
into leading agronomic cultivars would likely raise fewer concerns over public
acceptance of the modified crops, and fit more easily within traditional crop breeding
programmes.
One approach is to use gene sequences for marker-assisted breeding, and some of the
genes encoding regulatory proteins may be particularly useful in this regard.
However, an alternative approach, which retains the benefits of GM technology, is to
undertake metabolic engineering using only DNA sequences sourced from within the
target crop’s gene pool.
Such transfer of DNA sequences only between plants from within the same sexual
compatibility group has been termed ‘intragenics’ or ‘cisgenics’, as opposed to
traditional transgenics.
17. It is possible to replace all of the required sequences for function of the
Agrobacterium T-DNA with DNA sequences derived from the target crop, to give a
plant (P)-DNA fragment.
These vectors may be designed using bioinformatic searches of publicly available
DNA databases for the target crop to identify sequences that mimic the essential
components of the T-DNA, such as the T-DNA borders.
The selectable marker gene for identification of transformation events may also be of
plant origin, for example the acetohydroxyacid synthase gene conferring tolerance to
chlorsulfuron, or may be removed through a subsequent recombination process.
The effectiveness of such P-DNAs has already been demonstrated for crops, for down-
regulation of polyphenol oxidase activity in intragenic potatoes.
19. Protein
1.Protease inhibitor:-
Plant proteinase inhibitors (PIs) have been well established to play a potent defensive
role against predators and pathogens.
Although diverse endogenous functions for these proteins has been proposed, ranging
from regulators of endogenous proteinases to act as storage proteins, evidence for
many of these roles is partial, or confined to isolated examples.
On the other hand, many PIs have been shown to act as defensive compounds against
pests by direct assay or by expression in transgenic crop plants, and a body of
evidence for their role in plant defense has been accumulated consistently.
The role and mechanism of action for most of these inhibitors are being studied in
detail and their respective genes isolated.
These genes have been used for the construction of transgenic crop plants to be
incorporated in integrated pest management programmes.
This article describes the classes of protease inhibitors, their regulation and genes
used to construct transgenic plants against phytophagous insects.
20. Phytohaemoagglutinins/Lectins:-
Lectins are proteins in nature with molecular weight ranging from 60,000 to 100,000
Da. Many lectins contain covalently bound sugar moieties and are glycoprotein in
nature.
These are also called phytohemagglutins because they agglutinate red blood cells.
Lectins are widely distributed in the plant kingdom and have the unique property of
binding to carbohydrate-containing molecules, with a high degree of specificity toward
the sugar component.
Lectins inhibit growth of the animals.About 60% of the lectin survives intestinal transit
and becomes bound to the intestinal epithelium, where it causes disruption of the
brush border and atrophy of the microvilli, and reduces the viability of the epithelial
cells. As a consequence of the interaction of lectin with the epithelial surface of the
proximal small intestine, there is an increase in the weight of the small intestine.
This is because of the hyperplasia of the crypt cells, an effect that is believed to involve
the accumulation of polyamines, mostly spermidine, a known stimulant of cellular
proliferation.
21. Glycosides
Goitrogens
Goitrogens are foods which suppress thyroid function. In normals, goitrogens can
induce hypothyroidism and goiter.
In hypos, goitrogens can further depress thyroidal function and stimulate the growth
of the thyroid (goiter).
In hyperthyroids, goitrogens may help suppress thyroidal function until normal
thyroidal functioning can be restored.Goitrogens work by interfering with the
thyroidal uptake of iodine.
While many hypers try to limit thyroid output by iodine restriction, this strategy can
backfire.
Iodine restriction will cause the thyroid to increase in size (goiter) in an effort to filter
more blood to get more iodine.
When iodine is then re-introduced to the diet or accidentally ingested, the now larger
thyroid gland has the capacity for greater thyroid hormone production.
Therefore the consumption of goitrogens is not a good strategy. It is better to increase
copper metabolism by supplementation of copper and the assisting nutrients.
22. Saponins
Saponins comprise a large family of structurally related compounds containing a
steroid or triterpenoid aglycone (sapogenin) linked to one or more oligosaccharide
moieties by glycosidic linkage.
The carbohydrate moiety consists of pentoses, hexoses, or uronic acids.
The presence of both polar (sugar) and nonpolar (steroid or triterpene) groups
provides saponins With strong surface-active properties that then are responsible for
many of its adverse and beneficial effects.
The primary biological effect of Saponins is the interactions With cellular and
membrane components.
For example, saponins hemolyze red blood cells by nonspecific interactions with
membrane proteins, phospholipids, and cholesterol of erythrocytes.
23. Effects of saponin:-
Saponins are characterized by their hemolytic activity and foaming properties
and are responsible for imparting a bitter taste and astringency to plant materials
containing high concentrations of saponins.
Nonetheless, saponins are reported to affect the permeability of the small intestinal
mucosal cells and thus have effect on active nutrient transport. Saponins have also
been shown to inhibit various digestive enzymes,
Including trypsin and chymotrypsin, and are also known to inhibit protein degradation
by forming saponin-protein complexes.
On the other hand, positive nutritional effects of specific saponins such as
hypocholesterolemic effects and improvement of growth in various animal species
have also been reported.
Medicago sativa (Alfalfa, Lucerne) contains many saponins. Medicagenic acid is unique
to alfalfa.
Alfalfa saponins may lower growth rate in chicks and egg production of hens when
included in poultry diets above 5%.
24. Phenols
Gossypol
Nature and Biological Effects
Gossypol is a polyphenolic aldehyde that is an antioxidant and polymerization
inhibitor.
It is toxic to monogastric animals; pigs and rabbits are the most sensitive, whereas
poultry are relatively more tolerant.
The general symptoms of gossypol toxicity are constipation, depressed appetite, loss of
weight, and death, which usually results from circulatory failure.
Although acute toxicity is low, ingestion of small amounts over a prolonged period
can be lethal.
It is important to distinguish between free (soluble in 70–30 v/v aqueous acetone) and
bound gossypol since only the former is considered to be physiologically active.
They present in Gossypium spp.
26. Tannins:-
Tannins are polyphenolic compounds that are broadly categorized into two
major groups: (1) hydrolyzable tannins, consisting of a central core of carbohydrate
to which phenolic carboxylic acids are bound by ester linkage.
(2) condensed tannins, or proanthocyanidins, consisting of oligomers
of two or more flavan-3-ols, such as catechin, epicatechin, or the corresponding
Gallocatechin.
27. Mode of action:-
Tannins have a very high affinity for proteins and form protein-tannin complexes.
The ingestion of a plant containing condensed tannins decreases nutrient
utilization, protein being affected to a great extent, and decreases feed intake.
On the other hand, hydrolyzable tannins are potentially toxic to animals
Consumption of feeds containing high levels of hydrolyzable tannins cause liver and
kidney toxicity and lead to death of animals.
Oak and yellow wood poisonings are attributed to hydrolyzable tannins.
29. Miscellaneous
Antivitamin:-
Stresses Are Antivitamins
All kinds of stresses are vitamin antagonists. Drugs are serious stress producers in the
body because the body must exercise great effort in expelling them as quickly as possible,
lest they damage tissues and cells and interfere too much with normal functioning. In
addition, surgery, accidents, overly exhausting work or exercise, exposure to extreme's of
heat or cold, and emotions such as fear, hatred, anger, worry and grief all produce great
stress on the body. The B vitamins (thiamin, niacin, folic acid, pantothenic acid and
vitamin B12) and vitamin C, as well as proteins and minerals, are all depleted and/or
unassimilable as a result of stresses on the body.
Aspirin Is An Antivitamin
Aspirin interferes with digestive processes and can result in stomach bleeding. It
interferes with blood-clotting and lessens the ability of cells to absorb glucose for heat
and energy. It depletes most, if not all, nutrients and results in especially high losses of
vitamin C and the B vitamins plus the minerals calcium and potassium.
Antibiotics Are Antivitamins
Besides being a vitamin K antagonist, the antibiotic penicillin is also an antivitamin of
vitamin B6. The antibiotic streptomycin is a folic acid antagonist and the antibiotic
streptomycin inactivates manganese, a mineral which is needed for the functioning of
many enzyme systems.
30. Diuretics Are Antivitamins
Diuretics are drugs prescribed medically to promote weight reduction or to relieve
pressure of retained fluids. Even so-called "natural" diuretics, including herbal types, are
harmful, for all diuretics result in great losses of B vitamins, vitamin C, other vitamins,
and the minerals potassium and magnesium. Diuretics would never be prescribed to
anyone on a natural diet containing no rock salt or sea salt, as these salts are poisonous
and cause the body to retain fluids to hold the salt in suspension so it doesn't harm cells
and tissues.
Laxatives Are Antivitamins
All laxatives, including the herbal types, are vitamin antagonists. Mineral oil is perhaps
the most devastating laxative. It absorbs vitamin A and carotene, as well as the other fat-
soluble vitamins (vitamin D, vitamin E and vitamin K). It also absorbs calcium and
phosphorus, carrying them out of the body. (Hospitals today still use mineral oil as a
laxative for their patients, one of thousands of reasons why hospitals are antivital places.)
Laxatives will never be used by people on a natural all-raw diet of fruits, vegetables,
sprouts, nuts and seeds.
31. Minerals as antinutritional factor (antiminerals)
When excess minerals are present in the soil, the soil is said to be saline, and
plant growth may be restricted if these mineral ions reach levels that limit
water availability or exceed the adequate zone for a particular nutrient.
Sodium chloride and sodium sulfate are the most common salts in saline
soils.
Another important problem with excess minerals is the accumulation of
heavy metals in the soil, which can cause severe toxicity in plants as well as
humans.
Heavy metals include zinc, copper, cobalt, nickel, mercury, lead, cadmium,
silver, and chromium.
32. Anti-Enzymes
Enzymatic reaction continues fruits and vegetables get spoiled.
Bring browning in potato, brinjal and plantain.
Raw papaya contains an enzyme called papain.
Anthocyanase is responsible for loss of anthocyanins.
33. Food allergance & causal organism
a) Intentional additives
-binders and stabilizers (carboxymethyl cellulose, alginates, gums)
- therapeutic drugs (antibiotics, sulphonamides, nitrofurans, arsenilic acid)
- growth promotants (as above, plus anabolic steroids, synthetic androgens)
b) Toxic factors arising from processing
-solvent residues present in solvent extracted oilseeds (methylene chloride, ethylene
dichloride, trichloroethylene, acetone, iso-propyl alcohol)
- lipids spoiled by oxidation and/or heat (rancidity, oxidation products).
c) Contaminants of biological origin
-protozoan toxins from spoiled fish
- algal toxins from shellfish/fish
- fungal toxins in stored foods (i.e. aflatoxins)
- bacterial toxins from contaminated foodstuffs (i.e. botulinum toxin)
- pathogens (viable bacteria, viruses and fungi)
d) Synthetic contaminants
- pesticide residues (chlorinated hydrocarbons)
- organochlorine compounds (polychlorinated biphenyls)
- petroleum hydrocarbons
- heavy metals
34. Toxic Amino Acid
Mimosin
Properties
l-mimosine is a nonprotein amino acid.
Despite the consideration of Leucaena leucocephala as a promising alternate source of
protein for fodder, the presence of mimosine to the extent of 2% to 10% dry matter
in the leaf and 2% to 5% dry matter
In the seed has limited its use as a livestock feed since mimosine and its degradation
products 3-hydroxy-4-(1H)-pyridone (3,4-DHP) and 2,3-dihydroxypyridine (2,3-
DHP) have been known to be toxic to many species.
35. Mode of action
Ingestion of mimosine results in hair loss, goiter, reproductive disorders, epithelial
damage, reduced feed intake, and ultimately death in both nonruminants and
ruminants.
Certain segments of the human population are known to consume portions of the
leucaena in their diet, and a loss of hair has been frequently observed among those
individuals who eat the leaves, pods, and seeds in the form of a soup.
Resistance to mimosine toxicity in ruminants of certain geographical areas has been
attributed to the capability of their rumen microorganisms to restrictively metabolize
mimosine and DHP.
From the rumen of goats in Hawaii resistant to mimosine toxicity, a microorganism
(Synergistes jonesii), capable of metabolizing mimosine and DHP to innocuous
products has been successfully transferred to the rumen of cattle in Australia that
were susceptible to mimosine toxicity.
