Metabolic engineering involves redirecting enzymatic reactions in an organism to produce new compounds or improve existing ones. It focuses on intermediates or products like starch, vitamins, amino acids. Successful approaches introduce new pathways, like producing provitamin A in rice. Rate-limiting steps and multi-level modifications are important. Unexpected results can occur. Commercialization requires safety characterization. Goals include overproducing desired compounds, underproducing unwanted ones, and novel compounds. Engineering targets pathways for carbohydrates, amino acids, lipids, alkaloids, terpenoids and more. Important examples include high-lysine plants, nutritionally-improved cottonseed oil, and Golden Rice which produces beta-carotene in rice
General Principles of Intellectual Property: Concepts of Intellectual Proper...
Metabolic engineering
1. METABOLIC ENGINEERING……
Metabolic engineering is generally defined as the
redirection of one or more enzymatic reactions to produce
new compounds in an organism, improve the production
of existing compounds, or mediate the degradation of
compounds.
Dr. Soumitra Paul, MPP Lab, C.U.
2. • The use of term “engineering” implies that there is
some precise understanding of the system, that is
being modified
• Rate-limiting steps must be known
• A typical metabolic-engineering approach focuses on
a particular metabolic intermediate or product such as
starch, vitamin E, carotenoids, amino acids etc.
Metabolic engineering………..
Dr. Soumitra Paul, MPP Lab, C.U.
3. • The most successful metabolic-engineering approaches
are those that have introduced new pathways into plants
e.g. production of provitamin A in rice
• It’s a matter of great importance to perform a metabolite
profiling of transgenic plants (even in case of failure of
experiment) in order to diagnose the problem
• Unexpected results can happen e.g. dwarf tomato plants
instead carotenoid rich plants
• Commercial projects must characterize their plants in
order to release plant into a market
Dr. Soumitra Paul, MPP Lab, C.U.
4. BACKGROUND…….
• Over the past few years, significant advances have been made in metabolic
engineering through the application of genomics and proteomics technologies to
elucidate and characterize metabolic pathways in a holistic manner, rather than on a
step by step basis.
• Further work has been carried out on the modeling of metabolic pathways on a
genomic scale, which has shown that metabolic pathways are controlled at multiple
levels and any form of perturbation can have wide ranging effects at the whole
system level.
• Hence, the attention has now shifted towards more complex and sophisticated
strategies in which several steps in a given pathway are modified simultaneously to
achieve optimal flux.
Dr. Soumitra Paul, MPP Lab, C.U.
5. GOALS OF PLANT METABOLIC ENGINEERING …..
There are three basic goals of metabolic engineering in plants:
1. The production of more of a specific desired compound.
2. The production of less of a specific unwanted compound.
3. The production of novel compound.
PLANT METABOLIC ENGINEERING
DESIRED COMPOUND UNWANTED COMPOUND NOVEL COMPOUND
INCREASE
DECREASE
IMPORT
Dr. Soumitra Paul, MPP Lab, C.U.
6. Several ways to metabolically engineer an
organism
• Block a metabolic flux (re-channel)
• Channel a metabolic flux into new cell
compartments
• Induce a metabolic flux (can lead to
unexpected results)
• Introduce a new metabolic pathway into
organism (the most successful way)
Dr. Soumitra Paul, MPP Lab, C.U.
8. Incorporation of new pathway in plants
1. Engineering for VLC-PUFAs in Arabidopsis:
Incorporation of Δ9-elongase, Δ8-desturase and Δ5-desaturase genes from
bacteria, with seed specific promoter. Recently, reconstruction through
altering DHA metabolism by ɷ-3 desaturase from Phytopthora infestans into
Brassica juncea.
2. Engineering for folate metabolism,
Overexpression of ADCS (aminodeoxychorismate synthase) and GCHI
(GTP cyclohydrolaseI) gene increases PABA and pteridine molecules,
ultimately rises folate levels in tomato
3. Enhancing ascorbate bioosynthesis
Overexpression of rat L-gluconolactone oxidase in tobacco, myo-inositol
oxidase in Arabidopsis, supressing malate dehydrogenase in mitocondria in
tomato Dr. Soumitra Paul, MPP Lab, C.U.
10. CARBOHYDRATE METABOLISM…..
Starch:
• Starch is a storage carbohydrate that accumulates transiently in leaves and stably
in seeds, tubers and roots.
• It is a staple source of dietary carbohydrate for animals and also has a large
number of industrial uses.
• Metabolic engineering has been used in an attempt to increase starch yields and to
modify the properties of starch by changing the relative proportions of its two
components – amylose and amylopectin.
Dr. Soumitra Paul, MPP Lab, C.U.
11. ENGINEERING THE STARCH BIOSYNTHETIC PATHWAY……
• The allosteric enzyme ADP-glucose pyrophosphorylase (AGP) plays a key role in
regulating starch biosynthesis in cereal seeds and is likely the most important
determinant of seed sink strength.
• Plant AGPs are heterotetrameric, consisting of two large and two small subunits.
• Both wheat (Smidansky et al 2002) and rice (Smidansky et al 2003) have been
transformed with a modified form of maize (Zea mays L.) Shrunken2 gene (Sh2r6hs),
which encodes an altered AGP large subunit.
• This altered large subunit gives rise to a maize AGP heterotetramer with decreased
sensitivity to its negative allosteric effector, orthophosphate, and more stable interactions
between large and small subunits.
Dr. Soumitra Paul, MPP Lab, C.U.
12. • Both transgenic wheat and rice lines showed increase in seed weight and total plant
biomass. Results indicate increased availability and utilization of resources in response to
enhanced seed sink strength.
STARCH
B
I
O
S
Y
N
T
H
E
T
I
C
PATHWAY
Dr. Soumitra Paul, MPP Lab, C.U.
13. • Amylose content in wheat has been markedly increased with RNAi approach, by
suppressing simultaneously the expression of SBEIIa and SBEIIb, which are the
isoforms of the starch branching enzyme (Regina et al 2006).
Modifying Relative proportions of Amylose and Amylopectin
Scanning electron microscopy identified gross changes in granule size and structure as shown
in the figure above. Compared with the untransformed control, starch granules from
endosperms with reduced SBEIIa and SBEIIb expression displayed significant
morphological alterations.
Scanning electron micrographs of isolated starch granules. NB1 (nontransformed control wheat)
(a), 087 (hp-SBEIIa wheat) (b), and 008 (hp-SBEIIb wheat) (c).
Dr. Soumitra Paul, MPP Lab, C.U.
14. AMINO ACID METABOLISM:
• Amino acid synthesis pathways have been targets for metabolic engineering
predominantly to increase the abundance of essential amino acids- Lysine, threonine,
methionine and tryptophan.
• Lysine biosynthesis in plants is regulated primarily by a lysine-mediated feedback
inhibition of dihydrodipicolinate synthase (DHPS).
• Efforts have been made to improve lysine production in plants by metabolic
engineering, by utilizing bacterial DHPS enzymes that are much less sensitive to lysine
inhibition than their plant counterparts.
• Transgenic plants expressing the bacterial DHPS in the plastid, overproduced lysine by
upto more than 100-fold compared to control nontransformed plants (Galili 2002).
Dr. Soumitra Paul, MPP Lab, C.U.
15. The Aspartate family biosynthetic pathway of the essential amino acid.
Abbreviations: AK,aspartate kinase; DHPS,dihydrodipicolinate synthase; HSD,homoserine
dehydrogenase; HSK,homoserine kinase; TS,threonine synthase; CGS,cystathionine c-synthase;
CBL,cystathionine b-lyase; MS; methionine synthase; SAMS; S-adenosylmethionine synthetase.
Dr. Soumitra Paul, MPP Lab, C.U.
16. POLYAMINE METABOLISM:
• Polyamines are small aliphatic amines that are derived
from the amino acids ornithine and arginine by
decarboxylation.
• Polyamines have several important roles in plant
physiology and development. The three major polyamines
found in plants are putrescine, spermidine and spermine.
• Polyamine accumulation specifically in the rice grains was possible by expressing ODC
under the control of the wheat seed-specific, low molecular weight glutenin promoter, and
such lines could be very useful to improve the nutritional properties of rice grains without
affecting the growth or development of vegetative parts of plants.
• The primary targets in the polyamine biosynthesis pathway are the ornithine decarboxylase
(ODC) and arginine decarboxylase (ADC).
Putrescine
Spermidine
Spermine
Dr. Soumitra Paul, MPP Lab, C.U.
17. Polyamine
biosynthesis
in
plants
Another key enzyme in the polyamine biosynthesis, SAMDC, has also been targeted. The
Datura stramonium SAMDC gene was expressed in rice and the expected increase in SAMDC
activity increased the levels of spermidine and spermine accumulated in seeds (Thu-Hang et al.,
2002). Further, yeast SAMDC has also been expressed in tomato, increasing its nutritional value
(Mehta et al., 2002).
Dr. Soumitra Paul, MPP Lab, C.U.
18. LIPID METABOLISM:
• The modification of lipid metabolism to change the quantity and quality of fatty
acids in plants has important applications in the food industry.
• Plants represent a significant renewable source of fatty acids because many species
accumulate them in the form of triacylglycerol as major storage components in seeds.
• Recently, Liu et al 2002, utilized a hairpin RNA (hpRNA) mediated RNAi method to
downregulate two key fatty acid desaturase genes encoding stearoyl-acyl-carrier
protein 9-desaturase and oleoyl-phosphatidylcholine 6-desaturase.
Downregulation of these two genes in cotton resulted in nutritionally improved high
stearic and high oleic cotton seed oils which are essential fatty acids for better health
of human heart.
Dr. Soumitra Paul, MPP Lab, C.U.
19. ENGINEERING SECONDARY
METABOLIC PATHWAYS……
Alkaloids:
• Alkaloids are the largest group of secondary metabolite synthesized by plants and the
pathways, enzymes and regulatory genes involved in their synthesis have been extensively
studied.
• A very interesting example of metabolic engineering of alkaloid is replacement of
morphine with non-narcotic alkaloid reticuline in opium poppy (Papaver somniferum).
• In 2004, Allen et al, reported gene silencing in transgenic opium poppy using RNAi. They
interfered multiple steps of complex biochemical pathway with an hpRNA construct
designed to silence all members of the multigene Codeine reductase (COR) family.
Dr. Soumitra Paul, MPP Lab, C.U.
20. • After the gene silencing through RNAi , the transgenic plants accumulated (S)-
reticuline, a precursor non-narcotic alkaloid which occurs seven enzymatic steps
upstream of codeinone in the pathway. The (S)- reticuline accumulation in transgenic
opium poppy occurred at the expense of morphine, codeine, opium and thebaine.
Dr.SoumitraPaul,MPPLab,C.U.
21. TERPENOIDS
• The terpenoids, sometimes called isoprenoids, are a large and diverse class of naturally-
occurring organic chemicals, derived from five-carbon isoprene units assembled and
modified in thousands of ways.
• Despite their diversity, all terpenoids are synthesized from the common precursors
dimethylallyl pyrophosphate and isopentyl pyrophosphate. This occurs through two
distinct pathways – the mevalonate independent pathway and the mevalonate pathway,
both of which have been targets for metabolic engineering.
• Metabolic engineering of biosynthetic pathway for production of β-carotene in rice and
reduction of toxic gossypol in cotton have been done successfully.
Dr. Soumitra Paul, MPP Lab, C.U.
23. • In 2006, Sunilkumar et al. reported successful disruption of gossypol biosynthesis in
cottonseed tissue by interfering with the expression of the -cadinene synthase gene during
seed development. The gossypol content was reduced only in the seeds of cotton, without
affecting its levels in any other part of the plant.
Reduction of Gossypol in Cotton
• Cotton seeds have high quality protein content which
makes it a nutrient rich resource for food but it
cannot be utilized due to the presence of toxic
gossypol within the seed tissue.
• Gossypol is a cardio and hepatotoxic terpenoid, which
makes it unsafe for human and monogastric animal consumption. Hence any means, which
could yield gossypol-free cottonseed, would significantly contribute to human nutrition and
health.
Dr. Soumitra Paul, MPP Lab, C.U.
24. Structures and proposed biosynthetic pathway of gossypol and other
terpenoids in cotton plants. δ-cadinene synthase enzyme was targeted in the
seed through RNAi to interfere with gossypol biosynthesis
Dr. Soumitra Paul, MPP Lab, C.U.
25. Engineering the Carotenoid
Biosynthetic Pathway
• Much attention has been paid to the carotenoid metabolic pathway, which produces
pigments with roles in light harvesting and photoreception, and which form vital
components of human and animal diets.
• The principal example of carotenoid metabolic engineering in plants is of course the
synthesis of β-carotene in rice endosperm (Golden rice), which normally
accumulates GGPP (geranyl geranyl pyrophosphate) but lacks the subsequent
enzymes in the pathway.
• The four enzyme activities missing in the β-carotene synthesis pathway in cereal
grains are phytoene synthase, phytoene desaturase, ζ-carotene desaturase and
lycopene β-cyclase.
Dr. Soumitra Paul, MPP Lab, C.U.
26. Engineering the Provitamin A)b-Carotene)
Biosynthetic Pathway into (Carotenoid-Free) Rice
Endosperm
Xudong Ye, Salim Al-Babili, Andreas Kloti, Jing Zhang, Paola Lucca, Peter Beyer, Ingo Potrykus
* Vitamin A deficiency causes symptoms ranging from night blindness
to those of total blindness
* In Southeast Asia, it is estimated that a quarter of a million children go
blind each year because of this nutritional deficiency
* It is estimated that 125 million children worldwide are deficient in
vitamin A
* Oral delivery of vitamin A is problematic mainly due to the lack of
infrastructure Dr. Soumitra Paul, MPP Lab, C.U.
27. • No rice cultivars produce provitamin A in the endosperm
therefore recombinant technologies rather than conventional
breeding are required
• Immature rice endosperm is capable of synthesizing the early
intermediate geranylgeranyl diphosphate, which can be used to
produce the uncolored carotene phytoene by expressing the
enzyme phytoene synthase (psy) in rice endosperm
Continue….
Dr. Soumitra Paul, MPP Lab, C.U.
28. • The synthesis of b-carotene requires the
complementation with three additional
plant enzymes : phytoene desaturase
(pds) and z-carotene desaturase (zds),
each catalyzing the introduction of two
double bonds, and lycopene b-cyclase,
encoded by the lcy gene.
• To reduce the transformation effort, a
bacterial carotene desaturase ,(crt1)
capable of introducing all four double
bonds required, was used
• Transit peptide was attached to crt1
• A transit peptide exists in plant psy
Dr. Soumitra Paul, MPP Lab, C.U.
32. Vitamin E- Maize
HGGT catalyzes an analogous reaction to
HPT, only it is highly specific for GGDP
whereas HPT uses PDP as its prenyl
substitute.
Results from the expression of barley HGGT
in transgenic plants suggest that this enzyme
has strong substrate specificity for
geranylgeranyl diphosphate, rather than
phytyl diphosphate.
Expression of HGGT enzyme in tobacco calli
and Arabidopsis leaves resulted in
accumulation of Vitamin E antioxidants in the
form of tocotrienols ,principally as γ-
Tocotrienols, and generated little or no
change in the content of Tocopherols (Cahoon
et al, 2003)
Barley HGGT gene was over-expressed in
maize seeds, leading to a 20-fold increase in
tocotrienol level, which translated to an eight-
fold increase in total tocols (tocopherols and
tocotrienols) (Cahoon et al, 2003).Dr. Soumitra Paul, MPP Lab, C.U.
33. Genes
involved in
carotenoid
biosynthesis
Cloned/
transferred
Crop
species
Remarks Reference
Y1
cloned Maize
Importance of
such regulatory
gene in rice is
conceptualized
Buckner et al. 1990
crtI (Phytene
desaturase)
cloned/
transformed
Erwinia
uredovora/
Tobacco/
Herbicide
resistance;
Increased
Misawa et al 1990,
1993
crtE cloned
Erwinia
herbicola
coding for GGPP
synthase
Math et al 1992
A gene cluster cloned
Erwinia
herbicola
For complete
carotenoid
pathway
To et al 1994
psy transformed Tomato
Resulted in
dwarfism
redirecting the
metabolites from
gibberellin
pathway
Fray et al 1995
lcy cloned Daffodil
Lycopene to
beta-carotene
Al-Babili et al 1996
psy
cloned/
transformed
Daffodil/
Rice
Accumulation of
phytoene in rice
endosperm
Scheldz et al 1996;
Burkardt et al 1997
crtB
(phytoene
synthase)
transformed Brassica
Overexpression
led to increase
in carotenoids
and other
metabolites
Shewmaker et al
1999
Selected historical developments in carotenoid metabolism
in relation to plant metabolic engineeringDr.SoumitraPaul,MPPLab,C.U.
34. Co-transformationLBA4404/pZPsC
+
LBA4404/pZLcyH
Anther culture
Hemizygous T309 GoldenRice
(Ye et al. 2000)
Dihaploid homozygous T309 Golden Rice
(Baisakh et al. 2001b)
IR64
1st Backcrossing
F1IR64 x
IR64BC1F1
x
x
2nd Backcrossing
BC2F1
Marker-free
Selfing
BC2F2
Marker-free
PCR analysis
Molecular analysis
Phenotyping
Molecular
analysis
Selection of hph negative
transgenic progenies
PCR screening
and Southern confirmation
IR64 NILs
Marker-free
Phenotyping
HPLC
BC1F1 progenies
Marker-free
Flow chart for the
Development of
Marker-free
Near-isogenic golden
Rice lines of IR64
Dr. Soumitra Paul, MPP Lab, C.U.
37. ANTISENSE TECHNOLOGY
It is a loss of function technique where the genes are knocked out for investigation.
DNA
promoter
coding
sequence
messenger RNA
protein
ANTISENSE
promoter
coding
sequence
NO protein
Dr. Soumitra Paul, MPP Lab, C.U.
38. DOWNREGULATION OF PHYTIC ACID BIOSYNTHESIS…..
The biosynthesis of phytic acid is a complex
pathway controlled by various enzymes.
Of which myo inositol 3 phosphate
synthase (MIPS) and inositol phosphate
kinase (IPK1) plays the crucial role
(Suzuki et al. 2007).
Hence, suppression of any of these
enzymes by RNA interference
(RNAi) may prove to be effective
in reducing phytic acid levels in rice.
Proposed InsP synthetic pathways in plants. Left: the PLC-
independent pathway. Right: the PLC-dependent pathway (Suzuki et
al 2007).
Dr. Soumitra Paul, MPP Lab, C.U.
