Metabolic Engineering of Carbohydrates - (CUSAT) – Plant ...

Metabolic Engineering
of
Carbohydrates
By
Rajesh K V
III Sem MSc. Biotechnology

Genetic modifications of Plant
carbohydrate metabolism
• Molecular farming of carbohydrates
• Plants produce a wide range of commercially important
carbohydrates
• Cellulose and starch
- most abundant
- Some biotechnological effort towards the improvement of
yield and quality

Considerations when modifying Plant
carbohydrate metabolism
I. SOURCE – SINK INTERACTIONS
• Ensure the adequate translocation of sucrose from source to
sink tissues ( meristems , immature leaves, seeds, roots)
• is essential to ensure the optimal plant growth and crop yield
• For eg.
Introduction of antisense construct of the sucrose transporter
cDNA to potato plants
- reduction in the expression of the sucrose transporter (SUT1)
RNA
- hence reduction in the export of carbohydrates from the
source leaves
- reduced growth, root development & tuber yield.

II. Flexibility of the metabolism
• A characterisitic feature of plant metabolism,
is that alternative routes to the same product
often exist within the same tissue that makes
it harder to manipulate
• attempts to change flux through one pathway
may be compensated by changes in another.

III. Sugar- Induced Signaling In Plants
• Carbohydrates such as glucose, fructose,& sucrose also
function as signaling molecules
• many genes have been characterized of which transcription
is regulated by sugars
-Glucose induces Patatin gene expression
-Sucrose – chalcone synthase
• Sugars can also repress the gene transcription
-glucose – Chlorophyll a/b binding protein
-fructose- RuBp carboxylase smaller subunit

IV. Expression Of Introduced Genes
Promotor used – CaMV 35S type
constitutively expressed in all tissues throughout
development
the expression of a certain protein in particular cell
types might have detrimental effects
For eg;
Constitutive expression of E.coli enzyme
pyrophosphorylase in tobacco or potato stimulates
sucrose synthesis.

• To evade the “translocation” problems
the ectopic expression of genes should be
controlled either by
i) using a tissue specific promoter
Eg; granule-bound starch synthase promoter
ii) a developmentally regulated promoter
iii) a promoter which can be induced
(pathogen related promoter PR-1a).

Molecular pharming of Carbohydrates

STARCH

• The principal storage carbohydrate in plants
• A major product of photosynthesis
•
• Representing 65-80% of the dry weight in
cereals and potato tubers
• In USA and Europe, about 70% of the starch
produced is used for a variety of industrial
purposes
• With only 30% is used for human consumption and
animal feed.

Industrial purposes

Starch biosynthesis
• occur in the plastids of leaves & in amyloplasts
of storage organs
• Upon entry of sucrose in the sink cells , sucrose
is cleaved by sucrose synthase & converted to
Glu-1-P which can be used for the synthesis of
ADP-glucose, the major substrate for starch
biosynthesis
• reaction is catalysed by ADP –glucosepyrophosphorylase

Manipulation Of Starch Content
• The amount of starch deposited in the potato
tuber is affected by;
- the rate of photosynthesis
-the translocation of photoassimilates
-the sink strength (capacity to attract
photoassimilates).

Genetic Modification Of Starch
Structure & Properties
• The physical properties of starch granules are
mainly determined by;
-granule size
- lipid content
- amylose content
- chain length
- degree of branching
- amylase/amylopectin ratio

• The physical properties of the strach granules
can be modified by;
-altering the expression of endogenous
enzymes
- by introduction of foreign genes.

GENETIC MODIFICATION OF STARCH
• genes encoding the main enzymes of starch
biosynthesis have been cloned & used to
produce transgenic lines.

Granule-bound starch synthase I
genes(GBSS I)
• the main enzyme of amylose biosynthesis
• increased activity of GBSS I is expected to
increase the amylose content of starch, while
• suppression of GBSS I should enhance the
amylopectin content

Amylose-free or “waxy” starch
• Produced in potato
• Suppression of GBSS I activity by antisense
RNA technology
• the first successful genetic modification of
starch reported in 1991.

Soluble Starch Synthase (SSS)
• main enzyme of amylopectin biosynthesis
• has 2 isoforms - SSS I & SSS II
• A freeze-thaw stable potato starch has been
created by simultaneous down regulation of
all the 3 starch synthases, viz. GBSS I, SSS I &
SSS II, genes using anti sense RNA technology.
• yields an amylose - free short chain
amylopectin.

Starch Branching Enzyme (SBE)
• Enzyme required for amylopectin biosynthesis
• suppression should reduce the amount of
amylopectins.
• by using antisense constructs of the genes
concerned.
• resulted in a very high amylose starch

Manipulations Of Starch Metabolism
• Starch & its derivatives have various uses- in
making paper products, baking and other food
uses, textile manufacture, paints, rubbers etc.
• Many of the starch derivatives are obtained by
post- harvest modification of the isolated
starch
- is expensive & environmentally polluting
Transgenic production is cheaper by cost

CYCLODEXTRINS

• One type of high value product that could be
made from starch
• These compounds are typically 6, 7 or 8
membered rings comprising glucopyranose
subunits attached in α (1-4) linkage
• normally produced by bacterial fermentation
of maize starch
• Used for solubilization of hydrophobic
pharmaceuticals such as steroids

cyclodextrin

• Only a single attempt has been reported to
produce cyclodextrins(1991)
• Gene construct;
-a bacterial cyclodextrin glycosyl transferase gene –
from Klebsiella pneumoniae
-a plastid targeted sequence
- promotor from the patatin gene
Patatin is a protein that accumulates in potato tubers.

• the promoter directs high levels of expression
in the tubers of transgenic potatoes
• Transformation of potatoes with this construct
resulted in very little conversion (0.001-0.01%)
of starch to cyclodexrins.
• It was concluded that the insoluble starch
granules may have been inaccessible to the
bacterial enzyme, or that the enzyme became
trapped in the growing granule

POLYFRUCTANS

• are soluble polymers of fructose
• are synthesized& stored in the vacuole
• another carbohydrate targeted for production
in transgenic plants
• Have a typical structure of glu-fru-(fru) n
• Most abundant storage carbohydrates in
plants after starch and sucrose
• Inulin is water-soluble

Levan
Inulin

• Inulin:
-the major storage carbohydrate found in
bulbs of onions, storage roots of chicory
and Jerusalem artichoke
- formed by (1→2β)linkages
• Levans:
- present in leaves and stems of
cereal crops such as wheat
- possess (6→2β) linkages
Levan

Biosynthesis of Fructans
• two stage process
• The first step:
transfer of fructose from a donor sucrose
molecule to an acceptor sucrose molecule to
form kestose
the enzyme sucrose-sucrose
fructosyltransferase (SST)
• 2 G-F → G-F-F + G

• The Second step:
• kestose (GFF or GF2) acts as the fructose
donor to the growing fructan chain,
• via fructanfructan fructosyltransferase
activity & a sucrose molecule is recycled
• G-F-(F) n + G-F-F → G-F-(F) n+1 + G-F

Genetic manipulation:
• A number of transgenic plants producing
polyfructans have been developed
• In earlier experiments the sacB gene of B.
subtilis or B. amyloliquifaciens which encodes
a levan-sucrase catalyzing a 6-2β linkage was
transformed into maize, tobacco, potato,&
sugarbeet.
• sacB gene was modified with a vacuolar
targeting sequence from the yeast
carboxypeptidase gene(cpy).

Genetic manipulation:

In Maize;
• B. amyloliquifaciens sacB gene was
expressed in the endosperm under the
control of a zein promoter
• The enzyme was targeted to vacuole of
the endosperm cells using two different
vacuolar targeting sequences.
• In one construct the sweet potato
sporamin signal peptide & vacuolar
targeting sequences were fused to the N-
terminal end of the enzyme.

• In the other , the barley lectin signal
peptide was fused to the N-terminal end & the
barley lectin vacuolar targeting sequence was
fused to the C-terminal end of the enzyme
• Short oligofructans (GF2, GF3 &GF4) have been
produced in sugarbeet using a gene encoding
the 1-SST enzyme from Jerusalem artichoke.
• This enzyme catalyses the production of not
onlyGF2(ketose) but also GF3 &GF4.

Jerusalem artichoke

• Larger inulin molecules have been produced in
potato tubers transformed with the 1-SST & 1-
FFT genes of globe artichoke
• It has been possible to produce inulin of the
neoseries in chicory by transformation with
the barley 6-SFT gene to produce branched
fructans of the graminan type.
• The production of fructans in transgenic
plants has been critically reviewed by
Cairns(2003).

TREHALOSE

• A non-reducing di-saccharide
• Plays its part as an energy source.
• Produced in some plants and micro-organisms,
often in response to osmotic stress.
•
• Potential target for gene manipulation of
tolerance of abiotic stresses that create
water deficit.

Genetic manipulation:
• Genes for the synthesis of trehalose
from yeast and E.coli have already
been introduced into transgenic
tobacco.
• Purpose was to manipulate drought
tolerance.