Yogendra Pratap Singh

B.tech, 3rd year

Biotechnology.
WHAT ARE METABOLITES?

 Metabolites are the intermediates and products of

metabolism.
 Generally they are small molecules.

 A primary metabolite is directly involved in normal

growth, development, and reproduction.
 A secondary metabolite is not directly involved in
those processes, but usually has an important
ecological function.
WHAT IS METABOLIC ENGINEERING?

Optimization of genetic and

Regulatory processes

Increased
production of a certain
substance
 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)
METABOLIC ENGINEERING IN PLANTS

Prolific factories
for small molecules

More then 100.000

metabolites have been
identified

Richest with secondary

metabolites among
different organisms
EXAMPLES OF M.E.
Viral Resistance

Papaya

Resistant Transgenic (Left) and susceptible (Right)

VALUE ADDITION BY M.E.
 Value addition

Improved nutritional
content and delayed
ripening are transgenic
traits of interest in
tomatoes. Source: USDA
METABOLIC ENGINEERING TOWARDS
THE ENHANCEMENT OF
PHOTOSYNTHESIS
 PROBLEMS IN PHOTOSYNTHESIS

 Fact
Rubisco is a slow enzyme and fixes only few CO2 molecules.
it also fixes O2 ( competitive inhibition).
 Condition
In high CO2 environment & proper illumination the activity of
Rubisco is limited for C-fixation.
 Rubisco enzyme is activated by Carbamylation, the covalent
addition of a CO2 molecule to a Lysine side chain & subsequent
binding of a Mg+2 ion. It leads to reduction of further CO2
fixation capacity of the plant.
 Photosynthesis is often limited by the regeneration of CO2
acceptor molecule Ribulose-1,5 bisphosphate (RuBP) in the
photosynthetic carbon reduction cycle (PCRC) or Calvin Cycle.
 Under high light conditions maximum catalytic
efficiency of Rubisco becomes more important and it
limits photosynthesis.
 During drought, specificty is again more important as
CO2 supply is limited due to stomata closure.

Catalytic efficiency α 1
Specificity of Rubisco
 M.E. --- A SOLUTION.

 Escherichia coli cells that are dependent on Rubisco

activity in order to grow are selected
 Mutagenisis

 Mutant variants that showed increased level of carbon

fixation are selected
 Bacteria overexpressing the enzyme grow faster but
require CO2 enriched environment.
 RUBISCO ACTIVITY
 Rubisco is activated by carbamylation of a Lysine
residue & subsequent binding of Mg2+ ion. It is
facilitated by the enzyme Rubisco Activase(RCA).
 It is inactivated at high temperatures

 Thermostable variants are produced using gene

shuffling.
 M.E. IN PHOTOSYNTHETIC ELECTRON
TRANSPORT CHAIN

FACT

 Cyt c6 is present in cyt b6f complex.

 It participates in the e- transfer between cyt b6f complex & PSI
mediated by Plastocyanin.

Role of M.E.

 Over expression of cyt c6 resulted in faster stem elongation as

well as leaf and root growth at low light intensity and long day
duration.
 High content of metabolites.
 Improved chlorophyll.