innovative approach to metabolism via genetic engineering: desired products are made via changing the pathways

1. METABOLIC ENGINEERING
Maria K. John
M. Sc. Biotechnology

2. What shall we learn?
What is
Metabolic
engineering?
Biological
systems
used
Desired
products
Future
perspectives

3. • Metabolic engineering can be defined as purposeful modification of cellular
networks including metabolic, gene regulatory, and signaling networks to
achieve desirable goals such as enhanced production of metabolites
including pharmaceuticals, biofuels, and biochemical and other
biotechnology products.
• Simply, it is the intentional modification of cellular metabolism for the
production of desired compounds.

4.  Cellular metabolism comprises all of the biochemical reactions, that occur
within a cell, consisting of sequences of enzymatic steps called metabolic
pathways.
 Manipulation of metabolic pathways is achieved with the use of Recombinant
DNA technology.

5. VARIOUS DISCIPLINES LINKED WITH ME
Metabolic
Engineering
Biochemist
ry
Cell
physiology
Chemical
engineering
Genetics

6. Biological systems
• Commonly used host organisms include:
 Bacteria- eg. E.coli, Bacillus subtilis
 Fungi- eg.Saccharomyces cerevisiae, Aspergillus niger
 Plant cells- eg. Nicotiana tabacum, Physcomitrella patens
 Animal cells- Chinese hamster ovary cells, Spodoptera frugiperda

7. Desired products
 Phenyl alanine
o Raw material for the synthesis of aspartame.
o Chemically synthesized- but too expensive.
o Alternatively produced by
i)Removing feedback inhibition
By selecting strains resistant to Phe analogs.
ii)Removing competing pathways (synthesis of Tyr and Trp)

8.  Indigo blue, Melanin
o Microbial synthesis.
o From Pseudomonas putida, gene encoding naphthalene dioxygenase is cloned.
o Host organism- E.coli
o For overproduction, a strain of E. coli that overproduces either Trp or Tyr is
generated.
o Yields are improved by increasing the levels of cofactors.

9.  Vitamin C
o Conventionally produced by one microbiological and four chemical steps.
o Corynebacterium gene encoding 2,5-diketogluconic acid reductase is taken.
o Introduced into Erwinia.
o Thus steps reduced to one microbiological and one chemical step.

11.  Secondary metabolites from plants
o Secondary metabolic pathways of plants are so extensive and complex.
o Metabolic engineering in plants by using large scale plant cell cultures can be
used for the production of important phytochemicals.
1. Production of Vinblastine and Vincristine
 In Catharanthus roseus cell cultures, produced in low amounts.
 Too complex to synthesize in the laboratory.
 Terpene indole alkaloid biosynthetic pathway paved way for generating
vincristine and vinblastine.

13.  Vitamin A
o Mainly obtained from animal sources.
o Provitamin A (beta-carotene) present at high levels in certain fruits and
vegetables.
o But very little amount of β-carotene seen in cereal grains.
o Four enzymatic steps are needed for its synthesis in plants.
o But cereals lack all 4 steps.
o Represents an example of metabolic pathway extension.

15. o Rice plants were transformed with the Daffodil (Narcissus pseudonarcissus)
phytoene synthase gene.
o Later, transgenic rice plants expressing the daffodil genes encoding phytoene
synthase and lycopene β-cyclase was found out.
o These genes were expressed under the control of the rice glutelin-1 promoter,
which is endosperm specific.
o The crtI gene from the bacterium Erwinia uredovora, encodes an enzyme with
both phytoene desaturase and ζ-carotene desaturase activities.
o Bacterial gene was controlled by the constitutive cauliflower mosaic virus
(CaMV) 35S promoter.
o Resulted in golden rice grains.

16. Future Perspectives
 Paved way for alternative energy sources, chemical and antibiotic production,
nutrient-enriched food products, and therapeutic protein synthesis.
 Significantly evolved to meet human needs in a sustainable way through the
improvement of productivity, lowering of costs, and decrease of pollution.
 The future of metabolic engineering is tremendously exciting and its direction
will be strongly dictated by the expansion and acceptance of the emerging field
of synthetic biology.
 Synthetic biology aims to enable a systematic forward engineering of biology
for improved and novel applications.

17. References
o Metabolic Engineering- Comprehensive Biotechnology(Second edition), 2011.
o Principles of Gene Manipulation and Genomics- Old and Primrose( Seventh
edition).