PLANTS AS BIOREACTORS

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Lecture 3637 PLANTS AS BIOREACTORS

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WHAT IS A BIOREACTOR ?

• A device in which a substrate of low
  value is utilised by living cells to
  generate products of higher value.
• Plants are exploited as bioreactors for
  the production of biomolecules.

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WHY PLANTS ARE USED AS BIOREACTORS ?

• Post translational modifications
• Storage costs
• Ethical considerations

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COMPARISON WITH OTHER PRODUCTION SYSTEM

• Low cost alternative
• Post translational modifications
• Storage facilities
• Low upstream production cost

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PRODUCTION OF BIOMOLECULES

• Carbohydrates
• 1) Cyclodextrin biosynthesis
• Starch acts as the substrate
• Bacterial gene encoding cyclodextrin
  glycosyl transferase (cgt )

Contd...
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Cyclodextrin glycosyl transferase
Contd...
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HOW GENE CONSTRUCT IS MADE FOR cgt?
(Cyclodextrin glycosyl transferase)
a) Patatin gene promoter (tuber specific) b)
Sequence encoding peptide of RUBP
carboxylase c) cgt gene from Klebsiella
pneumoniae d) 3' sequence of nos gene of
Agrobacterium
Contd...
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Gene construct of cgt gene
5'
3'
P c p
t
Patatin promoter cgt gene Sequence encoding
transit peptide of RuBP carboxylase nos
terminator
P c p
t
Contd...
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• Targeted to amyloplast
• Expressed in tubers
• Expression level 0.001-0.01
• (Goddijn and Janpen, 1995)

Amyloplast
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• 2) Increasing Starch accumulation
• ADP- Glucose pyrophosphorylase
• ADP- Glucose
  Starch
• A mutated bacterial gene (glgc16)
  encoding ADP glucose pyrophosphorrylase
• Expressed in potato tubers
• Targeted to amyloplasts
• 60 increase in starch than control
• ( Verisser and Jaciobsen, 1993 )

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Rerouting the starch to produce fructan

• Fructosyl transferase gene from
  Bacillus subtilis
• Introduced in tobacco and potato
  plants
• Accumulation level
• 3-8 of dry
  wt. In tobacco leaves
• 1-30 in
  potato leaves
• 1-7 in potato
  microtubers

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Metabolic Engineering of Carbohydrate Metabolism
(Goddijn and Janpen,1995)
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PRODUCTION OF PROTEINS FROM PLANTS

• Expression of peptide relies on
• a) Stable intergration of transgene
• b) By transient expression of
  genetically
• engineered viruses

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HUMAN THERAPEUTIC PROTEIN-SOMATOTROPIN (hST)

• Synthesis of hST and ubiquitin fusion
  genes
• Cloning of chimeric hST genes using pPRV
  vectors
• Introduction to tobacco leaf chloroplast
  by biolistic process
• Leaves with different ages show different
  hST accumulation
• ( Jeffrey et al., 2000 )

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SEEDS AS BIOREACTORS
Why seeds are used as bioreactors ?

• Storage facility
• Transportation
• Oral consumption
• Existing agricultural facilities in seed
  handling
• 
  (Sun et al., 2002)

Seeds
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Production of recombinant Hirudin from seeds

• Hirudin has antithrombin activity
• Isolated from Hirudo medicinalis
• Limited availability (1 leech head contains
  20mg of hirudin)
• Construction of synthetic gene with amino acid
  sequence of hv2

3- D View of Hirudin
Contd...
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Oleosin - Hirudin fusion gene construct
5'
3'
P o X
H t
o x H
Oleosin gene Cleavage site Hirudin gene nos
terminator
t
Contd...
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• Fusion of gene with Arabidiopsis oleosin
  gene
• Introduction of fusion gene construct
  into Brassica napus
• Expression of seed specific oleosin hirudin
  transcripts
• Protein purified by oleosin partition
  technology
• ( Dana et al., 1996 )

Brassica napus
Contd...
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Oleosin based purification of heterologous
polypeptides
(Goddijn and Janpen, 1995)
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PRODUCTION OF BRYODIN IN TOBACCO PLANT

• Tobacco plants that are able to produce bryodin.
• This protein, which is produced in the roots of
  bryonia, deactivates ribosomes and is being
  tested for its effect against HIV infection.

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Plant Transformation

• The plant leaf disc is dipped in a solution of
  bacteria. The bacterial "Trojan Horse" infects
  the edges of the leaf disc and in the process
  integrates the pharmaceutical protein gone into
  the plant genome (pict 1).
• After infection the discs are placed on selection
  media that a flows only plant cells that carry
  the protein gene to survive and regenerate into
  plantlets. After about six weeks on selection
  media, a large number of plantlets that carry the
  pharmaceutical protein gene are visible at the
  edges of the original leaf disc (pict 2 3).

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• The plantlets are removed from the leaf disc and
  placed in clear plastic boxes that contain media
  that allows them to form roots (pict 4).
• The rooted plantlets are placed in pots and
  plants are allowed to grow and produce seed. This
  seed can then be used for large scale production
  of the pharmaceutical protein (pict 5).

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Protein Trafficking

• Following translation of the molecular ring gene,
  the protein will move through the endoplasmic
  reticulum and Golgi apparatus for processing,
  folding and glycosylation.

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PRODUCTION OF SPIDER SILK PROTEINS IN PLANTS
Production of transgenic plants
Spider silk protein
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Expression of spidroin-ELP-fusion proteins in the
ER of transgenic plants
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Purification of spider silk-ELP fusion proteins

• From transgenic plants Spidroin-ELP-fusions could
  be purified by addition of salt and by heat to
  95 purity.

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BIOPHARMACEUTICALS FROM PLANTS

• Plants constructed to express proteins
  like
• a-interferon, human serum albumin etc.
• Two expensive drugs are produced from
  plants
• A) Glucocerebrosidase
• B) Granulocyte macrophage colony
  stimulating factor

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GLUCOCEREBROSIDASE

• Lysosomal hydrolase
• Cause Gauchers disease
• Earlier this enzyme was purified from human
  placentas
• Now synthesized from tobacco plants
• (Giddings et
  al., 2000)

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Production of human lysosomal enzymes in
Nicotiana tabacum

• Gluco cerebrosidase- gaucher disease
• Alpha-hexosaminidase- Tay-Sachs disease
• Alpha-L-iduronidase- Hurler syndrome

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INDUSTRIAL ENZYMES

• Cellulase
• Isolated from bacterial and fungal organisms
• Expressed in potato plants
• Enzymes produced in foliage and vines

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• Phytase
• Isolated from Aspergillus niger
• Expressed in seeds
• Replace feed supplements for broiler chicken

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PRODUCTION OF BIOMOLECULES FROM PLANTS
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PLANT CELL SUSPENSION CULTURE AS BIOREACTORS

• Secondary metabolites recombinant
  proteins - production
• Antitumour agents like taxol can be
  produced
• Taxol from Taxus sp - treatment of breast
  and ovarian cancers
• (Seki et al., 1997)

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ADVANTAGES OF PLANTS AS BIOREACTORS

• Can produce high level of safe homogenous
  functional biomolecules
• Modern agriculture practice - easy scale up
  and processing
• Easy storage

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ADVANTAGES OF PLANTS AS BIOREACTORS

• Chimeric plant virus can be used to produce
  vaccines
• Administration safe and painless
• Long shelf life (seeds)
• Low cost

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LIMITATIONS AND REMEDIES

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FUTURE CHALLENGES

• Engineering challenges like maximization of
  expression levels
• Environmental safety
• Stability of product under storage
• Evaluation of dosage requirement
• Regulatory considerations and legal standards