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PLANTS AS BIOREACTORS

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Lecture 36&37 PLANTS AS BIOREACTORS PRODUCTION OF BRYODIN IN TOBACCO PLANT Tobacco plants that are able to produce bryodin. This protein, which is produced in the ... – PowerPoint PPT presentation

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Title: PLANTS AS BIOREACTORS


1
Lecture 3637 PLANTS AS BIOREACTORS

2
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.

3
WHY PLANTS ARE USED AS BIOREACTORS ?
  • Post translational modifications
  • Storage costs
  • Ethical considerations

4
COMPARISON WITH OTHER PRODUCTION SYSTEM
  • Low cost alternative
  • Post translational modifications
  • Storage facilities
  • Low upstream production cost

5
PRODUCTION OF BIOMOLECULES
  • Carbohydrates
  • 1) Cyclodextrin biosynthesis
  • Starch acts as the substrate
  • Bacterial gene encoding cyclodextrin
    glycosyl transferase (cgt )

Contd...
6
Cyclodextrin glycosyl transferase
Contd...
7
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...
8
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...
9
  • Targeted to amyloplast
  • Expressed in tubers
  • Expression level 0.001-0.01
  • (Goddijn and Janpen, 1995)

Amyloplast
10
  • 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 )

11
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

12
Metabolic Engineering of Carbohydrate Metabolism
(Goddijn and Janpen,1995)
13
PRODUCTION OF PROTEINS FROM PLANTS
  • Expression of peptide relies on
  • a) Stable intergration of transgene
  • b) By transient expression of
    genetically
  • engineered viruses

14
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 )


15
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
16
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...
17
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...
18
  • 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...
19
Oleosin based purification of heterologous
polypeptides
(Goddijn and Janpen, 1995)
20
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.

21
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).

22
  • 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).

23
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.

24
PRODUCTION OF SPIDER SILK PROTEINS IN PLANTS
Production of transgenic plants
Spider silk protein
25
Expression of spidroin-ELP-fusion proteins in the
ER of transgenic plants
26
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27
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.

28
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

29
GLUCOCEREBROSIDASE
  • Lysosomal hydrolase
  • Cause Gauchers disease
  • Earlier this enzyme was purified from human
    placentas
  • Now synthesized from tobacco plants
  • (Giddings et
    al., 2000)

30
Production of human lysosomal enzymes in
Nicotiana tabacum
  • Gluco cerebrosidase- gaucher disease
  • Alpha-hexosaminidase- Tay-Sachs disease
  • Alpha-L-iduronidase- Hurler syndrome

31
INDUSTRIAL ENZYMES
  • Cellulase
  • Isolated from bacterial and fungal organisms
  • Expressed in potato plants
  • Enzymes produced in foliage and vines

32
  • Phytase
  • Isolated from Aspergillus niger
  • Expressed in seeds
  • Replace feed supplements for broiler chicken

33
PRODUCTION OF BIOMOLECULES FROM PLANTS
34
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35
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36
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37
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)

38
ADVANTAGES OF PLANTS AS BIOREACTORS
  • Can produce high level of safe homogenous
    functional biomolecules
  • Modern agriculture practice - easy scale up
    and processing
  • Easy storage

39
ADVANTAGES OF PLANTS AS BIOREACTORS
  • Chimeric plant virus can be used to produce
    vaccines
  • Administration safe and painless
  • Long shelf life (seeds)
  • Low cost

40
LIMITATIONS AND REMEDIES

41
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
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