Lecture 36&37 PLANTS AS BIOREACTORS by GBdf2Nr

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									      Lecture 36&37
PLANTS AS BIOREACTORS
        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.
WHY PLANTS ARE USED AS BIOREACTORS ?


   •   Post translational modifications


   •   Storage costs


   •   Ethical considerations
COMPARISON WITH OTHER PRODUCTION SYSTEM


     • Low cost alternative


     • Post translational modifications


     • Storage facilities


     • Low upstream production cost
      PRODUCTION OF BIOMOLECULES

Carbohydrates


1) Cyclodextrin biosynthesis


   • Starch acts as the substrate


   • Bacterial gene encoding cyclodextrin
   glycosyl transferase (cgt )


                                            Contd...
Cyclodextrin glycosyl transferase




                           Contd...
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...
     Gene construct of cgt gene


5'                                             3'
        P          c           p       t


       P    Patatin promoter

       c    cgt gene

       p    Sequence encoding transit peptide of
            RuBP carboxylase
        t   nos terminator

                                           Contd...
             • Targeted to amyloplast


             • Expressed in tubers


             • Expression level : 0.001-
             0.01%


             (Goddijn and Janpen, 1995)



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


 Expression of peptide relies on:


  a) Stable intergration of transgene


  b) By transient expression of genetically
     engineered viruses
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 )
            SEEDS AS BIOREACTORS
Why seeds are used as bioreactors ?
                           • Storage facility
 Seeds




                           • Transportation


                           • Oral consumption


                           • Existing agricultural
                           facilities in seed handling

                           (Sun et al., 2002)
  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
3- D View of Hirudin         sequence of hv2
                                                Contd...
     Oleosin - Hirudin fusion gene construct

5'                                                 3'
        P          o         X         H   t


              o        Oleosin gene
              x        Cleavage site
              H        Hirudin gene


               t       nos terminator
                                               Contd...
                 • 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...
Oleosin based purification of heterologous polypeptides

               (Goddijn and Janpen, 1995)
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.
Plant Transformation
         1.   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).
         2.   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).
3. 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).
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).
     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.
 PRODUCTION OF SPIDER
SILK PROTEINS IN PLANTS
     Production of transgenic plants




                     Spider silk protein
Expression of spidroin-ELP-fusion proteins
      in the ER of transgenic plants
 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.
BIOPHARMACEUTICALS FROM PLANTS

• Plants constructed to express proteins like
     α-interferon, human serum albumin etc.


• Two expensive drugs are produced from plants


    A) Glucocerebrosidase


    B) Granulocyte macrophage colony stimulating
       factor
          GLUCOCEREBROSIDASE

• Lysosomal hydrolase


• Cause Gaucher’s disease


• Earlier this enzyme was purified from human placentas


• Now synthesized from tobacco plants


                 (Giddings et al., 2000)
 Production of human lysosomal
 enzymes in Nicotiana tabacum

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

        Cellulase
        • Isolated from bacterial
              and fungal organisms


        • Expressed in potato
             plants


        • Enzymes produced in
             foliage and vines
Phytase


• Isolated from Aspergillus niger


• Expressed in seeds


• Replace feed supplements for broiler chicken
PRODUCTION OF BIOMOLECULES FROM PLANTS


S No    Compound      Origin of gene(s)        Appl.          Plant sp.



       LIPIDS

 1.    Medium-        California          Food,           Oilseed rape
       chain fatty    baytree             detergent,
       acids                              industrial
 2.    Mono-          Rat                 Food            Tobacco
       unsaturated
       fatty acids
 3.    Polyhydroxy    Alcaligenes         Biodegradable   Arabidopsis, oil
       butyric acid   eutrophus           plastics        seed rape,
                                                          soybean
 4.    Saturated      Brassica rapa       Food,           Oilseed rape
       fatty acids                        confectionaries
   CARBOHYDRATES

5. Amylose free       Solanum       Food,           Potato
   starch             tuberosum     industrial

6. Cyclodextrins      Klebsiella    Food,          Potato
                      pneumoniae    pharmaceutical

7. Fructans           Bacillus      Industrial, food Tobacco,
                      subtilis                       potato

8. Increased amount   Escherichia   Food,           Potato
   of starch          coli          industrial

9. Trehalose          E.coli        Food stabilizer Tobacco
      PHARMACEUTICAL
      POLYPETIDES
10.   Antibodies            Mouse       Various        Mainly
                                                       tobacco

11.   Antigens              Bacteria,   Orally         Tobacco,
                            viruses     administered   tomato,
                                        vaccines       potato,
                                                       lettuce

12.   Growth hormone        Trout       Growth         Tobacco,
                                        stimulation    Arabidopsis

13.   Hirudin               Synthetic   Thrombin       Oil seed
                                        inhibitor      rape

14.   Human serum albumin   Human       Plasma         Tobacco,
                                        expander       potato

15.   Interferon            Human       Anti-viral     Turnip
INDUSTRIAL
ENZYMES

Alpha-amylase   Bacillus       Liquefaction   Tobacco,
                licheniforms   of starch      alfalfa

Phytase         Aspergillus    Animal feed    Tobacco
                niger

Xylanase        Clostridium   Animal feed,    Tobacco
                thermocellum, paper and
                Cryptcoccus   pulp, baking
                albidus
    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)
ADVANTAGES OF PLANTS AS BIOREACTORS

• Can produce high level of safe homogenous
functional biomolecules


• Modern agriculture practice - easy scale up and
processing


•   Easy storage
ADVANTAGES OF PLANTS AS BIOREACTORS


• Chimeric plant virus can be used to produce
vaccines


•   Administration safe and painless


•   Long shelf life (seeds)


•   Low cost
    LIMITATIONS AND REMEDIES


      Limitations              Remedies

* Downstream           * Expressing the protein
processing expensive   with sturctured oil body
and difficult          protein
                        e.g: Hirudin
* Accumulation of      * Targeting the transgene
transgene product in   into proper cellular
plant                  compartment
             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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