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Lecture 36&37 PLANTS AS BIOREACTORS

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.

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Lecture 36&37 PLANTS AS BIOREACTORS

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  1. Lecture 36&37 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 Whyseeds 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 Brassicanapus • 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. 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.

  27. 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

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

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

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

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

  32. PRODUCTION OF BIOMOLECULES FROM PLANTS

  33. 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)

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

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

  36. LIMITATIONS AND REMEDIES

  37. 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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