Plants as bio reactors

Plant as bioreactors:

Plant as bioreactors Presented by M.Shafana Fatima Lady Doak College

Introduction:

Introduction Bioreactor an apparatus in which a biological reaction is carried out. Plant as bioreactor Plants are genetically engineered to make products that are not of plant origin (therapeutically & industrially important products)

Why plants are used as bioreactors?:

Why plants are used as bioreactors? Grow easily and inexpensively Easy for harvesting and processing Protein accumulation is easy Attractive expression systems Post translational modifications Lacking of contamination by animal pathogen.

What kind of plants are used as bioreactors?:

What kind of plants are used as bioreactors? Transgenic plants including Cereal plants Fruit plants Leguminous plants Vegetable plants

Key processes:

Key processes Transforming the recombinant proteins Expression Patenting

Types of plant bioreactors:

Types of plant bioreactors On the basis of compartmentation of recombinant proteins

Seed based plant bioreactors:

Seed based plant bioreactors Seeds provide a suitable environment High rate of protein synthesis Accumulate large amount of proteins during seed development. Eg. ά -L-iduronidase in Arabidopsis thaliana seeds

Seed based plant bioreactors:

Seed based plant bioreactors expression is controlled using seed specific promoters For eg. in maize globulin-1 , and in rice glutelin promoter Gt-1 . Two types Seed Protein Storage Vacuole Bioreactors Seed Oil Body Bioreactors Main advantage : proteins do not degrade.

Seed Protein Storage Vacuole Bioreactors:

Seed Protein Storage Vacuole Bioreactors Protein storage vacuoles (PSVs) compartments for the storage of recombinant proteins in seed bioreactors three distinct sub compartments: the matrix, globoid and crystalloid. matrix :soluble storage proteins globoid : hydrolytic enzymes. crystalloid : BP-80 TMD and the CT of alpha-tonoplast intrinsic protein sequences.

Transporting pathways of subcompartments:

Transporting pathways of subcompartments For eg : transmembrane domain (TMD) + cytoplasmic tail (CT) of BP-80 a reporter from prevacuolar compartment (PVC) to PSV globoid BP-80 TMD + CT of alpha-tonoplast intrinsic protein (TIP) sequences target the same reporter to PSV crystalloid in transgenic tobacco seeds Similarly, AFVY, a vacuolar sorting determinant of phaseolin, can deliver reporter or recombinant protein to PSV matrix.

Membrane anchors for PSV targeting:

Membrane anchors for PSV targeting

Seed Oil Body Bioreactors:

Seed Oil Body Bioreactors seed oil body (OB) are surrounded by oleosin proteins that function in maintaining the structural integrity of OBs. provides a recognition signal for lipase binding during oil mobilization in seedlings. can store large amount of macromolecules Eg. manufacture of the anticoagulant hirudin in the oil body of seeds Brassica napus and Brassica carinata

Oil bodies:

Oil bodies

PLANT SUSPENSION CULTURES:

PLANT SUSPENSION CULTURES provide a fast system for producing secondary metabolites, biologically active recombinant proteins and antibodies The recombinant proteins sub cellular organelles or into extra cellular space Eg: expression of 80-kDa human lysosomal protein (controlled by 35s CaMV and a signal peptide) in transgenic tobacco BY-2 cells culture media.

Hairy Root System Bioreactor :

Hairy Root System Bioreactor The Hairy Root System with its rhizosecretion is due to infection of the soil bacterium- Agrobaterium rhizogenes . biosynthetic stability suitable for making biopharmaceuticals Eg : scopolamine in Hyoscyamus muticus L. hairy root culture

Chloroplast bioreactor :

Chloroplast bioreactor Insulin, interferons and other biopharmaceutical proteins can be made using Chloroplast bioreactor. One method : foreign genes are inserted into nuclear chromosomes and then target the expressed proteins into chloroplast. Other: direct targeting in chloroplast genome Eg: high yield in the expression of human serum albumin protein in chloroplast .

Plant bioreactor system:

Plant bioreactor system

Slide 18:

ά -L-iduronidase in Arabidopsis thaliana seeds ά -L-iduronidase in Arabidopsis thaliana seeds

Targeting mechanisms:

Targeting mechanisms

Comparative study of plant bioreactor systems :

Comparative study of plant bioreactor systems

Antibodies in plants :

Antibodies in plants Plants can assemble heavy and light chains into complete antibodies Permit appropriate post translational modifications Targeting the Abs through ER to apoplast. Extracting the Abs is easy

CONT….:

CONT…. Production of full sized Abs is difficult But production of Fab and F(ab')2 fragments Single chain Fv fragments chimeric antibodies such as IgG/A, Single domain antibodies and antibody fusion protein is easy with plant bioreactor system.

Examples :

Examples Recombination anti-sperm antibody (RASA) Gene : scFv1 9 from horse Expressed in transgenic tobacco. an anti- Salmonella enterica single-chain variable fragment (scFv)antibody in transgenic tobacco

Abs expressed in transgenic plants:

Abs expressed in transgenic plants Variable Light chain Variable Heavy chain Recombinant hinge region Constant domains

Slide 26:

Production Costs for Antibodies Production costs cost in $ / gram hybridomas 1000 transgenic animals 100 transgenic plants 10 Source: Daniell et al. (2001) TIPS 6, 219-226 E. coli & yeast Tr. animals and animal cells Transgenic plants

Foreign proteins:

Foreign proteins Recombinant proteins are among the best candidate products for commercial production in plant bioreactors. Production of cytokine enzyme, biologically active peptides and other pharmaceutical proteins.

Slide 28:

Production of recombinant Hirudin from seeds Hirudin has antithrombin activity Isolated from Hirudo medicinalis Construction of synthetic gene with amino acid sequence of hv2 Contd... 3- D View of Hirudin

Slide 29:

P o X H t o x H t Oleosin gene Cleavage site Hirudin gene nos terminator Oleosin - Hirudin fusion gene construct Contd... 5' 3 '

Slide 30:

Brassica napus 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

Slide 31:

Oleosin based purification of heterologous polypeptides

GFP in plants:

GFP in plants jellyfish gfp cDNA in Citrus sinensis protoplasts Expressed in Arabidopsis thaliana C24 seedlings

Slide 33:

Transgenic Arabidopsis thaliana C24 seedlings were produced by Agrobacterium-mediated root transformation and contain an altered form of the Aequorea victoria green fluorescent protein

POLYMERS :

POLYMERS A polymer is any chainlike molecule composed of a repeating unit called a monomer Monomers coalesce into polymers in a process called polymerization A biopolymer is any organic polymer.

Types of Biopolymers :

Types of Biopolymers There are two main types of biopolymers: from living organisms; and, those which need to be polymerized but come from renewable resources. Both types are used in the production of bioplastics

Biopolymers From Living Organisms:

Biopolymers From Living Organisms These biopolymers are present in, or created by, living organisms. These include carbohydrates and proteins. Biopolymer Natural Source What is it? Cellulose Wood, cotton, corn, wheat, and others the main component of plant cell walls. Soy protein Soybeans Protein which naturally occurs in the soy plant. Starch Corn, potatoes, wheat, tapioca, and others polymer made up of glucose.

Polymerizable Molecules :

Polymerizable Molecules These molecules come from renewable natural resources, and can be polymerized to be used in the manufacture of biodegradable plastics Eg ; lactic acid and triglycerides Lactic acid is polymerized to produce poly lactic acid.

Production of Polymers:

Production of Polymers Feedstock (Carbon Source) (such as Natural Gas, Oil, Corn, Soybeans, Sugar Cane) ↓ Monomer (such as Ethylene, Propylene, Lactic Acid) ↓ Polymer (such as Polyethylene, Polypropylene, Polylactic acid) ↓ Package (such as Bottle or Pouch)

Polyhydroxyalkanoates (PHAs):

Polyhydroxyalkanoates (PHAs ) Poly hydroxy alkanoates (PHAs) comprise a class of biodegradable polymers. . Genes encoding the two enzymes, aceto -acetyl-CoA reductase (PhbB) and PHB synthase (phbC), involved in the PHB synthesis The precursor acetyl-CoA is transferred from the bacterium Alcaligenes eutrophus and expressed in Arabidopsis thaliana .

BIOPLASTICS:

BIOPLASTICS Bioplastics are a form of plastics derived from plant sources such as sweet potatoes, sugarcane, hemp oil, soy bean oil and corn starch Bioplastics are environmentally friendly because, emission of less carbon dioxide, which is thought to cause global warming.

Uses of bioplastics:

Uses of bioplastics AUTOMOTIVE Components, Coatings, Interiors

Biodegradability of PHB plastics:

Biodegradability of PHB plastics