PLANT AS

BIOREACTOR

Submitted by : Komal

Verma

Roll no.3599

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Plant as Bioreactor

Introduction

A device in which a substrate of low value is utilized by living cells to

generate products of higher value.

From earlier days - Microbes & animal cell culture used to produce

biomolecules.

Advancement in Plant genetic engineering : Possible to use as Bioreactors.

Plants are important food resources from the earliest times.

Plants as bioreactors :Plants are genetically engineered to make products

that are not of plant origin (Therapeutically & industrially important

products, Biomolecules, chemical & pharmaceutical etc.

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Plant as Bioreactor

Why plants are used as bioreactors?

Fast biomass build up

Post translational modifications

Easy storage and distribution

Low upstream production cost

Lacking of contamination by animal pathogen.

Advantages: Plant as bioreactor

Low cost source.

Simple & Cost effective.

Plant pathogens do not infect humans or animals.

Easy scale up & rapid harvesting.

Chimeric plant viruses can be used in production of vaccines.

Produce large biomass.

Easy storage for long time

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Plant as Bioreactor

Key process…

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Design gene for high level

expression

Plant transformation

Regeneration of Cell

Selection of transgenic

Growth of plants in field

Harvesting of plant materials

Purification of product

Biosafety & Functionality

test

Plant as Bioreactor

Types of PLANT BIOREACTORS

5

plant

bioreactors

Seed based

plant

bioreactors

Plant

suspension

culture

Hairy root

system

bioreactor

Chloroplast

bioreactor

Protein

storage

vacuoles

Oil bodies

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

• 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

• 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

• 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.

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

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

Contd...

3- D View of Hirudin

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'

• 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 )

•an anti-coagulant for treatment of

thrombosis in rapeseed by SemBioSys(Boothe et al., 1997)

Brassica napus

Contd...

Oleosin based purification of heterologous polypeptides

(Goddijn and Janpen, 1995)

Plant as Bioreactor

Oil bodies

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.

• Antitumour agents like taxol can be produced from

Taxus sp - treatment of breast and ovarian cancers.

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

• 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 as Bioreactor

Production platforms

Cereals: Maize, Rice, Barley and Wheat are used.

o High seed protein content (7 to 10%).

o High biomass yield.

o Ease of transformation.

o Ease of scale-up.

Major disadvantage of Maize is: Cross pollination

But rice has advantage of self-pollination.

Rice as bioreactors : Oral delivery system for vaccine antigens,

immunotherapy and therapeutic proteins are recent advances.

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Streatfield SJ et al., 2003

Plant as Bioreactor

Year Recombinant protein Source Importance Reference

1989 Mouse

immunoglobulinTobacco

1st report on the production of antibody in

plants.Hiatt et al., 1989

1997 Chicken avidin Maize 1st commercialized plant-derived protein. Hood et al., 1997

2003 Bovine Trypsin Maize 1st marketed plant-derived protein. Woodard et al., 2003

2006

HN proteins of

Newcastle disease

virus

Rice 1st plant-based vaccine (for poultry) approved

by the USDA

Dow AgroSciences,

2006

2007 Antibody against

Hepatitis BTobacco

1st commercialized plant-derived antibody

(marketed in Cuba)Pujol et al., 2007

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Ventria Bioscience (US Based Company) : Two rice-derived proteins

Human lactoferrin and lysozyme

Have received regulatory approvals, and lactoferrin has already been marketed in

bulk for bioprocessing.

Production platform Cont…

Plant as Bioreactor

Production platforms (Cont.…)

Legumes:

Soybean and pea: Seeds have high protein content (20–40%).

Both self-pollinating plants & low risk of contamination.

Soybean : Express a humanized antibody against Herpes simplex virus.

Bovine casein, and a Human growth hormone. (Russell et al., 2005)

Oil seeds:

Safflower and rapeseed : Rich in seed oil & inexpensive downstream.

Safflower : High protein yield, Low acreage, and Self-pollinating.

Oilseed-derived protein : Commercial production of Hirudin, an anti-

coagulant for treatment of thrombosis in rapeseed by SemBioSys (Boothe et

al., 1997)

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Plant as Bioreactor

a. Shown are various Intracellular

organelles or Extracellular

spaces (ES) that can be used to

store the recombinant proteins

expressed in a plant bioreactor.

b. Targeting strategies in plant

bioreactors.

G - Golgi; PSV - Protein storage vacuole;

OB - Oil body; C - Chloroplast;

ES - Extracellular space;

PVC - Prevacuolar compartment.

Where they are produced?

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Plant as Bioreactor

Products obtained…

Plants genetically engineered to make products that are not of

plant origin

Products:

Vaccines antigens

Therapeutics products

Nutritional components

Industrial products

Bio plastics

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Tree depicting biotechnological advances using plants as bioreactors

Source: www.plantbioreactor.co.in/images/00_112.jpg

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

1.

LIPIDS Medium-chain fatty acids

California baytree

Food, detergent, industrial

Oilseed rape

2. Mono-unsaturated fatty acids

Rat Food Tobacco

3. Polyhydroxy butyric acid

Alcaligenes eutrophus

Biodegradable plastics

Arabidopsis, oil seed rape, soybean

4. Saturated fatty acids

Brassica rapa Food, confectionaries

Oilseed rape

PRODUCTION OF BIOMOLECULES FROM PLANTS

5.

CARBOHYDRATES Amylose free starch

Solanum tuberosum

Food, industrial

Potato

6. Cyclodextrins Klebsiella pneumoniae

Food, pharmaceutical

Potato

7. Fructans Bacillus subtilis

Industrial, food Tobacco, potato

8. Increased amount of starch

Escherichia coli

Food, industrial

Potato

9. Trehalose E.coli Food stabilizer Tobacco

10.

PHARMACEUTICAL POLYPETIDES Antibodies

Mouse

Various

Mainly tobacco

11. Antigens Bacteria, viruses

Orally administered vaccines

Tobacco, tomato, potato, lettuce

12. Growth hormone Trout Growth stimulation

Tobacco, Arabidopsis

13. Hirudin Synthetic Thrombin inhibitor

Oil seed rape

14. Human serum albumin Human Plasma expander

Tobacco, potato

15. Interferon Human Anti-viral Turnip

INDUSTRIAL ENZYMES Alpha-amylase

Bacillus licheniforms

Liquefaction of starch

Tobacco, alfalfa

Phytase Aspergillus niger

Animal feed Tobacco

Xylanase Clostridium thermocellum, Cryptcoccus albidus

Animal feed, paper and pulp, baking

Tobacco

Products : Plant as bioreactor

PRODUCTION OF BIOMOLECULES

Carbohydrates

1) Cyclodextrin biosynthesis

• Starch acts as the substrate

• Bacterial gene encoding cyclodextrin glycosyl transferase

(cgt ) And Targetted to amyoplast.

•Expressed in tubers.

Contd...

Cyclodextrin glycosyl transferase

Contd...

HOW GENE CONSTRUCT IS MADE FOR cgt?

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

(Cyclodextrin glycosyl transferase)

Contd...

5' 3'

Patatin promoter

cgt gene

Sequence encoding transit peptide of

RuBP carboxylase

nos terminator

P c p t

Gene construct of cgt gene

P

c

p

t

Contd...

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

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.

Plant as Bioreactor

Vaccine antigens:

It is an antigenic preparation : Immunity against a disease.

Recent Development : “Edible Vaccines” now more popular as “Plant Vaccines”

From Seeds, freeze-dried fruits and leaves: powder form vaccines are produced.

Antigenic determinants for Pathogens causing diseases have been produced

from plants. (Diarrhea, anthrax, rabies, cancer, HIV, tuberculosis etc)

Antigens like Insulin, rotavirus enterotoxin, anthrax lethal factor, HIV antigen,

foot and mouth disease virus antigen, heat stable toxin have been produced in

plants as a fusion partner of CTB or LTB.

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http://www.dowagro.com/uk/media/General/20061017.htm, Khandelwal et al., 2003; Sharma et al., 2004, Streatfield and

Howard, 2003, Tiwari et al., 2009 and Youm et al., 2008.

Plant as Bioreactor

Products : Plant as bioreactor

Therapeutic products:

Many form of recombinants antibodies are produced in plants.

• (Full size recombinant antibody, chimeric antibody, secretory antibody)

• 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

Sub cellular destination - high level expression.

Further research is concentrated on Humanizing the Plant N-glycans.

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Schillberg et al., 1999, Ma et al., 2003, Goldstein and Thomas, 2004

Plant as Bioreactor

Products : Plant as bioreactor (Cont.…)

Therapeutic products:

Diagnostic proteins (antibodies and enzymes), replacement

proteins, immune system stimulator/suppressants, biopolymers

and adhesive proteins are produced in plants.

Production of immunoglobulin fragments and their assembly in

plants –reported in tobacco.

Recently known as “plantibodies” – immunochromatography or

medical therapy.

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Schillberg et al., 1999, Ma et al., 2003, Goldstein and Thomas, 2004

Plant as Bioreactor

Products : Plant as bioreactor (Cont.…)

Nutritional components:

It can provide most of the nutrients required in the human diet.

Plant have been engineered to increase accumulation of:

• β-carotene (Naqvi et al., 2009), Lycopene (Fraser et al., 2002), Flavonoid (Butelli et al., 2008),

Nutraceuticals (Kang et al., 2009), Fatty acid (Hoffmann et al., 2008), Vitamins (Nunes et al., 2009),

Minerals (Lee & An, 2009) & Carbohydrates (Regina et al., 2006).

Biodegradable plastics:

Polyhydroxyalkanoates: biodegradable polymers which occur naturally in plants.

• Plant was engineered to produce PHAs or PHBs in the various plant cell compartments.

When PHB expression targeted to cytoplasm, accumulation level was low.

Expression was increased by targeting plastids, (40% of dry weight was obtained).

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Plant as Bioreactor

Products : Plant as bioreactor (Cont.…)

Industrial products:

First produced protein : Human growth hormones in tobacco.

Most expensive Drug – hGC (Human glucocerebrosidase) in tobacco (Kaiser

2008).

hST (Human somatotropin) was produced in tobacco (Staub et al. 2000).

Which treats : Turner syndrome, Chronical renal failure & Dwarfism in Children.

rHLF (Recombinant human lactoferrin) : Produced from dehusked rice

grain (Nandi et al., 2002). Which is identical to native HLF.

Synthetic fiber: Produced from Potato and tobacco. (Scheller et al., 2001)

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LIMITATIONS AND REMEDIES

Limitations Remedies

* Downstream processing expensive and difficult

* Expressing the protein with sturctured oil body protein e.g: Hirudin

* Accumulation of transgene product in plant

* Targeting the transgene into proper cellular 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.

Plant as Bioreactor

Conclusion…

More and more uses of plant bioreactors are coming up these

days.

Plant bioreactors : Investigated for making enzymes (Suitable for Food).

Another use of plants is to make genetically engineered plants that can

produce seeds which can function as a delivery mechanism for various

industrial enzymes.

As you can see these processes go far beyond the application of

biotechnology in traditional agriculture, and so today,

transgenic plants can produce on a mass scale proteins for

agricultural, veterinary and pharmaceutical use.

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Plant as Bioreactor

Conclusion (Cont.…)

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Table source: Yansong miao et al., 2008

Plant as Bioreactor

Problems need to be addressed

Storage issues related to transgenic fruits or leaves.

Most inserted genes are expressed at very low level in plants.

Enhancing the stability of products obtained.

Standardization of dosage in case of edible vaccine.

Examining issues related to commercialization.

Issues relating to the ethical, social, biosafety and environmental

impact.

Some plants produce allergenic compounds.

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Plant as Bioreactor

References

• Boothe JG, Parmenter DL, Saponja JA. (1997) Molecular farming in plants: oilseeds as vehicles for the production of

pharmaceutical proteins. Drug Dev Res, 42:172–81.

• Butelli E, Titta L, Giorgio M, Mock HP, Matros A, Peterek S, (2008). Enrichment of tomato fruit with health-promoting

anthocyanins by expression of select transcription factors. Nat Biotechnol, 26:1301–8.

• Dow AgroSciences. Dow AgroSciences achieves world's first registration for plant-made vaccines. 2006 Press release

(http://www.dowagro.com/animalhealth/resources/news/20060131b.htm).

• Fraser PD, Romer S, Shipton CA, Mills PB, Kiano JW, Misawa N (2009). Evaluation of transgenic tomato plants

expressing an additional phytoene synthase in a fruit specific manner. Proc Natl Acad Sci USA , 99: 1092–7.

• Goldstein DA, Thomas JA. (2004), Biopharmaceuticals derived from genetically modified plants. QJM, 97: 705–16.

• Hiatt A, Cafferkey R, Bowdish K. (1989), Production of antibodies in transgenic plants. Nature, 342: 76–8.

• Hoffmann M, Wagner M, Abbadi A, Fulda M, Feussner I. (2008), Metabolic engineering of omega3-very long chain

polyunsaturated fatty acid production by an exclusively acyl-CoA-dependent pathway. J Biol Chem, 283: 22352–62.

• Hood EE, Witcher DR, Maddock S, Meyer T, Baszczynski C, Bailey M, (1997), Commercial production of avidin from

transgenic maize: characterization of transformant, production, processing, extraction and purification. Mol Breed, 3:

291–306.

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Plant as Bioreactor

References(Cont.…)

• Kang K, Lee K, Sohna S, Parka S, Lee S, Kima S, (2009), Ectopic expression of serotonin hydroxycinnamoyltransferase

and differential production of phenylpropanoid amides in transgenic tomato tissues. Sci Hortic, 120: 504–10.

• Khandelwal A, Sita GL, Shaila MS. (2003), Expression of hemagglutinin protein of rinderpest virus in transgenic

tobacco and immunogenicity of plant-derived protein in a mouse model. Virology, 308: 207–15.

• Lee S, An G. (2009), Over-expression of OsIRT1 leads to increased iron and zinc accumulations in rice. Plant Cell

Environ, 32: 408–16.

• Ma JK, Drake PM, Christou P. (2003), the production of recombinant pharmaceutical proteins in plants. Nat Rev Genet,

4: 794–805.

• Naqvi S, Zhu C, Farre G, Ramessar K, Bassie L, Breitenbach J, (2009), Transgenic multivitamin corn through

biofortification of endosperm with three vitamins representing three distinct metabolic pathways. Proc Natl Acad Sci

USA, 106: 7762–7.

• Nunes AC, Kalkmann DC, Aragão FJ.(2009), Folate biofortification of lettuce by expression of a codon optimized

chicken GTP cyclohydrolase I gene. Transgenic Res, doi:10.1007/s11248-009-9256-1.

• Pujol M, Ramírez NI, Ayala M, Gavilondo JV, Valdés R, Rodríguez M, (2005), An integral approach towards a practical

application for a plant-made monoclonal antibody in vaccine purification. Vaccine, 23: 1833–7.

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Plant as Bioreactor

References (Cont.…)

• Regina A, Bird A, Topping D, Bowden S, Freeman J, Barsby T, (2006). High-amylose wheat generated by RNA

interference improves indices of large-bowel health in rats. Proc. Natl. Acad. Sci. USA, 103: 3546–51.

• Russell DA, Spatola LA, Dian T, Paradkar VM, Dufield DR, Carrol JA, (2005). Host limits to accurate human growth

hormone production in multiple plant systems. Biotechnol Bioeng, 89: 775–82.

• Schillberg S, Zimmermann S, Voss A, Fischer R. (1999), Apoplastic and cytosolic expression of full-size antibodies

and antibody fragments in Nicotiana tabacum. Transgenic Res. 8:255–63.

• Sharma AK, Jani D, Tyagi AK. (2004), Transgenic plants as bioreactors. Ind J Biotechnol. 3: 274–90.

• Streatfield SJ, Howard JA. (2003), Plant-based vaccines. Int J Parasitol, 33: 479–93.

• Tiwari S, Verma PC, Singh PK, Tuli R. (2009), Plants as bioreactors for the production of vaccine antigens. Biotech

adv, 449–67.

• Woodard SL, Mayor JM, Bailey MR, Barker DK, Love RT, Lane JR, (2003), Maize (Zea mays)- derived bovine

trypsin: characterization of the first large-scale, commercial protein product from transgenic plants. iotechnol

Appl Biochem, 38: 123–30.

• Youm JW, Jeon JH, Kim H, Kim YH, Ko K, Joung H. (2008). Transgenic tomatoes expressing human beta-amyloid

for use as a vaccine against Alzheimer's disease. Biotechnol Lett, 30: 1839–45.

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Plant as Bioreactor

THANKS FOR YOUR ATTENTION!

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