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?

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...

• 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 )

• 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

Rerouting the starch to produce fructan

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

• Storage facility

• Transportation

• Oral consumption

• Existing agricultural facilities in seed handling

(Sun et al., 2002)

Why seeds are used as bioreactors ?

See

ds

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 )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 Transformation1. 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 TraffickingFollowing 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

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

1.

LIPIDS

Medium-chain fattyacids

Californiabaytree

Food,detergent,industrial

Oilseed rape

2. Mono-unsaturatedfatty acids

Rat Food Tobacco

3. Polyhydroxybutyric acid

Alcaligeneseutrophus

Biodegradableplastics

Arabidopsis, oilseed rape,soybean

4. Saturatedfatty 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.

PHARMACEUTICALPOLYPETIDESAntibodies Mouse Various Mainly

tobacco

11. Antigens Bacteria,viruses

Orallyadministeredvaccines

Tobacco,tomato,potato,lettuce

12. Growth hormone Trout Growthstimulation

Tobacco,Arabidopsis

13. Hirudin Synthetic Thrombininhibitor

Oil seedrape

14. Human serum albumin Human Plasmaexpander

Tobacco,potato

15. Interferon Human Anti-viral Turnip

INDUSTRIALENZYMES

Alpha-amylase Bacilluslicheniforms

Liquefactionof starch

Tobacco,alfalfa

Phytase Aspergillusniger

Animal feed Tobacco

Xylanase Clostridiumthermocellum,Cryptcoccusalbidus

Animal feed,paper andpulp, baking

Tobacco

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

* Downstreamprocessing expensiveand difficult

* Expressing the proteinwith sturctured oil bodyprotein e.g: Hirudin

* Accumulation oftransgene product inplant

* Targeting the transgeneinto proper cellularcompartment

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