Plants As Bioreactors

Lecture 36& 37

Plants as Bioreactors

A bioreactor is a device in which a substrate of low value is utilized by living cells or enzyme to generate a products of higher value. In earlier days and till now, microbes, and animals cell culture are used in production of biomolecules. Here the cost of production is high and so scientist turned their attention towards ‘plants’ to utilize them as bioreactors which turned fruitful. Advances in biotechnology are enabling plants to be exploited as bioreactors for production of Carbohydrates, proteins, edible vaccines etc (Goddijn and Janpen, 1995). The novelty, advantages and potential of this technology and its wide range of applications have generated much interest and investigation. The number of products that have successfully been produced in plants using this ‘molecular farming’ approach is steadily increasing.

WHY PLANTS ARE USED AS BIOREACTORS?

1. Post Translational Modifications 2. Storage costs 3. Ethical considerations

1. Post Translational Modifications

Microbes do not provide a good cellular environment for post-translational modifications of eukaryotic proteins, which are often recovered from bacteria as insoluble aggregates. Solubilization of these aggregates and refolding of the polypeptides to obtain native proteins involves considerable costs. The cellular environment of plants, on the other hand, is as good as that of animals for post – translational modification and folding of the proteins so that animal proteins recovered from plants are usually atleast as good as the native animal proteins.

2. Storage Costs

Microbial biomass has to be immediately processed and the purified proteins have to be ordinarily stored under cold storage. Plant seeds could be stored under ambient

1 conditions from which the proteins may be isolated as per need. This would greatly reduce storage costs of the products.

3. Ethical Considerations

Biochemical production from transgenic animals raises several public and ethical concerns, while no such problem is associated with plants.

These novel reasons paved the way for scientists to engineer plant as Bioreactors for production of biomolecules like Carbohydrates, lipids, proteins and edible vaccines etc.

PRODUCTION OF BIOMOLECULES FROM PLANTS

A. Carbohydrates from plants

Starch is one of the major carbohydrates stored by plants and has many commercial uses either untreated or in modified forms. Since plants are a source of pre- cursor molecules that can act as substrates for introduced enzymatic activities, more carbohydrates production is possible. Also plants provides the optimal environment for the enzymes. A few important examples are discussed below. a. Producing cyclodextrins using starch as a substrate

Cyclodextrins are cyclic oligosacharides comprising 6 (), 7() or 8()  -1, 4 linked glucopyranose units. Their cylindrical structures enables them to carry ‘guest’ molecules and hence can be used in pharmaceutical delivery. The cost of production, in invitro limits its use. Large scale cyclodextrin biosynthesis in plants is feasible. The bacterial gene encoding cyclodextrin glycosyltransferase was transferred into potato and expressed in tubers. The chimaeric gene construct consisted of : (1) patatin gene promoter (for tuber-specific expression), (ii) the sequence encoding the transit peptide of the small subunit of the ribulose bis phosphate carboxylase (for ensuring the transport of the protein encoded by the chimaeric gene into the starch accumulating amyloplasts) (iii) the cgt gene, and (iv) the 3’ sequence of nopaline synthase (nos) gene of Agrobacterium. The expression of cyclodextrin glycosyltransferase (cgt) gene from Klebsiella

2 pneumoniae in potatotuber amyloplasts resulted in synthesis of  - and  - cyclodextrin at a level of 0.001 – 0.01%. These low levels may result from poor enzyme expression or limited access of enzyme to starch substrate. On commercial scale, it can be circumvented by pre-hydrolyzation which results in increased starch solubility that allows better access of enzyme to substrate . b. Increasing starch accumulation

Genetic Engineering has been used to increase the amount of starch in plant storage organs (Verisser and Jaciobsen, 1993). A mutated bacterial gene (glg c16) encoding ADP glucose pyrophosphorylese was expressed in potato tubers and targetted to amyloplasts. Low-starch, potato lines that were transformed, produced tubers with almost 60% more starch than control. c. Rerouting the starch to produce other carbohydrates i. Fructan biosynthesis

Starch precursors can be rerouted into the synthesis pathways of other storage carbohydrates. Non-fructan storing tobacco and potato plants have been included to accumulate fructan by introduction of B.subtilis fructosyltransferase gene. It was found that fructan accumualted to 3-8% of dry wt of leaves in tobacco, 1-30% in potato (leaves), 1-7% in microtubers. Total non-structural carbohydrates in leaves increased to 35% in transgenic plants. ii. Sugar alcohols

Tobacco plants transformed with mannitol 1-Po4 dehydrogenose gene (mtlD) from E.coli synthesise marnnitol at >6 mol per g fresh wt of tissue in leaves and roots. Similarly, pinitol, a myo inositol derived cyclic sugar alcohol has been synthesised in tobacco by introduction of myoinositol O – methyl transferase gene from Mesembryanthemum crystallinum (ice plant).

3 Also the additive, trehalose being extracted from yeast is also reported to be synthesised in small quantities in transgenic tobacco plants by Plant Biotech Companies MOGEN and CALGENE

Figure 1 Metabolic Engineering of Carbohydrate Metabolism (Goddijin and Janpan, 1995)

B. Proteins from plants

Plants are commercially attractive systems for the production of heterologous proteins. The expression of polypeptide relies on stable integration of transgene in plant genome. Another alternative is to produce novel proteins in plants by transient expression of genetically engineering viruses such as Cauliflower Mosaic Virus (CaMV), Tobacco Mosaic Virus (TMV) and Cowpea Mosaic Virus (CPMV).

1. Human therapeutic protein in Tobacco Chloroplasts: (Jeffrey et al., 2000)

The human therapeutic protein, namely human somatotropin (hst) can be expressed in a biologically active, disulfide – bonded form in tobacco chloroplast. hst is used in treatment of hypopituaitory dwarfism in children and Turner Syndrome. The hst and ubiquitin fusion genes were synthesised using E.coli preferred codons and yeast

4 ubiquitin sequences respectively. Later the chimeric hst genes were cloned into chloroplast transformation p PRV vectors linked to selectable spectinomycin resistance (aadA genes). The vectors target insertion of foreign genes into the plastid genome between the plastid trnv gene and rps 7/3’ – rps 12 operon in duplicated inverted repeat region. The chimeric genes were introduced into tobacco leaf chloroplast using biolistic process. Transformants identified by growth on spectinomycin are indeed plastid transformed. Southern blot analysis was done which proved positive. Accumulation of hst in transformed plants was monitored by western blot analysis. Leaves in different ages has different hst accumulations, matured and old leaves have similar amounts and young leaves with less hst.

Seeds as bioreactors

So far with the concerned production strategies only the plastids were used as targeted vehicles. Interests have turned towards the seeds which can be used as bioreactors for reasons including their convenience in transportation and storage, suitable either for oral consumption or purification and the existing agricultural facilities for seed handling (Sun et al., 2002).

The most attractive approach for protein expression and in field production is to target the protein to the ER and if long term storage is required, to target it for seed specific expression with seed specific promoters. To develop an efficient seed bioreactor platform, various promoters including seed specific and constitutive, host plants such as are Arabidiopsis, tobacco and rice, and target products including health related and important proteins are under research.

2. eg : Production and purification of recombinant Hirudin from plants seeds (Dana et al., 1996)

The antithrombin agent heparin is widely used in clinical therapy. This protein binds to the endogenous blood protein antithrombin III, enhancing its antiprotease activity and cause the rapid inactivation of thrombin in addition to factor Xa and number

5 of other factors. But this Heparin has side effect, cause haemorrhaging, so there has been a driving force in search of specific thrombin inhibitor like, the active agent responsible for anticoagulant effect namely “hirudin” which was isolated from salivary glands of the leech, Hirudo medicinalis. Hirudin is a small (7000 Da), highly acidic protein. Advances in recombinant DNA technology have elucidated the hirudin isoforms. This hirudin apart from its antithrombin activity, is pharmecodynamically inert.

The limited availability of hirudin in leeches (1 leech head contains 20 mg of hirudin) has necessitated its production in heterologous production systems. Biologically active recombinant hirudin (rHir) variants have been cloned and expressed in E. coli, yeast. In recent yrs, plant seeds represent a particularly alternative host for production of recombinant proteins since storage proteins naturally accumulate to very high levels in most seeds. Since purification is expensive, oleosin partition technology is followed.

Construction of Oleosin – Hirudin fusion

A synthetic gene with amino acid sequence of HV2 utilising the coden preferences for Arabidopsis and Brassica is constructed, which is shown below :

HIS THR THR ala ile glu gly arg ile thr tyr thr asp cys thr glu ser CAC ACT ACT GCG~ ATC GAA GGG AGA ATC ACT TAC ACT GAC TGT ACT GAA TCT Pvu 1 OLEOSIN 1------1 CLEAVAGE SITE HIRUDIN ------ gly gln asn leu cys leu cys glu gly ser asn val cys gly lys gly asn GGA CAG AAC CTC TGT CTC TGT GAA GGA TCT AAC GTT TGT GGA AAG GGA AAC HIRUDIN lys cys ile leu gly ser asn gly lys gly asn gln cys val thr gly glu

6 AAG TGT ATC CTC GGA TCT AAC GGA AAG GGA AAC CAG TGT GTT ACT GGA GAA HIRUDIN gly thr pro asn pro glu ser his asn asn gly asp phe glu glu ile pro GGA ACT CCA AAC CCA GAA TCT CAC AAC AAC GGA GAC TTC GAA GAA ATC CCT HIRUDIN glu glu tyr leu gln OCH val asp GAA GAA TAC CTC CAG TAA GIC GAC GG Sal 1

Figure - 3Oleosin – hirudin fusion gene construct.

Following PCR amplification, the synthetic HV2 was fused in frame to the 3’ end of fragment from an Arabidopsis oleosin gene, (as oleosins acts as potential carriers for recombinant proteins) which had previously been isolated from genomic library and modified to remove translation stop codon. In addition to the coding region, the genomic fragment also comprise the intron and 800 bp of Arabidopsis oleosin promoter which directs seeds – specific expression of fusion protein. Between the oleosin and hirudin genes, a recognition site for the protease factor Xa was engineered which enables cleavage and recovery of recombinant hirudin from fusion protein in course of purification. The gene was terminated with nopaline synthese transcriptional terminator sequence derived from Agrobacterium.

7 The fusion gene construct was introduced into Brassica napus plants along with selectable marker (NPT –II) for kanamycin resistance via Agrobaterium – mediated transformation. The stable integration of gene was confirmed by southern analysis, while Northern analysis of RNA revealed the expression of oleosin hirudin transcripts is seed specific. The stable accumulation of oleosin hirudin protein in transformed seeds is seen and the recombinant hirudin is purified from the seeds.

Figure 2Oleosin based purification of heterologous polypeptides (Goddijn and Janpen, 1995) D. Biopharmaceuticals from plants

Transgenic plants have been constructed that express proteins such as enkephalins, -interferon, human serum albumin, and two of the most expensive drugs; glucocerebrosidase and granulocyte-macrophage colony-stimulating factor (Giddings et

8 al., 2000). Applied Phytologics (API; Sacramento,CA) has modified rice plants to produce human -1-antitrypsin, a protein of therapeutic potential in cystic fibrosis, liver disease, and hemorrhage. Trials of -1-antitrypsin transgenic rice commenced in 1998, with protein extracted from malted grain. API hopes to have regulatory approval for transgenic plant medical products by 2004.

Lysosomal enzymes

Gaucher’s disease is a recessively inherited lysosomal storage disorder resulting from deficiencies of lysosomal hydrolase glucocerebrosidase enzyme. A drug developed from enzyme purified from human placentas is highly effective at reducing clinical symptoms. However, 10-12 tons/year of placentas are required to produce enough glucocerebrosidase for the average type I Gaucher's patient, making it one of the world’s most expensive drugs. A recent switch to production in mammalian cell culture systems has reduced this cost, but didn’t remove the drug from the “most expensive” league. Glucocerebrosidase production in transgenic tobacco was patented by Cramer and colleagues at Virginia Tech and State University (Blacksburg, VA) and CropTech (Blacksburg, VA) in 1999. Their studies “strongly support” the future commercial viability of transgenic plants for the production of glucocerebrosidase, and of other lysosomal enzymes, for enzyme replacement therapy (Giddings et al., 2000).

Industrial enzymes from plants Cellulase from potato

Transgenic potato plants have been modified to produce cellulase enzymes in the foliage. The cellulase producing genes were isolated from bacterial and fungal organisms. Cellulase which breaks down plants material is used in a wide variety of application from food processing to ethanol production. This process can be adopted to create additional enzymes such as lipases and proteases used in pharmaceuticals. Other than potato, corn can be modified to produce enzymes in the non-edible portions.

The same principle can be exploited in every process that required a protein or enzyme : and in which plant materials can be used. For example application of seed

9 formulated Amylase for starch liquefaction. Packaging phytase from Aspergillus niger in seeds by genetic engineering could completely replace feed Po4 as a supplement to the basal diet of broiler chickens.

PLANTS CELL SUSPENSION CULTURES AS BIOREACTORS

Recently plants cell suspension cultures are being used as bioreactors. These plants cell suspensions are normally derived from calli cultivated on solidified media and grown in liquid media. Most application of plant cell suspension cultures aim at the production of naturally occuring secondary metabolites. This has included production of shikonin, anthocyanins, ajmalicine and important anti-tumour agents like taxol, vinblastine and vincristine. Taxol is produced from cell cultures of Taxus species and is used in the treatment of breast and ovarian cancers (Seki et al., 1997)

Tobacco suspension cell lines exist as model system for recombinant protein (Fab fragment full size IgGs) introduction (Fischer et al., 1995) Here the recombinant proteins expressed in Plants Cell Suspension Culture are found in the culture supernatent or retained with the cell. The proteins are released from culture supernatent or from the cell by mild enzymic cell wall digestion. Expression level is found to be 2-20 mg of recombinant protein %g of fresh cell wt.

COMPARISON WITH OTHER PRODUCTION SYSTEM

Plants have several potential advantage over other production systems based on microbial fermentation, animal cells and transgenic cells. The very first thing is the fast biomass build up and low cultivation costs. It enables easy storage and distribution (transgenic seed material). Microbial system have a limited capacity for the accurate post translational modification of eukaryotic proteins, where as plants systems are more attractive for production enkaryotic proteins because they produce fully folded functional molecules with practically identical properties to the original protein. Plants cells carry out all of the post translational modifications required for optimal biological activity of proteins. As an example, r ab production in tobacco plants have the same sensitivity specificity and importantly, the same affinity as monoclonal antibodies produced by the original hybridoma cell line. Bacterial fermentation often results in production of

10 insoluble aggregates and substantial costs are involved in solubilizing and refolding these aggregate into native proteins. Fermentation require huge capital investment and in case of animal cell cultures, expensive growth media, hence plants act as low cost alternative.

The use of transgenic animals as production systems raises more public and ethical concerns then does the use of plants. In plants the upstream production cost in cases of proteins are lower than other system.

ADVANTAGES

1. Foremost thing, plants are easy to grow like bacteria and animal cell which requires specialised equipment or expensive media supplements such as fetal calf serum. 2. Low-cost source 3. Large biomass 4. Can produce high level of safe, homogenous functional biomoleculas and can be easily expanded to agricultural levels to meet industrial demand. 5. Modern agriculture practice enables easy scale up, rapid harvesting and processing of large quantities of leaves or seeds 6. Chimeric plant viruses can be used in production of vaccines 7. They can be stored readily, (eg. Seeds and tubers) no special maintenance is required and have long shelf life 8. In case of vaccines, administration is safe and painless 9. High level of heterologous proteins can be accumulated in plants based on promoters, leader sequence, codon usage etc., and are easily in distinguishable from those produced by hybridoma cell. 10. The ability to assemble immunoglobulins is major advantage that plants have over bacterial expression systems

11 LIMITATIONS AND HOW TO OVER COME IT?

1. The downstream processing is costlier and difficult in plants due to the low concentration of recombinant protein in the total biomass. This could be overcome by. i. first concentrating the recombinant protein by a suitable process eg : affinity chromatography ii. expressing the recombinant protein as a fusion product with a structured oil body protein like oleosin. eg. : hirudin iii. purification of proteins could be altogether avoided wherever the plant produce can be utilized directly for achieving the objectives eg: in case of phytase enzyme and edible vaccines. 2. Sometimes, an accumulation of a transgene product may adversely affect the performance of plants. In some cases, this production could be resolved by targeting the transgene expression into proper cellular compartment. For eg: Tobacco plants expressing a fructosyl transferase gene construct having an apoplastic targeting signal showed severe necrosis. But when this gene was linked with vacuolar targeting signal sequences, fructan accumulation occured without an aberrant phenotype of the plants. Similarly experience of acetoacetyl – CoA transferase in the cytoplasm resulted in a growth reduction of Arabidopsis plant. But when the gene production was targeted into chloroplast, there was no adverse effect on plant growth or seed yield.

FUTURE CHALLENGES

1. Evaluation of dosage requirement in case of edible vaccines. 2. Quantification of amount of uptake 3. Stability of product under storage 4. Engineering challenges including maximisation of expression levels, post- harvest storage stability and enhancement of oral immunogenecity 5. Environmental safety needs to be evaluated 6. Regulatory considerations and legal standards have to be met

12 7. The use of plants cell suspension culture for production of recombinant proteins and secondary metabolites are still in the infancy stage. 8. Extent and safety of biopharmaceuticals .