Using plant to manufacture high value products

Molecular Farming

What is molecular farming/pharming? Why use plants? What are the risks and concerns? Model experiments

Biotechnology in agricultureBiotechnology in agricultureMainly two categoriesy g

1. “Improvements” to existing livestock and crops, and

2 Development of entirely new uses of both 2. Development of entirely new uses of both animals and plants (biopharming).

“Improvements", include ‘input traits’ such as crops with extra resistance to insect as crops with extra resistance to insect attack, improved weed control, increase the plants tolerance to cold, drought and other environmental factor with added character environmental factor, with added character for quality product.Example: "Roundup ready" soya, "Starlite" corn or "Frost tolerant" tomatoes golden corn, or Frost-tolerant tomatoes, golden rice.

Plant products source: from wild or fi ld lti t dfield cultivated

Draw backs for productiona bac s o p oduct oSeasonal harvestingShort growing season in temperateg g pDisease and insect problemHigh cost for labour and machinery

Plant cell culture can be alternative b d l dsource. It can be a model system to study

the regulation of natural product biosynthesis in plantbiosynthesis in plant

Plant with valuable phytochemicalsp y

Plant as a source of traditional products like food, fiber, shelter etc are also the source of other fiber, shelter etc are also the source of other productsPharmaceuticles- Drug sourceAgrochemicals- fungicides and insecticidesAgrochemicals fungicides and insecticidesNutrition-food flavouring and colouringCosmetics-natural fragrance and oilsAll th l bl d t b d d b ll All these valuable products can be produced by cell in in vitro culture- bioprocessCellular: Better culture system- limiting factorsGenetic: Selection of better cell line with high productive in the metabolite.Molecular: Metabolic engineering: induction and

i repression genesNovel compound production: introduction of new gene/s and expression in cell

M l l r f rminMolecular farming

Molecular farming includes method to integrate a foreign gene into plants.g g g pMolecular farming is the term used only for new use of plants and is different in that this does not affect and has nothing to do with Food production. There are two types of Molecular Farming: Medical and non-Medical.

Non-Medical Molecular Farming

Non-Medical Molecular Farming includes production of g pIndustrial Enzymes and Polymers.

• Industrial enzymes: for example laccase in transgenic maize, T h i l t i f h f l • Technical proteins for research purposes: for example avidin, which is also produced in maize,

• Milk proteins such as human beta casein, which is produced in transgenic tomatoes, produced in transgenic tomatoes,

• Protein polymers: collagens, which are used for medical as well as industrial purposes.

• Development of plants producing biodegradable p p p g gplastics.

• Industrial oils such as hydraulic oil or high yielding biodiesels, new Biofuels, new Fibres and y gPapers, and as agents for Bioremediation and Phytoremediation,

Molecular farming: Application of molecular biology techniques to the synthesis of commercial products

The use of agricultural plants for the production of g p puseful molecules for non food, feed or fiber applications- plant molecular farming

Plants are already grown to produce valuable molecules including many drugsmolecules, including many drugs.

Molecular farming is different because the plants areMolecular farming is different because the plants are genetically engineered (GE) to produce the molecules we want: Novel product or enhanced commercial product of plant

WHY PLANTS?WHY PLANTS?Plants are also very flexible and can produce a wide variety of proteins in large amount.variety of proteins in large amount.Crop plants can synthesize a wide variety of proteins that are free of mammalian toxins and pathogens.p gCrop plants produce large amounts of biomass at low cost also in limited facilities-industrial scale production possiblep pFunctional protein produced: Modification after synthesis as in eukaryote Protein in compartment or seed and remain stable Protein in compartment or seed and remain stable until used Crops are therefore well suited for the production of safe low-cost therapeutic proteins and other p pproducts.Possible to omit/simplify purification

Why use plants?Using plant for molecular farmingUsing plant for molecular farming

Advantages Disadvantages

Cost reduction Environment contamination

F d l t i tiStability

Safety

Food supply contamination

Health safety concernsSafety Health safety concerns

Table Comparison of Expression Systems

Expressions

SystemYeast Bacteria Plant

virusesTransgenic Plants

Animal Cell

Cultures

Transgenic

Animals

Cost of maintaini

ng

inexpensive

inexpensive

inexpensive

inexpensive expensive expensive

Type of Type of storag

e-2.0°C -2.0°C -2.0°C RT* N2** N/A

Gene size (protein) restrictio

n

Unknown Unknown Limited Not limited Limited Limited

P d tiProduction cost Medium Medium Low Low High High

Protein i ld High Medium Very high High Medium to

hi h Highyield High Medium Very high High high High

Therapeutic risk Unknown yes Unknown Unknown yes yes

Plant Products

1.Plant-derived pharmaceuticals (non-GE)2. Plant-made pharmaceuticals and

industrial products (GE)

Industrial productsproteins

PharmaceuticalsRecombinant human proteinsproteins

enzymesmodified starches

Recombinant human proteinsTherapeutic proteinsenzymes

fatsoils

Antibodies (plantibodies)vaccines

waxesplastics

The first recombinant plant-derived pharmaceutical protein (PDP) was human serum albumin initially protein (PDP) was human serum albumin, initially produced in 1990 in transgenic tobacco and potato plants. Concept has been established for the production of Concept has been established for the production of many therapeutic proteins, including antibodies, blood products, cytokines, growth factors, hormones, recombinant enzymes and human , yand veterinary vaccines in transgenic plants.Furthermore, several PDP products for the treatment of human diseases are approaching pp gcommercialization, including recombinant gastric lipase for the treatment of cystic fibrosis, and antibodies for the prevention of dental caries and the treatment of non Hodgkin's lymphoma the treatment of non-Hodgkin s lymphoma. There are also several veterinary vaccines in the pipeline; Dow AgroSciences announced to produce plant based vaccines for the animal health plant-based vaccines for the animal health industry.

HOW DONE?Use methodologies from molecular biology, biochemistry, genetics and biochemistry, genetics and biochemical engineeringMetabolic engineering redirection of enzymatic redirection of enzymatic reaction to produce high value compounds, new ompo nds deg ade to i compounds, degrade toxic

compound,or make resistant to stressGenetic transfer: Genetic enhancement is used to introduce and express new genes coding for the high value proteins.

Metabolic Enginering Metabolic flux analysis and modification geneticall ate ( h hi )

1. Carbon flux increased in branch point

genetically-rate limiting step

(Pathway architecture)

2. Increase flux by 2. Increase flux by inhibition of competitive pathway reduce diversion

Cross talk between pathways

Or TF

Metabolic engineeringProblem: 1000s of interacting pathways in plant regulated g ggenetically and environment

3. Metabolite yield regulated by Competitive pathway inhibition or by pathway inhibition or by introducing new pathway

4. Blocking catabolism by down regulation of catabolic enzyme or transport to vacuole

Field Production

Plants expressing Plants expressing the pharmaceutical protein gene- can then be produced in then be produced in the field. Following harvest the tissue, the protein extracted for later use.use.

Genetic engineering is basic tool

Plant genetic engineeringI t d ti f f l d tIntroduction of new gene for novel productCreate recombinant DNA with gene from same or different organism- overexpression and overproductionTransfer of DNA to plant cell by either Agrobacterium or ‘ballistic’ transformationIntroduced DNA and expression of foreign protein in l t t i d l tplant-transgenic development

Biotransformation

Precaution should be taken forEnvironment contamination via gene flowContamination of food supplyContamination of food supplySecondary metabolite – may introduce allergicity or toxicity.

Strategies for ‘Molecular Farming’

May cause adverse effects in planty p

Strategies of plant gene expressionTransient transformationStable transformationChloroplast transformationLocation of trans-gene and expressionSelection of suitable plantMode of reproduction

ld hYield, harvest, processing

Plants for molecular pharmingAdvantages

l ( lCorrectly process protein (Plant configure both animal and human proteins)p )Pathogen freeCheaper productionStability (S d d f it t ilStability (Seeds and fruits as sterile packaging)Safety

DisadvantagesEnvironment contaminationFood supply contamination

Moss photobioreactor with Physcomitrella patens

Food supply contaminationHealth safety concernsPlant proteins have different sugar residues

Why use plants?Carbohydrate and lipidsCarbohydrate and lipids

Novel and modified Commercial productCellulose (cotton paper paints and polymers) and

Advantages DisadvantagesCellulose (cotton, paper, paints and polymers) and starch (Food feed and industry)- most abundant carbohydrateBiotechnology for- improve in yield and quality Cost reduction Environment contamination

F d l t i ti

Biotechnology for- improve in yield and quality, and for new products

Stability

Safety

Food supply contamination

Health safety concernsSafety Health safety concerns

Metabolic pathways for carbohydrate synthesis

AmyloplastAmyloplast

* *

*ADP-Gl pyrophosphorylase

St r hStarch• Starch used as food, feed and in industries (in US70% in

industries and 30% for human consumption and feed)Strach synthase (SS) : form amylose chain α1-4 link add ADP-glucose (~1000), soluble & Granule bound SS:Strach branching enzyme: α1-6 linkage in amylopectin (~104 to 106 glucose): SBE-A and SBE-BHexose phosphate to ADP glucose by ADP glucose phosphorylase specific to starch synthesis Enzyme phosphorylase-specific to starch synthesis. Enzyme efficiency affect amount of starch synthesisAmylose:amylopectin-20-30:70-80%Antisense inhibition of GBSS1-amylose free starchAntisense inhibition of GBSS1-amylose free starchAntisense inhibition of SBE-A and SBE-B –High amylose starch, which also contain high phosphatePotato with freeze thaw stability starch produced by Potato with freeze thaw stability starch produced by i9nhibition of GBSS and SSII and SSIII produce short chain amylopectin

Target carbohydrate for molecular farming

Poly fructans: straight and Poly fructans: straight and baranched, vacuole targeted construct used but acumulation was found very

Stress Food preservative

acumulation was found very low.

Trehalose: in response to osmotic stress used in food posmotic stress, used in food processing, dehydration and flavour retention

Medicinal: solubilisation of small hydrophobic molecules

C l d triCyclodextrinsBiotransformation of starch to cyclodextrin by y ybacterial fermentation of starch6, 7 or 8 membered ring of α1-4 linked glucopyranose –the interior is not hydrophobic, glucopyranose the interior is not hydrophobic, but considerably less hydrophilic than the aqueous environment, external sufficiently hydrophiliichydrophiliicSolubilise hydrophobic pharmaceutical like

steroid in concentrated condition. At lower conc. l th h ti lrelease the pharmaceuticle

Klebsiella pneumonae cyclodextrin glycosyl transferase gene under control of potato patatin promoter resulted only conversion of upto 0.01% starch to cyclodextrins in potato.

Bi pl ti (bi d r d bl )Bioplastic (biodegradable)Produced by microbes-possible to produce in bulk by plantby plantPolyhydroxyalknoate: R group determines properties

R: H to 10C: affect the physical properties of plastic formed- 3hydroxypropionate, 3hydroxybutyratep y yp p , y y yPHB in Alkaligenes eutrophus accumulate PHB up to 80% of DW

PHB production in plantphaA, phaB, phaC three enzyme necessary are cloned from Alcaligenes cloned from Alcaligenes eutrophusAcetoacetyl-CoA is produced in cytoplasm of

pha Aproduced in cytoplasm of plant cell for isoprenoid synthesisOnly phaB and phaC pha BOnly phaB and phaCtransfer to Arabidopsis accumulated PHB in cytoplasm, vacuole and

pha B

y p ,nucleus- plant stunted and amount low 20-100µg/g FW

phaC

Triple transfer increased up to 40% of DM-but with stunted and fertility loss

PHB production in transgenic Arabidopsis

a.Cytoplasm targeted gtransformation

b.Chloroplast targeted transformation

Biopolymer production( 400 000 /(World production 400,000 Tons/year, it is only 0.45% of total polymer)

PLAPLA(=polylactic acid)

BIODEGRADABLEBIODEGRADABLE POLYMER FROM CORN

PlasticApparelHygiene productsHousehold, industrial & institutional fabricsCarpets

Bio-polymers are environment friendly(Compared to oil based plastics)(Compared to oil based plastics)

Polymer (biodegradable)

Total production

Energy consumption

CO2emission(biodegradable) production

(Tonnes/year)consumption emission

Solanyl (lactic acid 40.000 40% less NApolymer, Potato based)

NatureWork 140,000 20-50% less 50-70% atu e o(lactic acid polymer, Maize based)

0,000 0 50% ess 50 0%less

BioPDO (Maize based polytrimethylene terephthalate

45,000 16% less No differrence

terephthalate polymer,

M l l r f rmin Pr t inMolecular farming-ProteinProduction of enzymes, and high value oduct o o e y es, a d g a ueproteins (biopharmaceuticals, vaccines, protein antibiotics and antibodies)P l i difi i d Post translation modification and Functional protein production possibleSafe store in cellular compartmentSafe store in cellular compartmentSafe (no contamination) and low cost pharmaceuticle production.pharmaceuticle production.Industrial scale amount of productionCan be directly added in feed or foody

Enzyme production

E Bi t l iE Bi t l i

Enzymes in % of total•• Pancreas extract with Pancreas extract with enzyme used inenzyme used in

Enzymes as BiocatalysisEnzymes as Biocatalysis

Detergent 33%

Food production 30-45%

Pulp and paper 1-3%

enzyme used in enzyme used in detergent at beginning detergent at beginning of 20th century. Globalof 20th century. Global p p p

Textile finishing 8-14%

Chemical production

5%

of 20th century. Global of 20th century. Global Enzyme market in Enzyme market in 2004/05 is $ 2.5 billion2004/05 is $ 2.5 billion production

Enzyme based IndustriesEnzyme based Industries: : Production of various Production of various h i l i / d i ih i l i / d i ichemicals using enzymes or/and microorganismschemicals using enzymes or/and microorganisms

Enzymes and their usesy

Enzyme UsesEnzyme UsesProteases Detergent, food, chemical, pharma

C b h d F d f d l d t til Carbohydrase Food, feed, pulp and paper, textile, detergent

Lipases Food, effluent treatment, detergent, h ilpharma, textile

Pectinase Food, beverage (fruit juice)

Cellulases Pulp, textiles, feed, detergent, energy

Amylases Food, textile

A tib di pr d tiAntibodies productionPlants can be used to produce monoclonal pantibodiesTobacco, corn, potatoes, soy, alfalfa, riceFree from potential contamination of mammalian virusesExamples: cancer dental caries herpes Examples: cancer, dental caries, herpes simplex virus, respiratory syncytial virusGE Corn can produce up to 1 kg antibody/acre and can be stored at RT for up to 5 years

Plant made Pharmaceuticals

Therapeutic proteins Disadvantages:Blood substitutes –human hemoglobinP t i t t t

Disadvantages:

• Long time to produce the linesProteins to treat

diseases: CF, HIV, Hypertension Hepatitis

produce the lines

• Gene silencing

• Low expression Hypertension,Hepatitis B…..many others

• Low expression level

To date, no plant-produced pharmaceuticals are commercially available but there is greatare commercially available but there is great promise.

Plants used for expression

• Transformation efficiency of the plant-TobaccoEdible plants potato tomato maize etc

Plants used for expression

• Edible plants- potato, tomato, maize etc• Higher biomass yield• Stable expression- expression controlled by

35S t35S promoter• Targeted expression

Strategies for protein production(stable and transient expression)

Stable expression: t f ti d transformation under 35S promotor (strong and constitutive).Targeted production

in specific tissuein specific tissueTuber and seed maintain biological gintegrity over long periodSynthesis in edible Synthesis in edible part for vaccineEnzymes in feed

Oleosin system: l inovel protein

Oleosin is the surface layer protein of oil b dibodies.Pharmaceutical protein: Hirudin-against thrombosis like heparinpLow yield in E.coliHirudin at C-terminal of A.t. Oleosin separated by cleavage site Xa for cleavage site Xa for endoproteaseEndoprotease treated oil bodies cleaves hirudin

d i hand in aqueous phase.Cheaper alternative: this Oil body/oleosin tech. will be economically Not suitable for protein that needs yfeasibleNot for protein that need processing and modification

Not suitable for protein that needs extensive processing in ER

Insulin in safflower

Transient iexpression

• Plant virus capsid as ca ie of carrier of recombinant protein

• Fused with coat protein and expressed when virus particles are made.p

• Virus vector are also made so that the expressed novel expressed novel protein is targeted to ER for processing.

• The virus infected plant thus produce the protein

E z pr d tiEnzyme productionEnzymes produced in plantsGlobal enzyme market is of billions dollar

Ph t : f r ph ph t tiliz ti nPhytase: for phosphate utilization•Phytase release phosphate from phosphate from phytate normally indigestible to monogastric animalsmonogastric animals

•Release of phytate in faeces leads to eutrophication

•Transgenic plant producing Phytase in producing Phytase in seed

•Diet with such seed increased phosphate utilization in animal

• Solved nutritional and environmental problem

Pharmaceutical proteins: Antibodies, V i th t iVaccins, other proteinsAntibodies produced in Plant

Synthesis of Synthesis of secretory sIgA molecule in t i transgenic plant. Four separate pparental transgenic lines crossed lines crossed to produce SIgA

Tobacco mosaic virus (engineered) for the expression of pharmaceutical of pharmaceutical proteins

Vaccine produced in plant

(Edibl i ?)(Edible vaccine?)

Biopharmaceuticals(Yield and recovery problem)(Yield and recovery problem)

Novel purification system or chloroplast trnsformationtrnsformationTricosanthin: component of tuber of Trichosanthus kirilowi. A ribosome inactivating protein-inhibit t th d i R id l tumour growth and immune response. Rapid large scale production in tobacco (2%of TSP in 2 weeks) by viral RNA based infection.Glucocerebrosidase: Gaucher disease-defect in break doun of Glucocerebroside & accumulate in lysosyme and swelling of spleen, liver and bone gdamage. Most expensive drug purified from human placenta. Production in tobacco is patented-may reduce cost.HSA:in liver cirrhosis. HSA with tobacco extracellular PR-S signal was correctly processed.

Golden riceGolden rice

Rice is an important staple food especially of AsiaThe aleurone layer is rich in vitamins but The aleurone layer is rich in vitamins but removed in polished rice: mostly used as food in AsiaNo Vit. A in edible part endospermConsumption of such food in large amount may lead to Vit A deficiency if not substituted may lead to Vit A deficiency if not substituted by other sourceDeficiency may lead to complete blindness and other diseases

Golden rice provide Vit AGolden rice provide Vit A

1999 Swiss scientist Ingo Potrykus & 1999 Swiss scientist Ingo Potrykus & German Peter Beyer succeeded in developing β Carotene producing rice .β Carotene is the precursor of Vit. AThe Genetically modified rice produce βC t i dCarotene in endospermIn 2005 Syngenta laboratory developed rice that contain 20 times more βrice that contain 20 times more βcarotene, sufficent to provide human requirement

The golden riceThe golden rice

How developed?How developed?

Biosynthesis of β caroteneBiosynthesis of β carotene

Geranyl Geranyl Geranyl Geranyl pyrophosphate one of the precursor is present in Rice present in Rice endospermFour enzymes Ph h Phytoene synthase, Phytoene desaturase, Carotene desaturase

d land Lycopene cyclase needed are absent in endosperm cells

Synthetic pathway

Two genes transformationTwo genes transformation

Carotene desaturase from bacteria (Erwinia uredovera) can substitute two enzymes Phytoene desaturase, Carotene desaturaseGene for Phytoene synthase taken from Daffodil Gene for Phytoene synthase taken from Daffodil (Narcissus pseudonarcissus). Lycopene cyclase found in rice endospermThe genes under the control of the endosperm-The genes under the control of the endospermspecific glutelin (Gt1) and the constitutive CaMV 35S promoter, respectivelyThe original golden rice was called SGR1, and under e o g a go de ce as ca ed SG , a d u degreenhouse conditions it produced 1.6 µg/g of carotenoids In 2005, researchers from Syngenta, produced "Golden , y g , pRice 2“ (23 times more carotenoids than golden rice (up to 37 µg/g). using phytoene synthase gene from maize with crt1 from the original golden rice.

Examples of Current Research

• Genetically engineered Arabidopsis plants can sequester arsenic from the soil. (Dhankher et al. 2002 Nature Biotechnology)

• Immunogenicity in human of an edible vaccine forImmunogenicity in human of an edible vaccine for hepatitis B (Thanavala et al., 2005. PNAS)

• Expression of single-chain antibodies in transgenicExpression of single chain antibodies in transgenic plants. (Galeffi et al., 2005 Vaccine)

• Plant based HIV 1 vaccine candidate: Tat• Plant based HIV-1 vaccine candidate: Tat protein produced in spinach. (Karasev et al. 2005 Vaccine)

• Plant-derived vaccines against diarrheal diseases.(Tacket. 2005 Vaccine)

Chl r pl t pr i tChloroplast expression systemLarge copy number: 10,000/cell- high Large copy number: 10,000/cell high expression-as much as 47% of TSPGene silencing is not reportedGe e s e c g s ot epo tedProcessing also of complex proteinExpression of unusual gene possible. TetC Expression of unusual gene possible. TetC with high AT content (73%) problem of expression in E.coliMaternal inheritedDown stream Processing may be easierg y

EconomicsEconomicsco o cs

The expectation is for lower production costs however there is no evidence that pharming will p gproduce cheaper, safe drugs.

Th k t i t d ithThere are unknown costs associated with containment, litigation and liability, production others?production…..others?

E n mi f m l l r f rminEconomics of molecular farmingMost cases uneconomic compared to ost cases u eco o c co pa ed topresent production methodProduction using free solar energy should b h bbe cheaper butHigh cost for extraction of product from plantplantHigh price biopharmaceuticle- can be favorablefavorableCan be Promising in future- improvement in technology

Th t f pr d ti nThe cost of productionCrop selection: High volume/low cost production p g / pcrop like soybeanPlant used continuously in improvement- the l t h ld b ll k i tifi llplant should be well known scientifically

For molecular farming generally outdated cultivar reaches the field 3% penalty (reduction cultivar reaches the field 3% penalty (reduction in yield)Identity preservation 6% costExtraction and processing cost.Petroselenic acid in soybean: Soybean oil cost $ 0 53 kg-1 The cost of PS acid reaches $ 1 98 kg-10.53 kg 1, The cost of PS acid reaches $ 1.98 kg 1

R f rReferencesSlater, Scott and Fowler: Plant Slater, Scott and Fowler: Plant Biotechnology