Biotechnology Appoach to medicinal plants · “Metabolic Engineering” 02.07.2010 ISBMAP2009,...

Biotechnology Appoach to medicinal plants

Oliver Kayser

University of Groningen

Pharmaceutical Biology

[email protected]

02.07.2010 ISBMAP2009, Ljubljana

Metabolic engineering is possible, but what are the targets?

The six F’s

• Food for Humans

• Feed for Animals

• Fiber

• Fuel

• Feedstocks for the Chemical Industry

• Pharmaceuticals


Metabolic engineering and medicinal plants

Compound Need (to/y) Price US$/kg Cultivation in ha

Artemisinin 120 1000 29,000

Paclitaxel 0.3 28,000 Wild collection(750,000 yew trees/y)

Docetaxol 0.3 24,000 semisynthesis

Resveratrol 10,000 1,200 fermentation, synthesis

Vincristin 0.3 10,000 not known

Genistein 36,000 1400 not known (138 Mil to soybean per year)



What plants are interesting?

• Crop plants– Oryza sativa– Zea maydis– Nicotiana tabacum (Cyp‐450‐Trangenesis)

• Medicinal plants– Papaver somniferum (Knock Out‐Strategy)– Catharanthus roseus – Anthriscus sylvestris (Combinatorial biosynthesis)– Taxus bacata (Synthetic biology)– Artemisia annua (Synthetic biology)


Why is engineering plants important?

Artemisia annua as source for the antimalaria drug artemisinin

1 treatment = 0.5 g artemisinin

1 million treatments needed per year


Need for artemisinin


2003 2004 2005 2006 2007 2008 2009 2010 2011

Pilloy, J. 2008, Ensuring sustainable Artemisinin Production, Oral Presentation, Guilin, China, 24‐26.11.2008

Processes for biotechnological production

• Plant cell cultures

• Transgenic microorganisms

• Transgenic plants or plant cell cultures

• Isolated enzymes

• Regenaration of geneticaly modified plants

• Metabolic engineering

• Synthetic biology02.07.2010 ISBMAP2009, Ljubljana

But:

‐Is the technology feasible?‐Is the economy of the process competitive with existing production methods?

Metabolic Engineering


The application of recombinant DNA methods to restructure metabolic networks can improve production of metabolite and protein products by altering pathway

distributions and rates.

1991, Science

Publications containing“Metabolic Engineering”


Medicinal plants: 97 publications in total

Goals of metabolic engineering

• Increased levels of natural product biosynthesis in plants

• New compounds (biosimilars) for biological activity

• New flower or food colours• New taste and fragrancy of food• Improved nutritional and health promoting effect of food (Nutraceuticals)

• Reducing unwanted compound (toxic, allergic)• Improved resistance against pest and diseases


What is Synthetic Biology?

• New term introduced beginning 2000

• Using uniform and defined building blocks to construct artificial pathways or organisms


Simple Sugar

erg9::PMET3‐ERG9Met

Acetyl‐CoA

Acetoacetyl‐CoA

Mevalonate

Mevalonate‐P

Mevalonate‐PP

HMG‐CoA

IPP

GPP

IDI1

FPP

Squalene

Ergosterol

DMAPP

ERG10

ERG19

ERG13

ERG12

ERG8

ERG1,7,11,24,25,6,2,3,5,4

tHMGR X2

ERG20

ERG20

H

H

O

HO

Artemisinic acid

ADS

Amorphadiene

H

H

H

HHO

H

HHO

HO

H

H

O

HNon‐Enzymatic

AMO/CPR

AMO/CPR

AMO/CPR

H

H

O

HO

Artemisinic acid

SyntheticBiology

Purification

ChemicalConversions

Reduction

DihydroartemisinicAcid

DihydroartemisinicAcid EsterHydroperoxide

DihydroartemisinicAcid Ester

Peroxidation

Oxidation andRing‐Closure

Esterification

Microbially DerivedArtemisinin


Why introducing engineering to breeding ?

• only a few results of breeding research are available ‐e.g. on the genetics of certain traits and on breeding methods,

• MAP comprise a particularly great number of species,• often breeding aims differ on one and other species depending on the field of usage,

• Overcoming bottlenecks in biosynthesis (e.g. Artemsinin, paclitaxel)

• GMO attractive for increased production of single natural products for extraction purpose (e.g. Anthriscus sylvestris, Papaver somniferum)


Strategies for metabolic engineering


S

S1,2,3

S1,3

S3S2S1

S1,2 S2,3 S1,3

S1,2,3

E1E3E2

E5

E13

E7 E8

E10

E11 E10

E12

E11

E12

S1,2 S2,3

E0

S3

RNA

Promoter Gene

Increasing copy number and Over expression of the gene of interestProtein engineering

Block of competing pathways

S2

S1,2

E2

E5

E10

XX

Channeling to compartments

Introduction of anew pathway

E-A

E-C

E-B

S1,2

S2

S

Gene targeting

Strategies for metabolic engineering

• Block a metabolic flux (re‐channel) and channel a metabolic flux into new cell compartments (re‐direction)

• Induce a metabolic flux (can lead to unexpected results)

• Blocking competitive pathways (knock outs)• Overexpression of genes of interest• Increasing copy number• Improving promoter strength• Introduce a new metabolic pathway into organism by heterologous genes (combinatorial biosynthesis)


Agrobacterium tumefaciens

• the species of choice for engineering dicot plants; monocots are generally resistant (but

you can get around this)• some dicots more resistant than others (a

genetic basis for this)• complex bacterium – genome has been

sequenced; 4 chromosomes; ~ 5500 genes

Agrobacterium tumefaciens

Agrobacterium transformation


Ti Plasmid

1. Large (-200-kb)2. Conjugative3. ~10% of plasmid transferred to plant cell

after infection4. Transferred DNA (called T-DNA) integrates

semi-randomly into nuclear DNA 5. Ti plasmid also encodes:

– enzymes involved in opine metabolism– proteins involved in mobilizing T-DNA (Vir

genes)

auxA auxB cyt ocsLB RB

LB, RB – left and right borders (direct repeat)auxA + auxB – enzymes that produce auxincyt – enzyme that produces cytokininOcs – octopine synthase, produces octopine

T-DNA

These genes have typical eukaryotic expression signals!

Vir (virulent) genes

1. On the Ti plasmid2. Transfer the T-DNA to plant cell3. Acetosyringone (AS) (a flavonoid) released by

wounded plant cells activates vir genes.4. virA,B,C,D,E,F,G (7 complementation

groups, but some have multiple ORFs), span about 30 kb of Ti plasmid.

Vir gene functions (cont.)

• virA - transports AS into bacterium, activates virG post-translationally (by phosphoryl.)

• virG - promotes transcription of other vir genes• virD2 - endonuclease/integrase that cuts T-

DNA at the borders but only on one strand; attaches to the 5' end of the SS

• virE2 - binds SS of T-DNA & can form channels in artificial membranes

• virE1 - chaperone for virE2• virD2 & virE2 also have NLSs, gets T-DNA to

the nucleus of plant cell• virB - operon of 11 proteins, gets T-DNA

through bacterial membranes

Binary vector system

Strategy:1. Move T-DNA onto a separate, small plasmid.2. Remove aux and cyt genes.3. Insert selectable marker (kanamycin resistance) gene in

T-DNA. 4. Vir genes are retained on a separate plasmid.5. Put foreign gene between T-DNA borders. 6. Co-transform Agrobacterium with both plasmids.7. Infect plant with the transformed bacteria.

Binary vector system

Crown galls caused by A. tumefaciens on nightshade.

More about Galls: http://waynesword.palomar.edu/pljuly99.htmhttp://kaweahoaks.com/html/galls_ofthe_voaks.html

In summary: How to engineer metabolic pathways?• Downregulation of gene expression

– Anti‐sense expression (Papaver somniferum, morphine; Allen et al, 2004)

• Upregulating of pathways– Ectopic expression of transcription factors (Brown, 2004)

• Post transcriptional gene silencing (Larkin et al., 2007)– Sense suppression

– RNA interference (RNAi)– Virus induced silencing (VIGS)

– Chimeric repressor silencing

– Immunomodulation

• Heterologous multigene expression (Li and Heide, 2006)

– Single transgene (biotransformation) (A.sylvestris, PTOX; Julsing et al., 2006)– Multiple transgene insertions

• Multiple intron‐encoded endonuclease sites

• Polycistronic vectors (artifical chromosomes) (Felipe, de P., 2002)02.07.2010 ISBMAP2009, LjubljanaAllen et al., 2004, Nature Biotech12:1559; Brown, P. 2004. Curr Opin Plant Biol. 7:202; Larkin PJ et al. Plant Biotechnol J. 1:26; Li, SM and

Heide, L. 2006. Planta Med. 12:1093; Julsing, MK et al., 2006. Biomolecular Eng. 6:265‐279; Felipe de, P. 2002. Curr. Gene Ther. 2:355

DNA is bound to the microprojectiles, which impact the tissue or immobilized cells at high speeds.

J. Sanford & T. Klein, 1988

Original biolistic gun. A modified 22 caliber.

An Air Rifle for a DNA Gun –Circa 1990

A.Thompson, Bob ?, and D. Herrin

Repairing an organellar gene: ~ 1 x 107 cells of a mutant of Chlamydomonas that had a deletion in the atpBgene for photosynthesis was bombarded with the intact atpBgene. Then, the cells were transferred to minimal medium so that only photosynthetically competent cells could grow.

Control plate – cells were shot with tungsten particles without DNA

The Helium Gas Gun – Circa 2000

The Hand-Held Gas Gun

Purpose:Introduce DNA into cells that are below the top surface layer of tissues (penetrate into lower layers of a tissue)

One interesting use:Making DNA Vaccines in whole animals.

Hughes EH, Shanks JV Met. Eng. 2002, 4:41‐48

ODC: Ornithine decarboxylaseADC: Arginine decarboxylasePMT: Putrescine N‐Methyl transferaseH6H: Hyoscyamine 6β hydroxylase

02.07.2010

Metabolic Engineering of Nicotine BiosynthesisGlycyrrhiza echinata

Ornithine

Putrescine

N‐Metyl‐Pyrollinium

Tropinone

Tropine

N‐Methyl‐Putrescine

Hyoscyamine

ScopolaminePhenylalanine

Nicotine

Nicotinic acid

Spermidine

Arginine

AgmantineODC, 10xS. cereviseae

10‐20x

ADC, 10‐20x

PMT

PMT, 10x

H6H

N

O

O

O

H OH

N

O

O

H OH

N

OH

N

O

NH2 OH

O

NH2

OH

O

NH2

NH2 NH2

NH

NH2 NH2 NH2

NH

NH2

N

N

ISBMAP2009, Ljubljana

Metabolic engineering of scopolamine


Hyoscyamine Scopolamine

Cloning of H6H from Hyoscyamus niger to Atropa belladonna Complete bioconversion from Hyoscyamine to Scopolamine

Hashimoto et al, 1993, Phytochemsitry 32:713-718

N

O

O

O

H OH

N

O

O

H OH

H6H

H6H: Hyoscyamine 6β hydroxylase

Problems with metabolic engineering of plants

• Developmental stage of plant (Atropa)

• Unexplored regulation of secondary metabolism (Atropa, Nicotiana)

• Pathways often specied‐specific (limited help of A. thaliana as model organism)

• Enviromental influence (Atropa, Nicotina)

• Cell compartmentation (Nicotiana)

• Tissue differentiation

02.07.2010 ISBMAP2009, LjubljanaHughes EH, Shanks JV. 2002. Met. Eng. 4:41‐48

Pathway Architecture


3 different functional groups (S1,2,3)

Low substrate specificity: 3 enzymesHigh substrate specificity : 12 enzymes

S

S1,3

S3S2S1

S1,2 S1,2 S2,3 S1,3 S2,3

S1,2,3

E1 E3E2

E4 E5 E6 E7 E8 E9

E10

E11E10 E12

E11

E12

Verpoorte R, Memelink J. 2002. Curr Opin Biotechnol 2002, 13:181

Pathway Architecture


3 different functional groups (S1,2,3)

Low substrate specificity: 3 enzymesHigh substrate specificity : 12 enzymes

S

S1,3

S3S2S1

S1,2 S1,2 S2,3 S1,3 S2,3

S1,2,3

E1 E3E2

E4 E5 E6 E7 E8 E9

E10

E11E10 E12

E11

E12

Verpoorte R, Memelink J. 2002. Curr Opin Biotechnol 2002, 13:181

Engineering the morphine pathway


Allen RS et al. Nat. Biotechnol. 22 (2004), 1559–1566

COR: codeinone reductase

02.07.2010

Metabolic engineering of the morphine biosynthesis

Glycyrrhiza echinata

2x Tyrosine

Norcoclaurine

Thebaine

Codeine

R‐Reticuline

Morphine

EtorphineBuprenorphine

Naloxon

Papaverine

Synthesis

Noscapine

NH2

O

OH

OH

NHOH

OH

OH

H

NCH3

OH

OH

H3CO

H3CO

H

O

H

H3CO

H3CO Codeinone

Morphinone

ON

H

H3CO

O

O

NH

OH

O

ON

H

OH

OH

ON

H

H3CO

OH

Synthesis

3 steps

4 steps

2-3 steps

X COR


Hairpin RNA mediated Gene Silencing

• Transformation of plant with hairpin RNA consisting of an inverted repeat

Allen RS, Nature Biotechnology 2004, 22: 1559 - 1566

T-DNA from the transformation vector COR 1.1/2 hpRNA designed to produce hpRNA and initiate silencing of all members of the Cor family in P. somniferum

COR 1.1 cDNA fragment

COR 2 cDNA fragment

RB S4S4 Pr

PdK intron

COR 1.1 cDNA fragmentCOR 2 cDNA fragment

Me13´term

35SPr

cat-1 intronNpt II

35Sterm

LB


Other applications of RNA interference

• Gossypol reduction incotton seeds by blocking δ‐cadinene synthase*

• Linamarin reduction inCassavy plants

• Reduction of allergens in tomatoes

*Sunilkumar G, Proc Natl Acad Sci USA 2006, 48: 18054–9.

OH

OH

OH

OH

OOH

OHO


17.03.2009

Metabolic channeling in Sorghum bicolorGlycyrrhiza echinata

Tyrosine

p‐Hydroxymandelonitrile

Dhunin

2‐p‐Hydroxy‐phenylacetaldoxime

Cyp79A1

Cyp71E1

UGT85B1

NH2

O

OH

OH

NH

O

OH

OHOH

N

O

OH

OHOHOH

N

O

OH

OHOH

N

O

OH

OHOH

OH

CN

OH

CN

OH

OH

CN

OGlc

O2 + NADPH

O2 + NADPH

O2 + NADPH

NADP+

NADP+

NADP+


Muñoz-Bertomeu et al. 2009, Metabol. Eng. 10:166-177

Acetyl‐CoA

Acetoacetyl‐CoA

Mevalonate

Mevalonate‐P

Mevalonate‐PP

HMG‐CoA

IPP

GPP

FPP

DMAPP

Expression of spearmint limone synthase in lavender

Plastid

GPP

LDP

α‐terpinyl cation


OH

OH

O

O

OH

SQS

STSSterols

Sesquiterpenoids

H

trans caryophyllene

IPP DMAPP

GPP

myrcen

linalool

α‐pinene

borneol

camphor

limone

α‐terpineol

α‐terpineolSTS: Sesquiterpene synthaseSQS: Squalene synthaseLDP: Linalyl diphosphateMTS: Monoterpene Synthase

LS450%

LS: Limonene synthase from M. spicata

RNA gel blot of LS transcript in leaves (b) and

flowers (c) of transgenic liines

Liu, R., Hu, Y., Li, J., Lin, Z. 2007, Metabol. Eng. 9:17

Genistein production in transgenic tobacco, lettuce, and petunia


NH2

O

OH

O

OH

O

SACo

RO

OH

R

OH

OH

FSGenistein

Dihydroflavonol

Flavone

CHS

C4H4CL

PAL

Naringeninchalconep-Coumaroyl-CoA

Cinnamate

Phenylalanine

IFS

F3Htobacco

Soybean

Tnos barLB LacZ P35S RBpCAMBIA3300

P3300-IFS

P3300-IFS/F3HR

OH

OH

OH

O

OH

O

OOH

OH

OH

O

OOH

OH

OH

OR

IFS: Isoflavone synthaseF3H: Flavanone-3-hydroxylase

Genistein production in transgenic tobacco, lettuce, and petunia

02.07.2010 ISBMAP2009, LjubljanaLiu, R., Hu, Y., Li, J., Lin, Z. 2007, Metabol. Eng. 9:17

Transgenic line Tobacco Petunia Lettuce

Lines analysed 8 8 8

Average 0.9 ±0.7 ng/ml FW 0.4 ±0.2 ng/ml FW 0.9 ±0.4 ng/ml FW

Highest 2.3 ng/ml FW 0.9 ng/ml FW 1.4 ng/ml FW

PCR: HPLC:

Example for heterologous gene expression: Podophyllotoxin biosynthesis

• Lignan

• antineoplastic drug

• precursor for semi synthesis of etoposide und tenoposide

• isolated from P. hexandrum, P. peltatum and others

H3CO OCH3

OCH3

O

O

OH

O

O

Podophyllum hexandrum, Berberidaceae


Production of Podophyllotoxin

H3CO OCH3

OCH3

O

OO

O

Deoxypodophyllotoxin (DOP)

• Organic synthesis– expensive, not economic

• Plant cell cultures– With Podophyllum und Linum Spezies – expensive– Low yield

• Bioconversion– DOP as precursor required


Heterologous bioconversion approach


H3CO OCH3

OCH3

O

OO

O

H3CO OCH3

OCH3

O

O

OH

O

O

Deoxypodophyllotoxin

Plasmid

P450 3A4

A. sylvestrisGene

Expression

Podophyllotoxin

Anthriscus sylvestris L.


Deoxypodophyllotoxin concentration in Anthriscus sylvestris


Bioconversion of recombinant human Cytochrome P450 3A4 in A. sylvestris

• Metabolisation of drugs

• All genes are known and sequenced

• Biochemistry of enzymes widely known

H3CO OCH3

OCH3

O

OO

O

H3CO OCH3

OCH3

O

O

OH

O

O

Deoxypodophyllotoxin Podophyllotoxin02.07.2010 ISBMAP2009, Ljubljana

Expression von Cyt P 450 3A4

1A1 1A2 2C9 2D6 2E9 3A4

M C 2C9 3A41A2M C 2C9 3A41A2

reductase

CYP450

Spectrophotometrical determination (pH 7,4, PBS, CO‐saturation)

400 450 500 550 600

nm

A

Ori

2C9

3A4

1A2

Identity(restriction analysis, EcoRI)

Kayser et al. (2006) J. Biotech. 02.07.2010 ISBMAP2009, Ljubljana

Bioconversion of DOP

Ori

3A4

?


HPLC‐MS‐ Identification

H3CO OCH3

OCH3

O

O

OH

O

O

HPLC‐MS, RP‐18, H2O:MeOH (90:90 – 90:90, 40 min),2 sec/scan

TIC of +Q1: from Sample 3 (9 DOP 2nd) of 050524Vasilev-1.wiff (Turbo Spray) Max. 5.3e7 cps.

2 4 6 8 10 12 14 16 18 20 22 24 26 28Time, min

0.0

5.0e6

1.0e7

1.5e7

2.0e7

2.5e7

3.0e7

3.5e7

4.0e7

4.5e7

5.0e7

5.3e7

Inte

nsity

, cps

19.6

2.95.8 25.33.8

2.218.7 21.1

28.015.113.713.3 17.212.3

TIC of +Q1: from Sample 2 (8 DOP) of 050524Vasilev-1.wiff (Turbo Spray) Max. 5.3e7 cps.

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30Time, min

0.0

5.0e6

1.0e7

1.5e7

2.0e7

2.5e7

3.0e7

3.5e7

4.0e7

4.5e7

5.0e7

5.3e7

Inte

nsity

, cps

19.7

5.7

2.72.1

25.2

15.14.0

18.8 29.913.8 21.0

17.3 24.77.7

m/z 414m/z 398

H3CO OHOH

O

OO

O

H3CO OCH3

OCH3

O

OO

O

OH

+Q1: 13.172 to 13.406 min from Sample 3 (9 DOP 2nd) of 050524Vasilev-1.wiff (Turbo Spray) Max. 1.0e6 cps.

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200m/z, amu

5.00e4

1.00e5

1.50e5

2.00e5

2.50e5

3.00e5

3.50e5

4.00e5

4.50e5

5.00e5

5.50e5

6.00e5

6.50e5

7.00e5

7.50e5

8.00e5

8.50e5

9.00e5

9.50e5

1.00e6

Inte

nsity

, cps

432

279

332

388315

415234 296 371 453163 327185136

124 199 270 408174 399223 300 437159 320 536 846611460 558 685 746624521 867780711 806 934 1079987 11421053 1107 1174974


LC‐SPE‐NMR of unknown compound at Rt 13.3 min

(A)

H-6

H-3

H-2’,

H-6

’

H-7 H-

7’ H-9a

H-9b

H-8’

H-8

ppm 7 6 5 4 3

H3CO OCH3

OCH3

O

OO

O

OHH

Epipodophyllotoxine


Mechanistic studies

Binding site of Cytochrome P450 3A4 with superimposed O2 molecule

DOP molecule, the oxidation site (β-hydrogen atom) is colored green


Stereoselective bioconversion

Stereoselectivity of the solutions towards β-hydrogen atom (green color)at C-7 position of DOP compared to α-hydrogen atom (red color) 02.07.2010 ISBMAP2009, Ljubljana

PTOX production in transgenic N. tabacum and A. sylvestris


N. tabacum

A. sylvestris Callus culture

Callus culture

Plant regeneration

Cyp450 3A42000 kb

Proof oftransformation

Proof oftransformation

Cyp450 3A42000 kb

?

☺

In cooperation with H. Warzecha, TU Darmstadt, Germany

LB D35S P450 NOS CaMV NPTII NOS RB

Detection in callus culture

Detection of PTOX in Anthriscus sylvestris HPLC-UV

HPLC-MS/MS

Callus culture LeavesTransgenic line A. sylvestris, callus A. sylvestris, leaves

Lines analysed 5 3

Average 1.2 ±0.4 ng/ml FW 0.2 ±0.3 ng/ml FW

Highest 1.5 ng/ml FW 0.4 ng/ml FW

PTOX

PTOX

DOPDOP


Vaccine plants

• Pioneered by Charlie Arntzen • cheap vaccine-delivery system • use plants producing a pathogen protein (or DNA) to

induce immunity• potatoes, bananas• being developed for a number of human and animal

diseases, including measles, cholera, foot and mouth disease, and hepatitis B and C.

• Four plant vaccines were successful in phase I clinical trials.

C.J. Arntzen et al. (2005) Plant-derived Vaccines and Antibodies: Potential and Limitations. Vaccine 23, 1753-1756.

Antibodies: a compelling success story

high specificity: in vitro and in vivo diagnostics

low toxicity: therapeutic applications

high drug approval rates (24 approved mAbs)

major products in biotechnology (~240 in clinical trials)

inherently stable human proteins

injectable, topical and oral applications

applicable for chronic conditions

potential long-lasting benefits

Production Costs for AntibodiesProduction costs cost in $ /gram

hybridomas 1000transgenic animals 100transgenic plants 10

Source: Daniell et al. (2001) TIPS 6, 219-226

E. coli & yeast Tr. animals andanimal cells

Transgenicplants

Comparison of Mammalian and Plant‐produced Antibodies

peptide sequence: identical

correct cleavage of Ig‐derived signal peptides

kinetics & affinity: identical

stability in seeds > 30 months

antibody types: plant system more versatile (sIgA)

post‐translational processing: different

core glycan identical, terminal sugar different plus xylose & fucose

antigenicity & clearance: apparently identical (shorter half‐life)

Vaccines

• Hepatitis B virus surface antigen HBsAg

• Norwalk virus capsid

• Vibrio cholerae enterotoxin subunit

• Animal vaccines– Mink enteritis virus ‐MEV

– Rabbit haemorrhagic disease virus ‐ RHDV

– Foot and mouth disease virus ‐ FMDV

Engineering Edible Vaccines

Targeting strategy:

• attaching antigens to cells that bind with M cells in intestinal lining

• M cells take in materials that enter intestines and pass them down to other cells like antigen‐presenting cells.

• Macrophages degrade proteins/antigens into fragments and display them on the cell surface.

• When T lymphocytes recognize the foreign fragments they trigger the release of antibodies and help in bigger attack on the cells

Oral vaccines

• First thought edible vaccines good idea– Banana a day

• Oral vaccination– Might elicit antigenic tolerance

– ProdiGene ‐ Got contamination of maize and soybean harvest in 2002 with transmissible gastroenteritis virus TGEV

Past and Future of PMPs

Plant produced Vaccines

• HPV L1 – forms VLPs – can protect • Measles virus haemagglutinin• HBsAg• Porcine TGEV• Tetanus toxin• Vacinia virus B5 • Allergy vaccines• HPV• HIV gp41• Newcastle disease virus• Rotavirus VP7

Each Plant has pros and cons

• Potatoes: good because can be stored for long periods without being refrigerated

• Disadv.: needs to be cooked in order to eat and heat can denature proteins

• Most beneficial because some potatoes are eaten raw in some countries and heat does not completely destroy protein

Each plant has pros and cons

• Bananas: no cooking required and grow in most countries

• Disadv.: banana trees take years to mature and spoils rapidly

• Tomatoes: grow fast

• Disadv.: spoil rapidly

Edible Vaccines and Pregnant Women

• Can a vaccine taken by a pregnant women also vaccinate the unborn child?

• Vaccine and antibodies can be transferred from mother to child by milk or through the placenta

PMP Development ‐ Highlights

Series of plant-derived vaccines from Arizona State University have completed clinical trials

Prodigene has trialled two plant-derived vaccines

LSBC pipeline of cancer vaccines prior to insolvency

Guardian Bioscience coccidiosis vaccine, CFIA phase II ongoing

Fraunhofer CMB, rabies vaccine trialled in humans

DowAgro Newcastle disease vaccine, approved Feb 2006

Heberbiovac (Cuba) approved antibody for HepB vaccine purification

Current Challenges in Molecular Farming

yield of recombinant proteins

real quantitative comparison (TSP vs pg/cell/24h)

protein stability (proteases)

post-translational modification(s)

backcrossing in elite lines

extraction and downstream processing

QA, QC and substantial equivalence

clinical trials & regulatory approval

Regulatory Challenges for PMP

loci of transgene insertion

expression properties and levels, including PTM

effects of the transgene on the expression of flanking endogenous genes

master line banking to ensure product consistency

contamination with animal excreta, pesticides, organic fertiliser

procedures for detection and removal of weeds and pests

cultivation variables

Roadmap Plants for the Future

1997 2005 2015 2025

Efficient agriculture- Bt technology- Herbicide

resistance

Health food and quality- Amino acids- Oil- Starch

Plant protection- Viruses- Nematodes- Fungi- Insects

Plant production platforms- Vitamines- Fatty acids- Enzymes- Bio-polymers- Pigments- Pharmaceutical products- Fibers

Stress resistance- Cold- Drought- Salinization

Concerns that have been raised about cultivating and consuming GM crops

1. They may be toxic or allergenic.2. They may become established in the wild

and outcompete other plants.3. They may negatively affect insects or other

organisms that use crops. 4. They may outcross to a nearby wild relative

spreading the transgene into a wild population.

References on release of GM crops into the environment

• Nap et al. (2003) Plant Journal 33, 1-18– Focuses on current status and regulations

• Conner et al. (2003) Plant Journal 33, 19-46– Focuses on ecological risk assessment

Synthetic biology

• Artemisinic acid

• Flavonoids (Kaempferol)

• Resveratrol

• Curcuminoids

• Stilbenoids

• Vanillin


O

OO

O

O

H3C

CH3

CH3

H H

H

OH

OH

OH

The artemisinin story

02.07.2010 ISBMAP2009, LjubljanaMartin VJJ et al. Nature Biotechnology 2003, 21:796 – 802

Facts about Artemisia annua and Artemisinin1.5 to A. annua to per ha4.5 kg artemisinin per ha0.4‐1% average artemisinin content in leaves60% average at extraction50% extraction‐purification process efficacy

2$ for single treatment1‐1.5$ costs of arteminin

Simple Sugar

erg9::PMET3‐ERG9Met

Acetyl‐CoA

Acetoacetyl‐CoA

Mevalonate

Mevalonate‐P

Mevalonate‐PP

HMG‐CoA

IPP

GPP

IDI1

FPP

Squalene

Ergosterol

DMAPP

ERG10

ERG19

ERG13

ERG12

ERG8

ERG1,7,11,24,25,6,2,3,5,4

tHMGR X2

ERG20

ERG20

H

H

O

HO

Artemisinic acid

ADS

Amorphadiene

H

H

H

HHO

H

HHO

HO

H

H

O

HNon‐Enzymatic

AMO/CPR

AMO/CPR

AMO/CPR

H

H

O

HO

Artemisinic acid

SyntheticBiology

Purification

ChemicalConversions

Reduction

DihydroartemisinicAcid

DihydroartemisinicAcid EsterHydroperoxide

DihydroartemisinicAcid Ester

Peroxidation

Oxidation andRing‐Closure

Esterification

Microbially DerivedArtemisinin


02.07.2010ISBMAP2009, Ljubljana

SyntheticBiology

Recombinant FlavonoidsSimple Sugar

NH2

O

OHNH2

O

OH

OHPAL

Rhodotorula rubra

O

OH

O

OH

OH

4CLS. coelicor

O

SACo

R

O

OH

R

OH

OH

E. coli

Phenylalanine Tyrosine

Coumaric acid p‐Coumaric acid

C4H

Glycyrrhiza echinata

CHSGlycyrrhiza echinata

Naringenin (R=OH); Pirecimbrin (R=H)

Apigenin (R=OH); (R=H))

OOH

OH

OR

O

Forkmann G, Martens S:. 2001. Curr Opin Biotechnol, 12:155

PAL: Phenylalanine ammonia lyaseC4H: Cinnamate 4-hydroxylase4CL: 4-Coumarate:CoA ligaseCHS: Chalkone synthase

Horinouchi S 2009 Curr Opin Chem Biol 13:197‐20402.07.2010 ISBMAP2009, Ljubljana

02.07.2010

Recombinant ResveratrolGlycyrrhiza echinata

Horinouchi S 2009 Curr Opin Chem Biol 13:197‐204

PAL: Phenylalanine ammonia lyaseC4H: Cinnamate 4-hydroxylase4CL: 4-Coumarate:CoA ligaseCHS: Chalkone synthase



Understanding the target pathway• Are all pathway intermediates and enzymes/genes known?• Are precursors for an introduced pathway present and biochemically available ?• Which genes encoding the pathway enzymes should be selected in the case of multigene families?

• Are related competing pathways understood, and their cloned genes available?• Can spillover pathways be predicted?• What is the tissue or cell‐specificity of the pathway, and are suitable tissue‐specific promoters available?

• What are the inter‐ and intra‐cellular transport mechanisms for intermediates and end‐products of the pathway?

• What are the transcriptional regulators of the pathway, and their targets?• Are sites of flux control understood?

Tools for pathway manipulation• Flux‐determining pathway gene(s) or transcription factor(s).• Stable or transient expression systems (e.g. Agrobacterium mediated transformation, VIGS, etc)

• Selected method for gene downregulation (antisense, RNAi, etc)• Constitutive tissue‐ or cell‐specific promoters• Vector(s) and delivery systems for multiple genes (e.g. co‐transformation, crossing of individual transgenics, generation of self‐cleavable polyproteins).

Analysis of transgenic plants• Copy number of transgene insertions• Transcript profiling (specific [e.g. RT‐PCR, gel blot analysis], or global [e.g. microarray]).

• Trait performance (the targeted trait or full agronomicevaluation, including disease and pest pressure)?

• Metabolite profiling (targeted or global).

What can be learned when the predicted result is not obtained?• Technical problems (the transgene was not expressed, its end product or a pathway intermediate is toxic or degraded, etc)

• Substrate(s) not available to the introduced pathway (wrong cell types, metabolic channeling)

• Competing pathways siphon off intermediates• The pathway as understood from the literature is incorrect • The regulatory architecture of the pathway is not fully understood

Dixon, RA, Current Opinion in Plant Biology 2005, 8:329–336

Conclusions• Metabolic engineering of medicinal plants is / will be a future technology

• Engineering of medicinal plants will be a key technology for purified natural products

• Synthetic biology is still in its infancy

• Regulation on EU and national level is strict

• Do the consumer accept genetic modified medicinal plants

• Most approaches are economically not feasible today


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Biotechnology Appoach to medicinal plants · “Metabolic Engineering” 02.07.2010 ISBMAP2009,...

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