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Biotechnology Appoach to medicinal plants
Oliver Kayser
University of Groningen
Pharmaceutical Biology
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
02.07.2010 ISBMAP2009, Ljubljana
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)
02.07.2010 ISBMAP2009, Ljubljana
02.07.2010 ISBMAP2009, Ljubljana
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)
02.07.2010 ISBMAP2009, Ljubljana
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
02.07.2010 ISBMAP2009, Ljubljana
Need for artemisinin
02.07.2010 ISBMAP2009, Ljubljana
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
02.07.2010 ISBMAP2009, Ljubljana
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”
02.07.2010 ISBMAP2009, Ljubljana
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
02.07.2010 ISBMAP2009, Ljubljana
What is Synthetic Biology?
• New term introduced beginning 2000
• Using uniform and defined building blocks to construct artificial pathways or organisms
02.07.2010 ISBMAP2009, Ljubljana
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
02.07.2010 ISBMAP2009, Ljubljana
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)
02.07.2010 ISBMAP2009, Ljubljana
Strategies for metabolic engineering
02.07.2010 ISBMAP2009, Ljubljana
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)
02.07.2010 ISBMAP2009, Ljubljana
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
02.07.2010 ISBMAP2009, Ljubljana
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
02.07.2010 ISBMAP2009, Ljubljana
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
02.07.2010 ISBMAP2009, Ljubljana
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
02.07.2010 ISBMAP2009, Ljubljana
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
02.07.2010 ISBMAP2009, Ljubljana
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
ISBMAP2009, Ljubljana
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
02.07.2010 ISBMAP2009, Ljubljana
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
02.07.2010 ISBMAP2009, Ljubljana
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+
02.07.2010 ISBMAP2009, Ljubljana
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
02.07.2010 ISBMAP2009, Ljubljana
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
02.07.2010 ISBMAP2009, Ljubljana
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
02.07.2010 ISBMAP2009, Ljubljana
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
02.07.2010 ISBMAP2009, Ljubljana
Heterologous bioconversion approach
02.07.2010 ISBMAP2009, Ljubljana
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.
02.07.2010 ISBMAP2009, Ljubljana
Deoxypodophyllotoxin concentration in Anthriscus sylvestris
02.07.2010 ISBMAP2009, Ljubljana
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
?
02.07.2010 ISBMAP2009, Ljubljana
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
02.07.2010 ISBMAP2009, Ljubljana
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
02.07.2010 ISBMAP2009, Ljubljana
Mechanistic studies
Binding site of Cytochrome P450 3A4 with superimposed O2 molecule
DOP molecule, the oxidation site (β-hydrogen atom) is colored green
02.07.2010 ISBMAP2009, Ljubljana
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
02.07.2010 ISBMAP2009, Ljubljana
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
02.07.2010 ISBMAP2009, Ljubljana
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
02.07.2010 ISBMAP2009, Ljubljana
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
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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
ISBMAP2009, Ljubljana
02.07.2010 ISBMAP2009, Ljubljana
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
02.07.2010 ISBMAP2009, Ljubljana