METABOLIC ENGINEERING:
Microorganisms as tools in chemistry
Taz Cheema
January 27, 2011
http://scienceblogs.com/oscillator/2010/09/bacterial_lightbulb.php
1
WHAT IS METABOLIC
ENGINEERING?
Dr. James E. Bailey
1944-2001
Metabolic engineering is
the improvement of
cellular activities by
manipulation of enzymatic,
transport, and regulatory
functions of the cell with
the use of recombinant
DNA technology.
2
Bailey, J., Toward a science of metabolic engineering, Science, 252, 1668, 1991
WHAT IS METABOLIC ENGINEERING?
Compound
Enzyme
http://www.google.com/images
http://blogs.nature.com/nm/spoonful/2008/03/
3
ENGINEERING BIOSYNTHETIC ENZYMES
Sorangium cellulosum
Gene cluster
Steptomyces coelicolor
• Engineer entire biosynthetic cluster into an organism more suitable to laboratory
manipulation
• Done only if: 1) natural product expression low or 2) natural product difficult to culture
and ferment
• Engineering biosynthetic enzymes in an organism more desirable than to manipulate
each producer organism individually
1. Science, 2000, 287, 640-642
2. Org.Biomol.Chem.. 2003, 1, 1-4
4
EPOTHILONE
• New chemotherapeutic agent
• Arrests cell cycle by binding to microtubules and inhibits depolymerisation
• Active in Taxol resistant tumours and multidrug resistant cancer types
• Currently analogues are in clinical trials
5
METABOLIC PATHWAY
A
• Introduce new
pathways using
rDNA
Enzyme 1
B
Enzyme 2
Krebs cycle
• Modify existing
pathways
•Enzymes
expressed that
define overall
conversion process
C
Enzyme 3
D
NEW
E
1.Stephanopoulos, G (1999) Metabolic Engineering 1:1-11
2.Genetic and metabolic engineering. EJB(1998), Vol.1 No.3
6
SORBITOL PRODUCTION
LDH
Lactobacillus plantarum (lactic acid bacterium)
1. Hols, P., Applied and Environmental microbiology 2007, 73, 6.. 1864–1872
2. Stephanopoulos,G.; Chemical Engineering Science 2002, 57, 2596-2602
7
SORBITOL PRODUCTION
LDH
• Sorbitol production in Lactobacillus plantarum (lactic acid bacterium)
• Enzymes expressed that define overall conversion process along with totality of cellular
functions
1. Hols, P., Applied and Environmental microbiology 2007, 73, 6.. 1864–1872
2. Stephanopoulos,G.; Chemical Engineering Science 2002, 57, 2596-2602
8
GENES AND THEIR ENZYMES
Gene 2
Gene 3
Gene 4 Gene 5 Gene 6
ENZYME 2
ENZYME 1
ENZYME 1
X
ENZYME 6
ENZYME 4
ENZYME 3
DNA
Gene 1
ENZYME 5
Wild Type
Stephanopoulos,G.; Chemical Engineering Science 2002, 57, 2596-2602
Y
9
GENES AND THEIR ENZYMES
Gene 2
Gene 3
Gene 4 Gene 5 Gene 6
ENZYME 2
ENZYME 1
ENZYME 1
X
ENZYME 6
ENZYME 4
ENZYME 3
DNA
Gene 1
ENZYME 5
Y
10
Chemical Engineering Science 2002, 57, 2596-2602
GENES AND THEIR ENZYMES
Gene 2
Gene 3
Gene 4 Gene 5 Gene 6 Gene 7
ENZYME 2
ENZYME 1
ENZYME 1
X
ENZYME 6
ENZYME 4
ENZYME 3
DNA
Gene 1
ENZYME 5
Y
11
Stephanopoulos,G.; Chemical Engineering Science 2002, 57, 2596-2602
BIO SYNTHESIS OF BUTANETRIOL
Microbe B
gene 
Microbe A
gene 
Microbe C
gene 
D-xylose
Microbe A
D-1,2,4-butanetriol

L-arabinose
L-1,2,4-butanetriol
12
Frost, J. et al; J. Am. Chem. Soc. 2003, 125, 12998
WHY BUTANETRIOL?
H2SO4 , HNO3
D,L-1,2,4-butanetriol
D,L-1,2,4-butanetriol trinitrate
• Less shock sensitive
D,L-1,2,3-Trinitroxy propane
• More thermally stable
• Less volatile than nitro-glycerine
• Limited availability of 1,2,4-butanetriol
http://www.allbestwallpapers.com/military_wallpapers.html
13
Frost, J. et al; J. Am. Chem. Soc. 2003, 125, 12998
CURRENT SYNTHESIS OF BUTANETRIOL
H+, MeOH
Malic acid
Dimethyl malate
Metal cat. (Ru)
H2O
5000 psi H2
160 oC
NaBH4
MeOH/ THF
8%
+ NaB(OCH3)4
2.5 %
74 %
D,L-1,2,4-butanetriol
1 ton
11 %
2.5 %
6 tons
2%
14
Frost, J. et al; J. Am. Chem. Soc. 2003, 125, 12998
BIO SYNTHESIS OF BUTANETRIOL
Pseudomonas fragi ATCC 4973
a
D-Xylose (100g/L)
D-Xylonic acid (77g/L)
a. D-xylonate dehydrogenase (P. fragi)
70%
c
Pseudomonas putida
3-deoxy-D-glyceropentulosonic acid
3,4-dihydroxybutanal
c. benzoylformate decarboxylase (P. putida)
15
Frost, J. et al; J. Am. Chem. Soc. 2003, 125, 12998
BIO SYNTHESIS OF BUTANETRIOL
Microbe A
gene 
Pseudomonas fragi
Pseudomonas putida
D-xylose
D-1,2,4-butanetriol
L-arabinose
E. coli DH5α/pWN6.186A
L-1,2,4-butanetriol
16
Frost, J. et al; J. Am. Chem. Soc. 2003, 125, 12998
BIO SYNTHESIS OF BUTANETRIOL
E. coli DH5α/pWN6.186A
a
D-Xylonic acid
D-Xylose
100 g/L
c
d
D-1,2,4-butanetriol
12 g/L
b
3,4-dihydroxybutanal
( 25%)
a. D-xylonate dehydrogenase (P. fragi);
D-3-deoxy-glyceropentulosonic acid
b. D-xylonate dehydratase (E. coli);
c. benzoylformate decarboxylase (P. putida); d. alcohol dehydrogenase (E. coli)
17
Frost, J. et al; J. Am. Chem. Soc. 2003, 125, 12998
RECOMBINANT DNA
Plasmid DNA
DNA to be
inserted
Sticky Ends
Recombinant DNA
Sticky Ends
DNA ligase
• Isolate plasmid DNA
• Select DNA
• Cut with restriction enzymes
• Piece together fragments
1. Pray, L.(2008) Recomninant DNA technology and transgenic animals. Nature Education 1 (1)
2. http://www.istockphoto.com/stock-photo-1626198-bacteria.php
18
RECOMBINANT DNA
Metabolite from
fermentation
Copies of protein
isolated
• Transform DNA into cell of interest
• Cells grow and divide producing copies
• Isolate protein/metabolite of interest
• Isolate copies of rDNA
Copies of gene
isolated and
transferred to other
organism
1. Pray, L.(2008) Recomninant DNA technology and transgenic animals. Nature Education 1 (1)
2. http://www.istockphoto.com/stock-photo-1626198-bacteria.php
19
CELLULAR METABOLISM IS COMPLEX
Cofactors, Vitamins
and other substances
Complex
Carbohydrates
• Metabolic network describes which
products are made from which
substrates
Nucleotide
Complex Lipids
• Highly interconnected pathways
• Alterations in one path have
direct/indirect affects on other
pathways
Carbohydrates
Other amino
acids
Lipids
• Pathways analysed through
Metabolic Flux Analysis (MFA)
Amino acids
Energy
2.
1. Lee et al.; Tissue Engineering 1999, 5, 347
http://microbialgenomics.energy.gov/MicrobialCellProject/thrusts2.shtml
20
METABOLIC FLUX ANALYSIS (MFA)
Substrate
Flow (Flux))
Product
21
http://www.google.com/images
METABOLIC FLUX ANALYSIS (MFA)
Substrate
Product
Flow
Mr. Wong
22
http://www.google.com/images
METABOLIC FLUX ANALYSIS (MFA)
exchange flux
Internal flux
Internal metabolite concentration
Substrates /products
• Genomic data not sufficient to describe flux distribution
• Exchange flux easy to determine (uptake vs secretion)
• Internal fluxes hardest to determine
2.
1. Lee et al.; Tissue Engineering 1999, 5, 347
Stephanopoulos,G.; Chemical Engineering Science 2002, 57, 2596-2602
3. Tanis, J.; Metabolic engineering flux balance analysis, 2006
23
METABOLIC CELL FACTORY
D,L-1,2,4-butanetriol (12g/L)
Frost, J. et al; J. Am. Chem. Soc.
2003, 125, 12998
Indigo
J.Indu. Microbiology &
Biotchnology 2002,28,127
Riboflavin (B2)
Appl.Micro.Bio.2003,53,509
Artemisinin
ACS.Chem.Bio, 2007,3, 64
Polyhydroxyalkanoates (PHAs)
Microbiology and molecular bio. 1999, 61, 21
http://pi.lilly.com/us/humulin-n-ppi.pdf
Morphine
Nature Chem. Bio. 2010, 6,251
Codeine
Curr.Opion.Plant Bio. 2005, 8, 280
24
METABOLIC CELL FACTORY
Atrazine
Gallivan,P.; Nature Chem.Bio, 2010, 6, 464
Algae Biofuel
J.R.Soc.Interface, 2010, 7, 703
Doxorubicin
J.Bacteriol. 1999 Lomovskaya
et.al. 181, 1, 305
Bioluminescent E.coli
http://2010.igem.org/Team:Cambridge
Bioremediation of Oil spills
Technology in Society 2010, 32, 331
Taxol
MacMillan,D.;Synlett 2007, 10, 1477
25
METABOLIC CELL FACTORY
Oleic acid (Canola/Soybean oil)
Plant Physiology, 2001, 125, 160
Priming crops with transgenic plants
Curr. Opin. Biotech, 2008,19, 181
Vitamin A
Science, 2000, 287, 303
Vitamin E
Plant Cell, 2003, 15, 3007
Insect resistant Plants
Plant Physiology, 2008, 146, 881
Dhurrin (cyanogenic glucoside)
26
PNAS, 2005, 102, 1779
METABOLIC CELL FACTORY
Taxol
MacMillan,D.;Synlett 2007, 10, 1477
Priming crops with transgenic plants
Curr. Opin. Biotech, 2008,19, 181
Vitamin A
Science, 2000, 287, 303
27
TAXOL
• Potent anticancer agent
• Isolated in 1966 from bark of Pacific yew tree
• Binds to microtubules and inhibits depolymerisation into
tubulin
• 10 kg of dried bark = 1 g of Taxol
• Major supply crisis
• Challenging chemical synthesis
• 10-deacetylbaccatin III (10-DAB) (Leaves of European
yew) 1g / 1kg
Pacific yew tree
1. MacMillan,D.;Synlett 2007, 10, 1477
2. Nature 1979, 277, 665
3. http://itech.dickinson.edu/chemistry/?tag=michael-dalton
28
TAXOL VIA SYNTHETIC CHEMISTRY
51 chemical
steps
37 chemical
steps
Nicolaou (1994)
Isopyromucic acid
Wender (1997)
Verbenone
41 chemical
steps
47chemical
steps
Holton (1994)
Kuwajima (1998)
Propionaldehyde
Patchoulene oxide
47 chemical
steps
Danishefsky
(1995)
Wieland Miescher ketone
38 chemical
steps
Serine
Mukaiuama
(1999)
• 6 independent total synthesis of Taxol achieved
• Wender’s synthesis shortest and most efficient;
overall yield 0.4%
29
MacMillan,D.; Synlett 2007, 10, 1477
TAXOL VIA SYNTHETIC CHEMISTRY
KOtBu
Verbenone
O3
Prenyl
Bromide
79%
85%
h (450 W)
85%
500 g
1.LDA/
Ethyl propiolate
1.Me2CuLi
2. TMSCl
89%
2. AcOH
97%
95%
LiAlH4
KHMDS
N-(phenylsulfonyl)phenyloxazirdine
Wender,P.; J. Am. Chem. Soc. 1997, 119, 2755
30
TAXOL VIA SYNTHETIC CHEMISTRY
1.TBSCl
1.m-CPBA
1. O2, P(OEt)3
KOtBu
2.
2. DABCO
3.TIPSCl
85%
2. NaBH4
91%
PPTS
91%
1. H2,
Crabtree’s cat.
2. TMSCl
3. Triphosgene
98%
PCC
• 14 synthetic steps (AB Ring)
• All C, O in correct pattern
31
Wender,P.; J. Am. Chem. Soc. 1997, 119, 2755
TAXOL VIA SYNTHETIC CHEMISTRY
KHMDS
1N HCl, NaI
94%
91%
TESCl
92%
1.
ZnCl2 89%
2. BOMCl
3. NH4F; PhLi 93%
4. Ac2O 79%
1.
2.
, Et3N
Eschenmoser’s salt
97%
1.
80%
2. O3
86%
32
Wender,P.; J. Am. Chem. Soc. 1997, 119, 2755
TAXOL VIA SYNTHETIC CHEMISTRY
1.
1.HCl, NaI
2. MsCl
3. LiBr
72% (11:1)
2. TrocCl
67%
1.OsO4
2.Triphosgene
3. KCN
92%
1.∆
2. Ac2O
1.TASF
2. PhLi
89%
10-deacetylbaccatin III (46%)
+
Tetrahedron 1992, 48, 6985
Baccatin III (33%)
Taxol - 37 total steps(0.4%)
33
Wender,P.; J. Am. Chem. Soc. 1997, 119, 2755
TAXOL VIA SYNTHETIC CHEMISTRY
20 synthetic steps
14 synthetic steps
Aldehyde (A-B ring)
Baccatin III
3 steps
• 500 g Verbenone starting material used
• Overall yield of 0.4% (2 g)
• “Stop and go” synthesis not practical
for industrial scale
Taxol - 37 total steps
34
Wender,P.; J. Am. Chem. Soc. 1997, 119, 2755
TAXOL VIA SYNTHETIC BIOLOGY
Species: Taxus media
• Plant cells perceive environmental changes, generate biological response
• Accumulation of taxol is thought to be a biological response
•1996 Yukimune produced Baccatin III content with 100 µM Methyl Jasmonate addition to Taxus
media cell culture
• Methyl Jasmonate induces biological response in plants
• Taxol = 110.3mg/L/2 weeks, Baccatin III = 25.2 mg/L/2 weeks
• Issues: >100 µM M. Jasmonate decrease product production by 15 fold
Yukimune,Y., Nat. Biotech. 1996, 14, 1129
35
TAXOL VIA SYNTHETIC BIOLOGY
Endophytic Fungi: Fusarium mairei
• Taxus chinesis and fungis in 20 L co-bioreactor.
• Taxol production = 25.63 mg/L/15 days with fungis vs. 0.68
mg/L/15days no fungi
• Issues: 1. No co-biorecactor for industrial use available.
2. Biomass of fungi decreasses with inoculation time
Cheng, L.;Appl. Microbio Biotechnol (2009) 83, 233
36
TAXOL VIA SYNTHETIC BIOLOGY
• November 2010 Ajikumar & Stephanopoulos
optimized overproduction of taxadiene
CYP450
Taxadiene
Taxadien-5α-ol
• Taxadiene, first committed Taxol intermediate
• Used a multivariate-modular approach
• Used modified CYP450
Escherichia coli
• Produced 1g/L = 15, 000 fold increase in an
engineered E. Coli strain
37
Ajikumar,P. et al. Science 330, 70 (2010)
TAXOL VIA SYNTHETIC BIOLOGY
Taxadiene 5α hydroxylase
1 CoA-acylation
2. Sidechain
Taxol
CYP450
Baccatin III
Taxadien-5α-ol
• Removed bottlenecks
• Created two operons 1: (dxs-idi-ispDF), 2. (G-T)
• 300 mg/liter taxadiene
• Engineered CYP450 of E.coli
Ajikumar,P. et al. Science 330, 70 (2010)
38
TAXOL: SYNTHETIC CHEM OR BIO?
Wender,P.; J. Am. Chem. Soc. 1997, 119, 2755
Ajikumar,P. et al. Science 330, 70 (2010)
• 14 synthetic steps to AB ring
• Multivariate-modular pathway engineering
• 20 synthetic steps from AB ring to
Baccatin III
• Able to produce 300 mg/L of Taxadiene in
48 hours (30 Kg/ 100 K L)
• 37 total synthetic steps from
Verbenone to Taxol
• Taxadiene to Taxadiene-5α-ol via CYP450
• Overall yield of 0.4%
• Time to synthesize: months? Years?
• Further enzymatic reactions required
39
PLANT METABOLIC ENGINEERING
Herbivore
Extreme heat
http://www.physorg.com/news157900895.html
Pathogen
Flooding
40
www.google.com/images
PLANT METABOLIC ENGINEERING
• Plant secondary metabolites not essential for basic growth and development
•>200, 000 secondary metabolites produced
• For plants: pollination & seed dispersion, interactions with other plants, recognition of
herbivores, and defence by attracting predators of herbivores
• For humans: interesting applications in pharmacology, chemical industry, crops.
• Structural complexity makes it very difficult to synthesize chemically in adequate yields
Quinine
Taxol
Vincristine
41
A.G.Fett-Neto (ed.), Plant Secondary Metabolism Engineering, Methods in Mol. Bio. 643
PLANT PATHWAY BOTTLENECKS
Can manipulate the
Developmental control
Organ/tissue specific
Abiotic/biotic stresses
Circadian rhythms
42
A.G.Fett-Neto (ed.), Plant Secondary Metabolism Engineering, Methods in Mol. Bio. 643
PLANT PATHWAY BOTTLENECKS
• Some pathways entirely or partially introduced and
expressed in bacteria/ yeast
• Modifications done via recombinant DNA
• Transfer of pathways between plant species much more
difficult but can be done
Dhurrin in Arabidopsis plant
PNAS, 2005, 102, 1779
1.A.G.Fett-Neto (ed.), Plant Secondary Metabolism Engineering, Methods in Mol. Bio. 643
2.Image: http://en.wikipedia.org/wiki/Arabidopsis_thaliana
43
PRO-VITAMIN A
• Most successful metabolic-engineering done in plants:
Vitamin A
• Rice milled to remove outer layer which removes several nutrients such as provitamin A
• Vitamin A deficiency is a serious public health problem
• Symptoms ranging from night blindness to total blindness, diarrhea, respiratory
diseases, and measles
• Quarter of a million children go blind each year due to provitamin A deficiency
1. Ye, X; Science, 2000, 287, 303-305
2. Science, 2000, 287, 303
44
PRO-VITAMIN A
•Express pathway, in rice endosperm, to vitamin A precursor β-carotene
www.menurice.com/All_About_Rice
• Entire β-carotene biosynthetic pathway introduced into rice endosperm in single
transformation with three vectors
1.Ye, X; Science, 2000, 287, 303-305
2 .A.G.Fett-Neto (ed.), Plant Secondary Metabolism Engineering, Methods in Mol. Bio. 643
45
PRO-VITAMIN A
Immature rice endosperm
Geranylgeranyl diphosphate
phytoene desaturase
uncolored carotene phytoene
z-carotene desaturase
bacterial carotene
destaurase
lycopene β-cyclase
46
Ye, X; Science, 2000, 287, 303-305
PRO-VITAMIN A
Ye, X; Science, 2000, 287, 303-305
Pain, J.; Nature biotech., 2005, 23, 482-487
1.6 μg/g
37 μg/g
47
PLANT VOLATILES
1. Curr. Opin. Biotech, 2008,19, 181
2. http://healthsystemcio.com
48
PLANT VOLATILES
• In response to herbivore attack plants emit divers volatile blends
• > 200 different compounds
• Directly intoxicate, repel or deter herbivorous insects, or attract natural predators of
herbivors
• Potential in agriculture, preserve forest ecosystems, and crop protection
• Priming crops by planting few transgenic plants that constantly emit defence volatiles
1. Curr. Opin. Biotech, 2008,19, 181
2. http://healthsystemcio.com
49
Attracting Bodyguard to Arabidopsis
• M.E of Arabidopsis thaliana plants to emit two new isoprenoids
(3S)-(E)-nerolidol
(E)- DMNT
• These attracted carnivorous predatory mites that aid the plants’ defence mechanism
against herbivorous anthropods
Spider mites
Predatory Mites
http://www.entomology.wisc.edu/predatory-mites
http://www.hydro-gardens.com/spidermite.htm
50
Iris F. Kappers, et al. Science 309, 2070 (2005)
Attracting Bodyguard to Arabidopsis
Farnesyldiphosphate (FPP)
(3S)-(E)-nerolidol
(E)- DMNT
• Targeted FaNES1 in mitochondria using CoxIV
• Transgenic Arabidopsis plants with CoxIV-FaNES1 construct generated
51
Iris F. Kappers, et al. Science 309, 2070 (2005)
Attracting Bodyguard to Arabidopsis
wT
Clean
air wt
Clean
air/
Nerolidol/DMNT
CoxIV-FaNES1
wT/wT, spider
52
Iris F. Kappers, et al. Science 309, 2070 (2005)
SUMMARY
• Metabolic engineering
• Expression system
Steptomyces
•Manipulation of enzymatic functions
• Creation of biosynthetic pathway
D-1,2,4-butanetriol
53
SUMMARY
Plasmid DNA
DNA to be
inserted
Sticky Ends
DNA ligase
Sticky Ends
Recombinant DNA
54
SUMMARY
Wender,P.; J. Am. Chem. Soc. 1997, 119, 2755
Taxol – 37 synthetic steps (0.4%)
Ajikumar,P. et al. Science 330, 70 (2010)
Taxadiene – 300mg/L 48 hrs
55
SUMMARY
Substrate
Product
Flow
Mr. Wong
http://www.google.com/images
56
SUMMARY
Riboflavin (B2)
Artemisinin
Doxorubicin
Bioluminescent E.coli
Taxol
57
SUMMARY
Oleic acid (Canola/Soybean oil)
Vitamin E
Insect resistant Plants
Priming crops with transgenic plants
Vitamin A
Dhurrin (cyanogenic glucoside)
58
Acknowledgments
Dr. Robert Ben
Dr. Mathieu Leclere
Dr. Roger Tam
John Trant
Chantelle Capicciotti
Jackie Tokarew
Anna Balcerzak
Michela Febbraro
Ross Mancini
Devin Tonelli
Malay Doshi
59
Dr. Jay Keasling
"With the tools of synthetic
biology, we don't have to just
accept what Nature has
given us."
Amorphadiene
Artemisinin
Keasling, J., et al.; Nature biotech. (2003) 21, 796
60