Download Metabolic engineering Synthetic Biology

Metabolic engineering
Metabolic engineering
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Targeted and purposeful alteration of metabolic pathways found in an organism in
order to better understand and use cellular pathways :
- To increase the production rate of the existing products
- To produce new valuable products
- To expand the substrates that can be assimilated by organisms
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Practice of optimizing genetic and regulatory processes within the cells to
maximize the production of a target material by the cells.
- Expression and release of repression
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Metabolic engineers commonly work to reduce cellular energy use (i.e, the
energetic cost of cell reproduction or proliferation) and to reduce the waste
production.
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Direct deletion and/or over-expression of the genes that encode the metabolic
enzymes
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Current focus is to target the regulatory networks in a cell to efficiently engineer
the metabolism
Biosynthetic pathway of L-Thr in E. coli
Glucose
Phosphenolpyruvate
ppc
Pyruvate
metL L-Aspartate
thrA
aspC
Oxaloacetate
TCA cycle
aceBAK
lysC
mdh
L-Aspartyl phosphate
asd
L-Aspartate semidaldehyde
dapA
L-Lysine
thrA
metA
Homoserine
L-Methionine
thrB
Homoserine phosphate
thrC
L-Threonine
ilvA
Feedback repression
L-Isoleucine
Microbial production of fatty-acid-derived fuels and chemicals from plant biomass
• Biofuels: Production of ethanol from corn starch or sugarcane
 Harder to transport than petrol
Raise of global food prices
• Need for high-energy fuel : Fatty-acid derived fuels
 Energy-rich molecule than ethanol
 Isolated from plant and animal oils
• More economic route starting from renewable sources
- Engineering E. coli to produce fatty esters(bio-disel), fatty
alchols, and waxes directly from sugars or hemi-cellulose
- Cost-effective way of converting grass or crop waste into fuels
Fatty Acid Biosynthesis
• Synthesis takes place in the cytosol
• Intermediates covalently linked to acyl carrier protein
- Activation of each acetyl CoA.
- Acetyl CoA + CO2  Malonyl CoA
• Four-step repeating cycle, extension by 2-carbons /cycle
– Condensation
– Reduction
– Dehydration
– Reduction
Fatty Acid Synthase (FAS)
• Polypeptide chain with multiple domains, each with
distinct enzyme activities required for fatty acid biosynthesis.
• ACP( Acyl carrier protein ):
- Activator in the fatty acid biosynthesis
- Part of FAS complex
• FAS complex: The acyl groups get anchored to the CoA
group of ACP by a thioester linkage
• Condensing enzyme/β-ketoacyl synthase (K-SH): Part of FAS,
CE has a cysteine SH that participates in thioester linkage with the
carboxylate group of the fatty acid.
• The growing FA chain alternates between K-SH and ACP-SH
Nature Vol. 463 (2010)
Alternative biomass
• Corn starch, sugar cane: currently used
• Cheaper renewable sources
- Cellulose
- Macro algae : Multi-cellular marine algae, sea weed
(red, brown, and green algae)
- Switch grass
Ascophyllum nodosum
Synthetic Biology
• Design and construction of new biological entities such as enzymes,
genetic circuits, and cells or the redesign of existing biological systems.
• Synthetic biology builds on the advances in molecular, cell, and systems
biology and seeks to transform biology in the same way that synthesis
transformed chemistry and integrated circuit design transformed
computing.
• The element that distinguishes synthetic biology from traditional
molecular and cellular biology is the focus on the design and construction
of core components (parts of enzymes, genetic circuits, metabolic
pathways, etc.) that can be modeled, understood, and tuned to meet
specific performance criteria, and the assembly of these smaller parts and
devices into larger integrated systems that solve specific problems.
Production of the anti-malarial drug precursor artemisinic acid in engineered yeast
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US $ 43-million dollar grant from the Seattle-based Bill & Melinda Gates Foundation
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Artemisinin : extract from the leaves of Artemisia annua, or sweet wormwood.
- used for more than 2,000 years by the Chinese as a herbal medicine called qinghaosu.
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The parasite that causes malaria has become partly resistant to every other treatment tried so far.
Artemisinin is still effective, but it is costly and scarce. The supply of plant-derived artemisinin
is unstable, resulting in shortages and price fluctuations
• Artemisinin works by disabling a calcium pump in the malaria parasite, Plasmodium falciparum.
Mutation of a single amino acid confers resistance (Nature Struct. Mol. Biol. 12, 628–629; 2005).
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200 million people infected with malaria each year mainly in Africa, and at least 655,000 deaths
in 2010  Treatment : Intravenous or intramuscular quinine
Malaria
Mosquito-borne infectious disease of humans and other animals caused by
protists (a type of unicellular microorganism) of the genus Plasmodium.
Malaria causes symptoms that typically include fever and headache, which in severe cases
can progress to coma or death : No effective vaccine exists2
In 2012, 219 million documented cases. Between 660,000 and 1.2 million people died
It begins with a bite from an infected female Anopheles mosquito, which introduces
the protists through saliva into the circulatory system.
A motile infective form (called the sporozoite) to a vertebrate host such as a human
(the secondary host), thus acting as a transmission vector. A sporozoite travels
through the blood vessels to liver cells (hepatocytes), where it reproduces asexually (tissue
schizogony), producing thousands of merozoites.
These infect new red blood cells and initiate a series of asexual multiplication cycles (blood
schizogony) that produce 8 to 24 new infective merozoites (낭충)
Only female mosquitoes feed on blood; The females of the Anopheles genus of mosquito pr
efer to feed at night
A Plasmodium in the form that enters humans and other vertebrates from
the saliva of female mosquitoes (a sporozoite)
New pathway in yeast for artemisinic acid production
Strategy to engineer the yeast cell to
produce the artemisinic acid at cheaper
cost
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Engineering the farnesyl pyrophosphate
(FPP) biosynthetic pathway to increase FPP
production: HMG-CoA reductase (3-hydroxy-3-
methyl-glutaryl-CoA reductase); rate-controlling enzyme
in the mevalonate pathway that produces cholesterol and
other isoprenoids
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Introduction of the amorphadiene synthase
(ADS) gene from Artemisia annua,
commonly known as sweet wormwood
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Cloning a novel cytochrom P450 that
performs a three-step oxidation of
amorphadiene to Artemisinic acid
from A. annua
Production level : ~ 1.6 g/L by yeast
Improvement of production yield of artemisinic acid
- Production level is too low to be economically feasible
- Discovery of a plant dehydrogenase and a second cytochrome that provide an efficient
biosynthetic route to artemisinic acid, with fermentation titres of 25 g/L of
artemisinic acid by yeast.
- Practical, efficient and scalable chemical process for the conversion of artemisinic acid
to artemisinin using a chemical source of singlet oxygen, thus avoiding the need for
specialized photochemical equipment.
- The strains and processes form the basis of a viable industrial process for the production
of semi-synthetic artemisinin to stabilize the supply of artemisinin for derivatization
into active pharmaceutical ingredients.
- Because all intellectual property rights have been provided free of charge, the technology
has the potential to increase provision of first-line antimalarial treatments to the
developing world at a reduced average annual price.
Paddon et al., Nature (2013)
Overview of artemisinic acid production pathway
Overexpressed genes controlled by the GAL induction system are shown in green. Copper- or methionine-repressed
squalene synthase (ERG9) is shown in red. DMAPP, dimethylallyl diphosphate; FPP, farnesyl diphosphate; IPP,
isopentenyl diphosphate. tHMG1 encodes truncated HMG-CoA reductase. b, The full three-step oxidation of
amorphadiene to artemisinic acid from A. annua expressed in S. cerevisiae. CYP71AV1, CPR1 and CYB5 oxidize
amorphadiene to artemisinic alcohol; ADH1 oxidizes artemisinic alcohol to artemisinic aldehyde; ALDH1 oxidizes
artemisinic aldehyde to artemisinic acid.
Chemical conversion of artemisinic acid to artemisinin
Cell factory for valuable compounds from renewable biomass
Bio-Nylon
Production of Bio adipic acid from renewable source (C6 feed stock)
Petroleum
Adipic acid
Pretreatment of
biomass
Biomass
Sugars
Bioprocess
New Strain
Adipic acid
Chemical
process
Use and Applications
Bio Nylons production : World market $ 10 Billion
Raw material for Carpet
Raw material for Nylon
Raw material for various
polymers
Electronic materials
Muconic acid derivatives
Biosynthesis of cis,cis-muconic acid
tryptophan
phenylalanine
tyrB, aspC
Design of new metabolic pathway in Corynebacterium
4-hydroxy
phenylpyruvate phenylpyruvate
Glucose
pheA::aroFm
tyrA::aroGm
pyruvate
prephenate
Shikimic acid pathway
pps
PEP
Δcsm
aro, aroII
aroB
DAHP
E4P
aroD
DHQ
aroK
aroE
DHS
SA
aroA
S3P
tryptophan
trpC~A
trpG
ΔtrpE
aroC
EPSP
Chorismic acid
Dihydroxyacetone
phosphate
ubiC
pobA: p-hydroxybenzoate hydroxylase
cis,cis-muconic acid
pobA
Chemical
synthesis
catA
aroY
catechol
Adipic acid
protocatecheuate
p-Hydroxybenzoic acid
Design and construction of new strain
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Synthetic promoter
Incorporation of new enzymes
Deletion/knock-out of waste pathway
Incorporation of transporter
Control of carbon flux
Cofactor balance
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Critical point : Balanced synthesis of PEP and E4P
Glucose
Pentose phosphate pathway
PTS
Glucose 6-P
pgi
Glucono-1,5-lactone 6-P
zwf
6-P-Gluconate
pgl
Ribulose 5-P
gnd
ru5p
Fructose 6-P
tkt
pfk
Fructose 1,6-P
Erythrose 4-phosphate
(E4P)
Xylulose 5-P
tka
tal
Sedoheptulose 7-P
Digydroxy acetone P
Glycolysis
Glyceraldehyde 3-P
tis
pgk
3-P Glycerate
eno
Phosphoenolpyruvate
(PEP)
aroF,G
DHAP
Dihydroxyacetone phosphate