Metabolic engineering
Metabolic engineering
• Targeted and purposeful alteration of metabolic pathways found in an
organism in order to better understand and use cellular pathways for the
production of valuable products
• Practice of optimizing genetic and regulatory processes within cells to
increase the cells' production of a substance.
• Metabolic engineers commonly work to reduce cellular energy use (i.e,
the energetic cost of cell reproduction or proliferation) and to reduce
waste production.
• Direct deletion and/or over-expression of the genes that encode the
metabolic enzymes
• 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(biodisel), fatty alchols,
and waxes directly from sugars or hemi-cellulose
- Cost-effective way of converting grass or crop waste into fuels
Nature Vol. 463 (2010)
Alternative biomass
• 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
•
US $ 43-million dollar grant from the Seattle-based Bill & Melinda Gates Foundation
•
Artemisinin : extracted from the leaves of Artemisia annua, or sweet wormwood, and
has been used for more than 2,000 years by the Chinese as a herbal medicine called qinghaosu.
The parasite that causes malaria has become at least 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
200 million people infected with malaria each year mainly in Africa, and at least 655,000 deaths
in 2010  Treatment : Intravenous or intramuscular quinine
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).
•
•
•
•
Malaria
Mmosquito-borne infectious disease of humans and other animals caused by
protists (a type of microorganism) of the genus Plasmodium.
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
Malaria causes symptoms that typically include fever and headache, which in severe
cases can progress to coma or death.
Only female mosquitoes feed on blood; The females of the Anopheles genus of mos
quito prefer 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
Strategy to engineer the yeast cell to
produce the artemisinic acid at cheaper
cost
•
Engineering the farnesyl pyrophosphate
(FPP) biosynthetic pathway to increase FPP
production
•
Introduction of the amorphadiene
synthase (ADS) gene from Artemisia annua,
commonly known as sweet wormwood
•
Cloning a novel cytochrom P450 that
perform 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
- Discovery of a plant dehydrogenase and a second cytochrome that provide an efficient
biosynthetic route to artemisinic acid, with fermentation titres of 25 grams per litre 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
Strain
development
Adipic acid
Chemical
process
Use and Applications
High-value added compound for Nylon production : World market 10 조
Carpet 등 섬유 원료
Nylon 제조 원료
Polymer의 원료
기타 산업용 재료의 원료
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
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