metabolic engineering
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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 - PowerPoint PPT PresentationTRANSCRIPT
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 ener-getic 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
L-Aspartyl phosphate
Homoserine phosphate
Glucose
Phosphenolpyruvate
Pyruvate
TCA cycleOxaloacetate
ppc
mdh
aceBAKaspC
L-Lysine
L-Methionine
L-Aspartate
L-Aspartate semidaldehyde
Homoserine
L-Threonine
L-Isoleucine
thrA lysC
metL
asd
thrA
thrB
thrC
ilvA
dapA
metA
Feedback repression
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 bi-ology and seeks to transform biology in the same way that synthesis trans-formed 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 com-ponents (parts of enzymes, genetic circuits, metabolic pathways, etc.) that can be modeled, understood, and tuned to meet specific performance crite-ria, and the assembly of these smaller parts and devices into larger inte-grated systems that solve specific problems.
• 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).
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
MalariaMmosquito-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 mosquito prefer to feed at night
A Plasmodium in the form that enters humans and other vertebrates from the saliva of female mosquitoes (a sporozoite)
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 syn-thase (ADS) gene from Artemisia annua, commonly known as sweet wormwood
• Cloning a novel cytochrom P450 that per-form a three-step oxidation of amorpha-diene to Artemisinic acid
from A. annua
Production level : ~ 1.6 g/L by yeast
New pathway in yeast for artemisinic acid
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 ofartemisinic 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)
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.
Overview of artemisinic acid production pathway
Chemical conversion of artemisinic acid to artemisinin
Cell factory for valuable compounds from renewable biomass
Production of Bio adipic acid from renewable source (C6 feed stock)
Petro-leum
Biopro-cess
Pretreatment of biomass
Biomass
Sugars Strain devel-opment
Chemi-cal
process
Adipic acid
Adipic acid
Bio Nylon
Carpet 등 섬유 원료 Nylon 제조 원료
Polymer 의 원료 기타 산업용 재료의 원료
Use and Applications
High-value added compound for Nylon production : World market 10 조
Muconic acid derivatives
PEP
E4P
DAHP Chorismic acidDHQ
aro, aroII aroB aroD aroE aroK aroA aroCDHS SA S3P EPSP
p-Hydroxybenzoic acid
tryptophan
prephenate
phenylpyruvate4-hydroxyphenylpyruvate
trpC~AtrpG
Δcsm
pheA::aroFm tyrA::aroGm
phenylalaninetryptophan
tyrB, aspC
protocatecheuate
aroYcatA
catechol
cis,cis-muconic acid
Adipic acid
Chemical synthesis
ubiC
ΔtrpE
pobA
pyruvate
pps
Biosynthesis of cis,cis-muconic acid
pobA: p-hydroxybenzoate hydroxylase
Design of new metabolic pathway in Corynebacterium
Glucose
Shikimic acid pathway
Dihydroxyacetone phosphate
Critical point : Balanced synthesis of PEP and E4P
Glucose
Glucose 6-P
Fructose 6-P
Fructose 1,6-P
Digydroxy acetone P
Glyceraldehyde 3-P
3-P Glycerate
Phosphoenolpyru-vate(PEP)
Glucono-1,5-lactone 6-P 6-P-Gluconate Ribulose 5-P
Erythrose 4-phosphate(E4P)
Sedoheptulose 7-P
DHAP
Xylulose 5-P
PTS
zwf pgl gnd ru5p
tkt
tal
tka
pgi
pfk
pgk
eno
tis
aroF,G
Pentose phosphate pathway
Glycolysis
Dihydroxyacetone phosphate