Metabolic engineering Slide 2 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 Slide 3 Slide 4 Biosynthetic pathway of L-Thr in E.
coli L-Aspartyl phosphate Homoserine phosphate Glucose
Phosphenolpyruvate Pyruvate TCA cycleOxaloacetate ppc mdh aceBAK
aspC L-Lysine L-Methionine L-Aspartate L-Aspartate semidaldehyde
Homoserine L-Threonine L-Isoleucine thrAlysC metL asd thrA thrB
thrC ilvA dapA metA Feedback repression Slide 5 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
Slide 6 Nature Vol. 463 (2010) Slide 7 Alternative biomass Macro
algae : Multi-cellular marine algae, sea weed (red, brown, and
green algae) Switch grass Ascophyllum nodosum Slide 8 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. Slide 9 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, 628629; 2005). P roduction of the
anti-malarial drug precursor artemisinic acid in engineered yeast
US $ 43-million dollar grant from the Seattle-based Bill &
Melinda Gates Foundation Slide 10 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 Slide 11 A Plasmodium in the form that
enters humans and other vertebrates from the saliva of female
mosquitoes (a sporozoite) Slide 12 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 New pathway in yeast for artemisinic acid Slide 13
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) Slide 14
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
Slide 15 Chemical conversion of artemisinic acid to artemisinin
Slide 16 C ell factory for valuable compounds from renewable
biomass Production of Bio adipic acid from renewable source (C6
feed stock) Petroleum Bioprocess Pretreatment of biomass Biomass
Sugars Strain development Chemical process Adipic acid Bio Nylon
Slide 17 Carpet Nylon Polymer Use and Applications High-value added
compound for Nylon production : World market 10 Slide 18 Muconic
acid derivatives Slide 19 PEP E4P DAHP Chorismic acid DHQ aro,
aroII aroBaroDaroE aroK aroAaroC DHS SAS3P EPSP p-Hydroxybenzoic
acid tryptophan prephenate phenylpyruvate 4-hydroxy phenylpyruvate
trpC~A trpG csm pheA::aroF m tyrA::aroG m phenylalanine tryptophan
tyrB, aspC protocatecheuate aroY catA catechol cis,cis-muconic acid
Adipic acid Chemical synthesis ubiC trpE 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 Slide 20
Critical point : Balanced synthesis of PEP and E4P Glucose Glucose
6-P Fructose 6-P Fructose 1,6-P Digydroxy acetone PGlyceraldehyde
3-P 3-P Glycerate Phosphoenolpyruvate (PEP) Glucono-1,5-lactone
6-P6-P-GluconateRibulose 5-P Erythrose 4-phosphate (E4P)
Sedoheptulose 7-P DHAP Xylulose 5-P PTS zwf pglgndru5p tkt tal tka
pgi pfk pgk eno tis aroF,G Pentose phosphate pathway Glycolysis
Dihydroxyacetone phosphate
