Transcript Metabolic engineering Synthetic Biology

Metabolic engineering
Metabolic engineering
 Targeted and purposeful alteration of metabolic pathways 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 to achieve a goal.
 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
L-Isoleucine
Feedback repression
Feedback inhibition
Microbial production of fatty-acid-derived fuels and
chemicals from plant biomass
 Biofuels : Relied on manufacturing ethanol from corn
starch or sugarcane
 Harder to transport than petroleum, low energy density
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
alcohols, and waxes directly from sugars or hemi-cellulose
- Cost-effective way of converting grass or crop waste into
fuels
- Increased production of free fatty acids and Acyl-CoAs overexpressing
TES and ACL, and by eliminating b-oxidation (ΔfadE)
- Fatty alcohols are produced directly from fatty acyl-CoAs by
overexpressing FAR (fatty acyl-Coa reductase
- Esters are produced by overexpressing AT (acyltransferase)
Nature, 463 (2010)
Alternative biomass
Macro algae : Multi-cellular marine algae, sea weed
(red, brown, and green algae)
Switch grass
Ascophyllum nodosum
Direct biofuel production from Brown macro-algae
by an engineered microorganism
 Corn and sugarcane: industrial feedstock
 Food versus fuel concerns preclude their long-term use
 Lignocellulosic materials: Preferred feedstock,
but fermentation of the simple sugars in lignocellulose
are costly and complex
 Needs for energy-intensive pretreatment and
hydrolysis processes
Marine macro-algae(seaweeds) : next generation feedstock
 Brown macroalgae as an ideal feedstock for production of biofuels and
renewable commodity chemicals
 Requiring no arable land, fertilizer, or fresh water resources
 No economic concerns associated with land management
 Avoids adverse impact on food supplies
 Large-scale cultivation is practices in several countries,
yielding 15 million metric tons per year
 Contains no lignin, and sugars can be released by simple
operations such as milling or crushing
 Most abundant sugars in brown macroalgae : alginate, mannitol,
and glucan ( glucose polymers)
 Alginate : a linear block copolymer of two uronic acids,
Β-D-mannuronate and α-L-guluronate
 Potential of ethanol production from macroalgae :
 limited by the inability of industrial microbes to
metabolize the alginate components
 The discovery of the genes from Vibrio splendidus
encoding enzymes for alginate transport and metabolism
 Construction of a microorganism with the capacity
for degrading, up-taking, and metabolizing alginates
 Expression of the genes in E. coli for the production of
ethanol from macroalgae
Science, 335 (2012)
Production of the anti-malarial drug precursor artemisinic acid
in engineered yeast
 300 million to 500 million people infected with malaria each year
mainly in Africa
 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.
 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.
 Artemisinin works by disabling a calcium pump in the malaria
parasite
 Mutation of a single amino acid was sufficient to confer resistance
(Uhleman et al. Nature Struct. Mol. Biol. 12 (2005)
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 : ~ 100 mg/L
by yeast culture
Ro et al., Nature (2009)
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.