Genetically engineered bacteria: Chemical factories of the future?

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Genetically engineered bacteriaChemical
factories of the future?
Image G. Karp, Cell and molecular biology
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Gregory J. Crowther, Ph.D. Acting Lecturer
Mary E. Lidstrom, Ph.D. Frank Jungers Professor
of Chemical Engineering
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The chemical industry today

• supplies chemicals for many manufactured goods
• employs many scientists and engineers
• based on chemicals derived from petroleum
• not a renewable resource
• supplied by volatile areas of the world
• - many produce hazardous wastes

www.hr/tuzla/slike
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Possible solutionUse bacteria as chemical
factories
Value-added products
Starting materials

• Self-replicating multistage catalysts
• Environmentally benign
• Use renewable starting materials (feedstocks)

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Advantages of bacteria vs. other cells

• Relatively small and simple
• Reproduce quickly
• Tremendous metabolic / catalytic diversity

• - thrive in extreme environments
• - use nutrients unavailable to other organisms

www.milebymile.com/main/United_States/Wyoming/
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Potential products
Fuels
Engineered products
- hydrogen (H2) - methane (CH4) - methanol
(CH3OH) - ethanol (CH3CH2OH)
- starting materials for polymers (rubber,
plastic, fabrics) - specialty chemicals
(chiral) - bulk chemicals (C4 acids)
Natural products (complex synthesis)
- vitamins - therapeutic agents - pigments -
amino acids - viscosifiers - industrial enzymes -
PHAs (biodegradable plastics)
www.myhealthshack.net www.acehardware.com
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Limitations of naturally occurring bacteria
Bacteria are evolved for survival in competitive
natural environments, not for production of
chemicals desired by humans!
coolgov.com
- are optimized for low nutrient levels -
have defense systems - dont naturally make
everything we need
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Redesigning bacteria
Goal optimize industrially valuable parameters.
Redirect metabolism to specific products
Remove unwanted products - storage products
- excretion products - defense systems
pyo.oulu.fi
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Metabolic engineering(a form of genetic
engineering)
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Deleting a gene
X
Gene 1
Gene 2
Gene 3
DNA
DNA
X
X
Enzyme 1
Enzyme 2
Enzyme 3
A
B
C
D
A
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Adding a new gene
Gene 1
Gene 2
Gene 3
DNA
DNA
Enzyme 1
Enzyme 2
Enzyme 3
A
B
C
D
A
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Adding a new gene
Gene 1
Gene 2
Gene 3
Gene 4
DNA
Enzyme 1
Enzyme 2
Enzyme 3
A
B
C
D
Enzyme 4
A
E
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How are genetic changes made?

• Most common approach
• Put a gene of interest into a stable carrier
  (vector), a circle of DNA called a plasmid.
• 2. Put the plasmid into a new cell.

Gene 4
plasmid
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How are genetic changes made?

• Most common approach
• Put a gene of interest into a stable carrier
  (vector), a circle of DNA called a plasmid.
• 2. Put the plasmid into a new cell.

plasmid
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How are genetic changes made?

• Most common approach
• Put a gene of interest into a stable carrier
  (vector), a circle of DNA called a plasmid.
• 2. Put the plasmid into a new cell.

Gene 4
plasmid
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How are genetic changes made?

• Most common approach
• Put a gene of interest into a stable carrier
  (vector), a circle of DNA called a plasmid.
• 2. Put the plasmid into a new cell.

plasmid
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How are genetic changes made?
Gene 1
Gene 2
Gene 3
DNA
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How are genetic changes made?
Gene 1
Gene 2
Gene 3
X
X
DNA
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How are genetic changes made?
Gene 1
Gene 2
Gene 3
Gene 4
DNA
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Metabolic engineering mishaps maximizing
ethanol production
glucose
ethanol
PFK
PFK was thought to be the rate-limiting enzyme of
ethanol production, so its levels were increased
via genetic engineering.
Problem rates of ethanol production did not
increase!
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Metabolic engineering mishaps maximizing
PHA production
CH3OH
To maximize PHA production in M. extorquens, one
might try to knock out the right-hand pathway.
H4MPT
H4F
HCHO
X
CH2H4F
CH2H4MPT
Serine Cycle
CO2
PHA
Problems HCHO builds up and is toxic Cells
cant generate enough energy for growth
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Cellular metabolism is very complicated!
Lots of molecules Highly interconnected
Mathematical models can help us predict the
effects of genetic changes
opbs.okstate.edu/leach/Bioch5853/
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Flux balance analysis
0
C
A
10
0
10
A
B
10
D
10
10
E
In a steady state, all concentrations are
constant. For each compound, production rate
consumption rate.
To get a solution (flux rate for each step),
define an objective function (e.g., production of
E) to be maximized.
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Edwards Palsson (2000)
Reference PNAS 97 5528-33, 2000. Used flux
balance analysis to predict how well E. coli
cells would grow if various genes were
deleted. The graph at left shows their
predictions of how fluxes are rerouted in
response to gene deletions.
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Edwards Palsson (2000)
Fraction of normal growth rate
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Edwards Palsson (2000)
Predictions of whether various E. coli mutants
should be able to grow were compared with
experimental data on these mutants. In 68 of 79
cases (86), the prediction agreed with the
experimental data.
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Ethical issues
Is it OK to tamper with the genes of living
organisms? What are the possible effects on
those organisms? What are the possible effects
on human health? What are the possible effects
on the environment?
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Summary
Bacteria have great potential as
environmentally friendly chemical factories.
Much additional research will be
needed for this potential to be
fulfilled. Further progress will
require knowledge of biology,
chemistry, engineering, and
mathematics.
www.elsevier.com
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More informationabout metabolic engineering
depts.washington.edu/mllab web.mit.edu/bamel www
.genomatica.com www.metabolix.com
Lidstrom lab (UW)
Stephanopoulos lab (MIT)
Company founded by Palsson (UCSD)
Well-written background info and examples
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Contacts for theme interviews
Xiaofeng Guo (4th-year grad student) xfguo_at_u.washi
ngton.edu Project studying metabolic shifts of
methanol-consuming bacteria by quantifying enzyme
activities and metabolite concentrations under
various conditions. Alex Holland (4th-year grad
student) aholland_at_u.washington.edu Project
manipulating polyphosphate metabolism in
radiation-resistant bacteria to generate an
organism that can precipitate heavy metals.