1. Genetically engineered bacteria: Chemical factories of the future?
Relocation mechanism
Assembly line
Central computer
Security fence
Outer and internal walls
Image: G. Karp, Cell and molecular biology

2. 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

3. Possible solution: Use bacteria as chemical factories
Starting materials
Value-added products
Self-replicating multistage catalysts
Environmentally benign
Use renewable starting materials (feedstocks)

4. 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

5. 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)

6. 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
- don’t naturally make everything we need

7. Redesigning bacteria Goal: optimize industrially valuable parameters.
• Redirect metabolism to specific products
• Remove unwanted products
- storage products
- excretion products
- defense systems
pyo.oulu.fi

8. Metabolic engineering (a form of genetic engineering)
DNA
Gene 1
Gene 2
Gene 3
Enzyme 1
Enzyme 2
Enzyme 3 A B C D

9. X X X Deleting a gene A B C D A DNA DNA Gene 1 Gene 2 Gene 3 Enzyme 1

10. Adding a new gene A B C D A DNA DNA Gene 1 Gene 2 Gene 3 Enzyme 1

11. Adding a new gene A B C D A E DNA Gene 1 Gene 2 Gene 3 Gene 4 Enzyme 1

12. 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

13. 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

14. 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
Gene 4
plasmid

15. 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

16. How are genetic changes made?
DNA
Gene 4

17. How are genetic changes made?
DNA X X
Gene 4

18. How are genetic changes made?
DNA

19. 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!

20. 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
CH2=H4F
CH2=H4MPT
Serine Cycle
CO2
Problems:
• HCHO builds up and is toxic
• Cells can’t generate enough energy for growth
PHA

21. 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/

22. Flux balance analysis C A A B D E 10 10 10 10 10C A 10 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.

23. 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.

24. 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?

25. 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.

26. More information about metabolic engineering
depts.washington.edu/mllab
web.mit.edu/bamel
Lidstrom lab (UW)
Stephanopoulos lab (MIT)
Company founded by Palsson (UCSD)
Well-written background info and examples