Genetically engineered bacteria: Chemical factories of the future?

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

2. Gregory J. Crowther, Ph.D. Acting Lecturer Mary E. Lidstrom, Ph.D. Frank Jungers Professor of Chemical Engineering

3. 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

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

5. 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/

6. 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

7. 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

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

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

10. 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

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

12. 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

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. Gene 4 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

15. Gene 4 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. Gene 4 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

17. Gene 4 How are genetic changes made? Gene 1 Gene 2 Gene 3 DNA

18. Gene 4 How are genetic changes made? Gene 1 Gene 2 Gene 3 X X DNA

19. How are genetic changes made? Gene 1 Gene 2 Gene 3 Gene 4 DNA

20. 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!

21. 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 PHA Problems: • HCHO builds up and is toxic • Cells can’t generate enough energy for growth

22. 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/

23. 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.

24. Edwards & Palsson (2000) Reference: PNAS97: 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.

25. Gene deletions that should not affect growth. Gene deletions that should slow growth. Gene deletions that should stop growth. Edwards & Palsson (2000) Fraction of normal growth rate

26. 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.

27. 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?

28. 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

29. 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

30. Contacts for theme interviews Xiaofeng Guo (4th-year grad student) xfguo@u.washington.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@u.washington.edu Project: manipulating polyphosphate metabolism in radiation-resistant bacteria to generate an organism that can precipitate heavy metals.