Genetically engineered bacteria: chemical factories of the future?
1. Genetically engineered bacteria: Chemical factories of the future? Image: G. Karp, Cell and molecular biology Relocation mechanism Assembly line Central computer Security fence Outer and internal walls
2. Gregory J. Crowther, Ph.D. Acting Lecturer Mary E. Lidstrom, Ph.D. Frank Jungers Professor of Chemical Engineering
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. Metabolic engineering (a form of genetic engineering) DNA Gene 1 Gene 2 Gene 3 Enzyme 1 Enzyme 2 Enzyme 3 A B C D A DNA
10. Deleting a gene DNA Gene 1 Gene 2 Gene 3 Enzyme 1 Enzyme 2 Enzyme 3 A B C D A DNA
11. Deleting a gene DNA Gene 1 Gene 2 Gene 3 Enzyme 1 Enzyme 2 Enzyme 3 A B C D A DNA X
12. Deleting a gene DNA Gene 1 Gene 2 Gene 3 Enzyme 1 Enzyme 2 Enzyme 3 A B C D A DNA X X
13. Deleting a gene DNA Gene 1 Gene 2 Gene 3 Enzyme 1 Enzyme 2 Enzyme 3 A B C D A DNA X X X
14. Adding a new gene DNA Gene 1 Gene 2 Gene 3 Enzyme 1 Enzyme 2 Enzyme 3 A B C D A DNA
15. Adding a new gene DNA Gene 1 Gene 2 Gene 3 Enzyme 1 Enzyme 2 Enzyme 3 A B C D A Gene 4
16. Adding a new gene DNA Gene 1 Gene 2 Gene 3 Enzyme 1 Enzyme 2 Enzyme 3 A B C D A Gene 4 Enzyme 4
17. Adding a new gene DNA Gene 1 Gene 2 Gene 3 Enzyme 1 Enzyme 2 Enzyme 3 A B C D A Gene 4 Enzyme 4 E
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22. How are genetic changes made? DNA Gene 1 Gene 2 Gene 3
23. How are genetic changes made? DNA Gene 1 Gene 2 Gene 3 Gene 4
24. How are genetic changes made? DNA Gene 1 Gene 2 Gene 3 Gene 4
25. How are genetic changes made? DNA Gene 1 Gene 2 Gene 3 X X Gene 4
26. How are genetic changes made? DNA Gene 1 Gene 2 Gene 3 Gene 4
27. Metabolic engineering mishaps: maximizing ethanol production PFK ethanol glucose 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!
28. Metabolic engineering mishaps: maximizing PHA production CH 2 =H 4 F Serine Cycle CH 2 =H 4 MPT H 4 MPT CH 3 OH HCHO H 4 F CO 2 PHA To maximize PHA production in M. extorquens , one might try to knock out the right-hand pathway. Problems: • HCHO builds up and is toxic • Cells can’t generate enough energy for growth X
29. 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/
30. Flux balance analysis A A B C D 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. 10 10 10 10 0 0 10
31. 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.
32. Edwards & Palsson (2000) Fraction of normal growth rate Gene deletions that should not affect growth. Gene deletions that should slow growth. Gene deletions that should stop growth.
33. 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.
34. 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?
35. 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
36. More information about 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