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

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

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

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/

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

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

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

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

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

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

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

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

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

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

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

How are genetic changes made? DNA Gene 4

How are genetic changes made? DNA X X Gene 4

How are genetic changes made? DNA

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!

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

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/

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

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.

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?

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

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