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

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

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

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

Potential products Fuels Natural products (complex synthesis) Engineered products hydrogen (H 2 ) - methane (CH 4 ) - methanol (CH 3 OH) - ethanol (CH 3 CH 2 OH) - starting materials for polymers (rubber, plastic, fabrics) - specialty chemicals (chiral) - bulk chemicals (C 4 acids) - vitamins - therapeutic agents - pigments - amino acids - viscosifiers - industrial enzymes - PHAs (biodegradable plastics)

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 1Gene 2Gene 3 Enzyme 1 Enzyme 2 Enzyme 3 A BCD A DNA

Gene 1Gene 2Gene 3 Enzyme 1 Enzyme 2 Enzyme 3 A BCD A Deleting a gene DNA X X X

Adding a new gene DNA Gene 1Gene 2Gene 3 Enzyme 1 Enzyme 2 Enzyme 3 A BCD A DNA

Adding a new gene DNA Gene 1Gene 2Gene 3 Enzyme 1 Enzyme 2 Enzyme 3 A BCD A Gene 4 Enzyme 4 E

How are genetic changes made? Most common approach: 1.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? plasmid Gene 4 Most common approach: 1.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.

How are genetic changes made? Gene 4 plasmid Gene 4 Most common approach: 1.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.

How are genetic changes made? Gene 4 plasmid Most common approach: 1.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.

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

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

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

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!

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 H4FH4F 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

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 A AB 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

Edwards & Palsson (2000) Reference: PNAS 97: , 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.

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.

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.

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

Contacts for theme interviews Xiaofeng Guo (4 th -year grad student) Project: studying metabolic shifts of methanol- consuming bacteria by quantifying enzyme activities and metabolite concentrations under various conditions. Alex Holland (4 th -year grad student) Project: manipulating polyphosphate metabolism in radiation-resistant bacteria to generate an organism that can precipitate heavy metals.