Genetically engineered bacteria: chemical factories of the future?

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

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

www.milebymile.com/main/United_States/Wyoming/

- thrive in extreme environments

- use nutrients unavailable to other organisms

Potential products

• Fuels

• Natural products (complex synthesis)

• Engineered products

www.myhealthshack.net; www.acehardware.com

- hydrogen (H2)- methane (CH4)- methanol (CH3OH)- ethanol (CH3CH2OH)

- starting materials for polymers (rubber, plastic, fabrics)- specialty chemicals (chiral)- bulk chemicals (C4 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 systemspyo.oulu.fi

Metabolic engineering(a form of genetic

engineering)

DNAGene 1 Gene 2 Gene 3

Enzyme 1 Enzyme 2 Enzyme 3A B C D

A

DNA

DNAGene 1 Gene 2 Gene 3

Enzyme 1 Enzyme 2 Enzyme 3A B C D

A

Deleting a gene

DNA

DNAGene 1 Gene 2 Gene 3

Enzyme 1 Enzyme 2 Enzyme 3A B C D

A

Deleting a gene

DNA X

DNAGene 1 Gene 2 Gene 3

Enzyme 1 Enzyme 2 Enzyme 3A B C D

A

Deleting a gene

DNA X

X

DNAGene 1 Gene 2 Gene 3

Enzyme 1 Enzyme 2 Enzyme 3A B C D

A

Deleting a gene

DNA X

X X

Adding a new gene

DNAGene 1 Gene 2 Gene 3

Enzyme 1 Enzyme 2 Enzyme 3A B C D

A

DNA

Adding a new gene

DNAGene 1 Gene 2 Gene 3

Enzyme 1 Enzyme 2 Enzyme 3A B C D

A

Gene 4

Adding a new gene

DNAGene 1 Gene 2 Gene 3

Enzyme 1 Enzyme 2 Enzyme 3A B C D

A

Gene 4

Enzy

me 4

Adding a new gene

DNAGene 1 Gene 2 Gene 3

Enzyme 1 Enzyme 2 Enzyme 3A B C D

A

Gene 4

Enzy

me 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?

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?

DNAGene 1 Gene 2 Gene 3

How are genetic changes made?

DNAGene 1 Gene 2 Gene 3

Gene 4

How are genetic changes made?

DNAGene 1 Gene 2 Gene 3

Gene 4

How are genetic changes made?

DNAGene 1 Gene 2 Gene 3

Gene 4X X

How are genetic changes made?

DNAGene 1 Gene 2 Gene 3Gene 4

Metabolic engineering mishaps: maximizing ethanol production

PFKethanolglucose

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

CH2=H4F

Serine Cycle

CH2=H4MPT

H4MPT

CH3OH

HCHOH4F

CO2

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

AA 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.

1010

10

10

0

0

10

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.

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.

www.elsevier.com

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