Genetically engineered bacteria:

Chemical factories of the future?

Relocation mechanism

Assembly line
Central computer
Outer and
internal walls
Image: G. Karp, Cell
and molecular biology

Security fence

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
- dont 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

Enzyme 1

Gene 2

Gene 3

Enzyme 2

Enzyme 3

Deleting a gene

DNA

Gene 1

Enzyme 1

Gene 2

Gene 3

Enzyme 2

D
X X

Enzyme 3

Adding a new gene

DNA

Gene 1

Enzyme 1

Gene 2

Gene 3

Enzyme 2

Enzyme 3

Adding a new gene

DNA

Gene 1

Enzyme 1

Gene 2

Gene 3

Enzyme 2

C
Enzyme 4

Gene 4

Enzyme 3

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?

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?

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

Gene 4

plasmid

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?

DNA

Gene 1

Gene 2

Gene 4

Gene 3

How are genetic changes made?

DNA

Gene 1

Gene 2

Gene 3
Gene 4

How are genetic changes made?

DNA

Gene 1

Gene 2

Gene 4
3

Metabolic engineering of bacteria

Increasing biological production of small
molecules
Random screening for overproducing
strains (genome shuffling)
Rational engineering of pathways

Many biological small molecules are

useful
Antibiotics
Vitamins
Amino acids and derivatives (indigo,
aspartame)
secondary metabolites from plants-alkaloids (caffeine, theobromine, etc.)
Etc.
Synthesis often requires multiple steps and
enzymes

Increasing production of antibiotics (and

other small molecules)--traditional
methods
Obtain organism that produces a specific
compound--Penicillium mold originally made
micrograms per liter of culture
Randomly mutagenize the organism and
screen for increased production, repeat using
top producing organism
Outcome: grams of penicillum per liter of
culture (1000-fold increase in production)
Time consuming and expensive process!

An alternative to simple random

mutagenesis: genome shuffling

The shuffling advantage: simultaneous recombination of

entire genomes (breeding) with multiple parents

(old way)

(new way)

The set-up
Compare classical strain improvement (CSI) to
genome shuffling
Streptomyces sp.: produce polyketide antibiotics
Induce recombination by recursive protoplast fusion:
Fuse protoplasts
Regenerate cell walls, grow as a population (F1)
Make protoplasts with F1, repeat until F4
Test with 4 auxotrophy markers (next page)
Test for increased antibiotic production

Test of recursive shuffling

4 parental strains

Supplements required:

Strain

Description

S. coelicolor 268412

proA1 argA1 uraA1

pro, arg, ura (not cys)

S. coelicolor 268512

proA1 cysD18 uraA1

pro, cys, ura (not arg)

S. coelicolor 268612

argA1 cysD18 uraA1

arg, cys, ura (not pro)

S. coelicolor M124

12

proA1 argA1 cysD18

pro, arg, cys (not ura)

Can strains be isolated that can grow in the absence of pro, arg,
ura, and cys (indicating progeny with all 4 genes wild type)?
YES.

Indicates increased efficiency

of recombination

Test case: increase tylosin production by S. fradiae?

SF1 was treated with NTG, 11 strains selected (22000

screened), those 11 strains were shuffled once (GS1) and
then again (GS2)

Comparing CSI to genome shuffling

Genome shuffling
Technique has also been used to generate
acid-tolerant strains of Lactobacillus (useful
for production of lactic acid)
Applicable to eukaryotic microbes?
Still dont know the mutations that have
occurred, or what the state of the genome is
following several fusion events

Increasing production of a biological

compound: rational design
1) Increase production of a naturally produced
commercial compound
Modify existing genes
2) Obtain a new organism that can convert an
existing compound into a commercial
compound
Introduce new genes
Modify existing genes

Engineering E. coli to produce

indigo
Mutate tryptophan synthase
complex to release indole
Introduce napthalene
dioxygenase (from Pseudomonas
putida)

natural source of indigo:

woad [Isatis tinctoria]

woad

Pict (painted--with woad)

Introduce isatin hydrolase (from a

soil microbe) to prevent
production of indirubin (color)
from isatin

blue

burgundy

Potential routes for overproducing

biological compounds
Remove rate-limiting transcriptional
controls
Remove rate-limiting enzyme allostery
controls
Kinetically enhance rate-limiting enzymes
Genetically block competing pathways
Enhance commitment of carbon to the
pathway of interest
Enhance transport of compound out of cell

How to overproduce phenylalanine?

1) Remove feedback
inhibition (select strains
resistant to
phenylalanine
analogues)
2) Avoid repression (place
genes under control of
non-phe controlled
promoters)
3) Remove pathway
competition (delete tyr
and trp specific genes)
4) Overexpress phespecific genes
5) Increase E4P and PEP
synthesis

Rational metabolic engineering

Requires at least some knowledge of the
biochemical pathway required for
compound synthesis
Trial and error--try something, see if it
works, or where new block is (and focus
on the new block)
Potentially very labor intensive
But high degree of control over the
organism

Non-E.coli Bacterial Cloning

Homologous recombination

Cloning in bacteria
other than E.coli?
Utility:
Study bacterial processes and pathways
that may not be correctly expressed in E.
coli, eg. pathogenesis, antibiotic production
properties not available in E.coli, eg.
natural transformation

Disadvantages:
Often a poor selection of specialized
vectors
Transformation (by the usual techniques)
may be difficult

Necessary components for non-E.coli

cloning

Method for introducing DNA

Transformation (spontaneous)
Transformation (chemical, electroporation)
Conjugation
Method for replicating DNA
Plasmid replicon
Integration into chromosome (homologous
recombination)
Cloned gene must be expressed in the non-E.
coli host (if you want to use the new host as an
expression vector)

Natural transformation
Spontaneous uptake of DNA from the
environment
(Likely to be a major route for
horizontal gene transfer)
Fairly common in bacteria-- but this is
one thing E. coli cannot do!

Conjugation as a method of transfer

Promiscuous plasmids--selftransmissible to many hosts
(not a complete substitute for
transformation, since DNA must often
be manipulated in vitro, then
reintroduced)

Plasmid Host Range

Host-range of plasmid replicons is highly
variable
E. coli specialized vectors:
have narrow host range
But their range can be increased by
creating hybrid plasmids that replicate in
E. coli and in new host: Shuttle Vectors

Integration by recombination
If transformed DNA has homology to
chromosome (or other plasmid), this DNA can
be integrated by homologous recombination
Two pieces of DNA with the same sequence:
RecA protein guides a complex that causes
strand exchange between homologous
sequences
Homologous recombination is rare but
spontaneous (with a highly predictable
frequency: ~ 1/1000 cells will recombine)

Homologous recombination: portrait of a single cross-over

Recombination (single
crossover)

Transfer plasmid (or linear piece of DNA) into host in

which it cannot replicate
Select for antibiotic marker

Recombination in genome engineering:

(PCR product)

Tet r

recombination
(genome)
flank

gene

flank

(engineered
genome)
Cell is Tet r, and red gene is knocked out

Things that can be easily done with PCR products,

transformation, and recombination.

Gene deletions (with or without the antibiotic

resistance gene)
Addition of tags to chromosomal proteins
Gene replacement (targeted mutagenesis)

Recap
Non-E.coli bacteria can be useful for recombinant
DNA studies, though not as versatile as E. coli
Natural transformation is an important feature of
some species
Shuttle vectors: hybrid plasmids with more than
one type of replicon to increase host range
Recombination is an important tool for
maintaining recombinant DNA and for
manipulating the genome

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.

H4F
CH2=H4F

HCHO

H4MPT

XCH =H MPT
2

Serine Cycle

Problems:

PHA

HCHO builds up and is toxic

Cells cant generate enough energy for growth

CO2

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

10

10

10

C
10
10

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: 552833, 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
stop growth.

Gene
deletions
that
should slow
growth.

Gene deletions that should not

affect 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 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