Transcript Directed Evolution - University of Illinois at Urbana

Jonathan Sun
University of Illinois at Urbana Champaign
BIOE 506
February 15, 2010
http://www.sliceofscifi.com/wp-content/uploads/2008/02/nc_evolution_080103_ms.jpg
Outline
Introduction
 Motivation
 Methods
 Applications
 Conclusions

February 15, 2010
University of Illinois
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Evolution
Darwin => natural selection
 1970 – John Maynard Smith

 Evolution is a walk from one functional
protein to another in the landscape of all
possible sequences
 “Fitness” of protein based on
favorability for reproduction or
based on experimenter in
artificial selection
Romero and Arnold: Exploring Protein Fitness Landscapes by Directed Evolution
February 15, 2010
University of Illinois
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Picture (not many more to come)
Screening
criteria is
important
 Stability can be
used instead of
improvement
 Allows for
functionally
neutral
mutations

Romero and Arnold: Exploring Protein Fitness Landscapes by Directed Evolution
February 15, 2010
University of Illinois
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What is Directed Evolution?
An engineering strategy used to improve
protein functionality through repeated
rounds of mutation and selection
 First used in the ‘70s
 Around .01-1% of all random mutations
estimated to be beneficial
 Based off natural evolution processes,
but in a much quicker timescale

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University of Illinois
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Another (more direct?) Method
Rational design – modify protein
function based on understanding
consequences of certain changes
 We are still relatively ignorant as to how
a protein’s gene sequence encodes
functionality
 Directed evolution avoids this problem
by creating libraries of variants
possessing desired properties

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University of Illinois
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Why is it Needed?
Biotechnology – increased demand for
specific properties that don’t necessarily
occur naturally
 Can be used to improve existing
proteins’ functionality
 Can be applied as far as the ideas come
– enzymes and catalysts to
pharmaceuticals or crops

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University of Illinois
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Successful Directed Evolution

Desired function should be/have:
 Physically feasible
 Biologically or evolutionarily feasible
 Libraries of mutants complex enough to
contain rare beneficial mutations
 Rapid screen to find desired function

Increases understanding of protein
function and evolution – disconnects
protein from natural context
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University of Illinois
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Basic Method
Bloom and Arnold: In the light of directed evolution: Pathways of adaptive protein evolution
A parent gene is selected
 Mutations/diversity are induced
(mutagenesis or recombination)
 Selection criteria applied
 Repeat with new parent genes selected

February 15, 2010
University of Illinois
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Random Mutagenesis
Traditional method
 Point mutation based – error prone PCR
 Frequency of beneficial mutations very
low
 Multiple mutations virtually impossible to
come out positive

February 15, 2010
University of Illinois
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DNA Shuffling
Recombination used to create chimeric
sequences containing multiple beneficial
mutations
 “Family shuffling” of homologous genes
 “Synthetic shuffling” – oligonucleotides
combined to create full-length genes
 Whole-genome shuffling – accelerated
phenotypic improvements
 Drawback – high homology required

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University of Illinois
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RACHITT
Random Chimeragenisis on Transient
Templates
 Small DNA fragments hybridized on a
scaffold to create a chimeric DNA
fragment
 Incorporates low-homology segments

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University of Illinois
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Even More Methods






Assembly of Designed Oligonucleotides
(ADO)
Mutagenic and Unidirectional Reassembly
(MURA)
Exon Shuffling
Y-Ligation-Based Block Shuffling
Nonhomologous Recombination – ITCHY,
SCRATCHY, SHIPREC, NRR
Combining rational design with directed
evolution
February 15, 2010
University of Illinois
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ADO
Nonconserved regions with conserved
parts as linkers
 PCR with dsDNA without primers
 Full length genes in expression vector
 Creates large diversity of active variants
without codon bias for parental genes

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University of Illinois
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MURA
Random fragmentation of parental gene
 Reassembled with unidirectional primers
for specific restriction site
 Generates N-terminally truncated DNA
shuffled libraries

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University of Illinois
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Exon Shuffling
Similar to natural splicing of exons
 Chimeric oligos mixed together,
controlling combination of which exons
to be spliced
 Protein pharmaceuticals based on
natural human genes – less immune
response

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University of Illinois
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Nonhomologous Recombination
Creation of new protein folds
Structures not present in nature – useful for
evolution of multifunctional proteins
 Incremental truncation for the creation of
hybrid enzyme (ITCHY) – two genes in
expression vector with unique restriction
sites, blunt end digestion, ligated >SCRATCHY
 Nonhomologous random recombination –
potentially higher flexibility in fragment size
and crossover frequency


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University of Illinois
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A Combination
Rational design with directed evolution
 Success depends on ability to predict
fitness of a sequence
 Computationally demanding
 Kuhlman et al created a new protein fold
 Focuses library diversity for directed
evolution

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University of Illinois
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Directed Evolution in Action
Has been applied to improve
polymerases, nucleases, transposases,
integrases, recombinases
 Applications in genetic engineering,
functional genomics, and gene therapy
 Optimized fluorescent proteins and
small-molecule probes for imaging and
techniques like FRET

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University of Illinois
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The Case of a Fluorescent Protein

dsRED – parent protein evolved to have
better solubility and shorter maturation
time
dsRed
February 15, 2010
mCherry
University of Illinois
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Biochemical Catalysts
Useful in industry because of high
selectivity and minimal energy
requirements
 Need for high availability at low costs
 Active and stable under process
conditions – not naturally occuring
 Some reaction enzymes still yet to be
identified and produced

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University of Illinois
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Application to Enzymes
Improve stability and activity of biochemical
catalysts
 Can modify pH or temperature dependence
 Substrate specificity or catalytic activity
 MANY applications:

 Proteolytic – Subtilisin in detergents
 Cellulolytic and esterases – biofuel production
 Cytochrome P450 superfamily – catalyze
hydroxilation

Whole metabolic pathway evolution
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University of Illinois
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Whole Metabolic Pathways
Closer to natural compound production
 Single enzyme activity upregulation
does not necessarily lead to increase in
final product
 Different methods:

 Whole genome shuffling
 Key enzymes targeted
 Naturally expressed operons targeted
 Target gene regulation factors
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University of Illinois
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Pharmaceuticals
Therapeutic proteins
 Antibodies – natural somatic
recombination
 Vaccines – improved effectiveness, less
side effects
 Viruses – gene therapy and vaccine
development

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University of Illinois
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Agriculture
Plants with increased tolerance for
herbicides or expression of toxins
 Golden rice

 Expresses elevated beta-carotene (Vitamin
A precursor)
 Directed evolution
- 23 times more in
second version
 Not approved for
distribution
http://en.wikipedia.org/wiki/Golden_rice
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University of Illinois
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Conclusions
Directed evolution can be a powerful
tool taking advantage of nature’s power
to improve upon itself
 Used in a wide variety of applications for
protein improvement – stability, activity,
substrate specificity, etc
 Potential for genetically engineering
improved drugs or crops
 Ultimately, combining tools will lead to
better understanding and applications

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University of Illinois
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Thank You!
Questions?
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University of Illinois
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