Directed Evolution of
a Fungal Peroxidase
Joel R. Cherry et al.
Irene Woo
Enzong Yap
Question

Explain the difference between initial activity
and residual activity.
Presentation Outline
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Introduction
Methods and Results
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Primary Rounds of Random Mutagenesis
Site-specific Randomization
Secondary Round of Random Mutagenesis
Primary Round of In Vivo Shuffling of Mutations
Secondary Round of In Vivo Shuffling
Discussion
Q&A
What is Directed Evolution?
• Genetically alter enzymes to improve their
performance under application-specific
conditions
Coprinus cinereus (CiP)
Heme Peroxidase

Removes H2O2 created by Superoxide dismutase

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
2O2- + 2H-  H2O2 + O2
H2O2  H2O + O2
[SOD]
[CiP]
Peroxidase catalyzes the oxidation of dyes that
leach out of colored clothing in the wash
rendering them colorless
Cyclic Redox Reaction

Cyclic Redox Reaction
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CiP + H2O2  Cpd 1 + H2O
Cpd1 + Dye (reduced)  Cpd2 + Dye (oxidized)
Cpd3 + Dye (reduced)  CiP + Dye (oxidized) + H2O

Dye (oxidized)= colorless
Goal of Directed Mutations
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Improved stability and activity of the dyetransfer inhibitor CiP peroxidase
• Screen with different wash conditions
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High pH 10.5
High temperature 500C
High peroxide concentration 5-10mM
How to Create Directed Mutations
•
•
•
Mutations are accumulated in successive
generations
Sequential mutation coupled with random
recombination.
DNA shuffling
Experiment Flowchart
Site Directed
Mutagenesis (SD)
1st round Random
mutagenesis (R1)
Site Specific Mutagenesis
2nd round Random mutagenesis (R2)
In Vivo Shuffling
Presentation Outline
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
Introduction
Methods and Results
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Site Directed Mutagenesis
Primary Rounds of Random Mutagenesis
Site-specific Randomization
Secondary Round of Random Mutagenesis
Primary Round of In Vivo Shuffling of Mutations
Secondary Round of In Vivo Shuffling
Discussion
Q&A
Site Directed (SD) Mutagenesis
http://www.web-books.com/MoBio/Free/Ch9G.htm
Site directed mutagenesis to
target protein structural features
Stability
 Solvent exposed amino acids – potential
unstable sites
 Salt bridge and disulfides –stabilizing
structures
Activity
 Active site charge and accessibility
Selection of Mutant
Generate
Mutations
Initial activity
assay [ABTS]
Enzyme inactivation step
(screening/selection)
Residual
activity assay
Initial Activity, %Residual Activity
Mutant A
Mutant B
Initial Activity “mph”
10 POXU/ml
5 POXU/ml
Activity after
screening/inactivation “mph”
Residual Activity “%
max speed”
1 POXU/ml
3 POXU/ml
10%
60%
POXU where 1 U is the amount of peroxidase required to oxidze one 1umol H2O2 per min in pH7 buffer
SD Data
Oxidizable residue sites
 M242I, Y272F, and M166F
Destabilizing Interaction

E239
Presentation Outline
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Introduction
Methods and Results
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Site Directed Mutagenesis
Primary Rounds of Random Mutagenesis
Site-specific Randomization
Secondary Round of Random Mutagenesis
Primary Round of In Vivo Shuffling of Mutations
Secondary Round of In Vivo Shuffling
Discussion
Q&A
Random mutagenesis (R1)
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Error Prone PCR on wild type genome
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Identify regions to target distinct from sitedirected mutagenesis
Random Mutagenesis 1 Data
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V53A increased initial activity
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E239 responsible for increased residual activity
 Prove by inserting each individual mutation into wild type CiP
Presentation Outline
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Introduction
Methods and Results
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Site Directed Mutagenesis
Primary Rounds of Random Mutagenesis
Site-specific Randomization
Secondary Round of Random Mutagenesis
Primary Round of In Vivo Shuffling of Mutations
Secondary Round of In Vivo Shuffling
Discussion
Q&A
Site specific randomization
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Mutations from Site directed mutagenesis [ie: E239K]
Mutations from 1st round random mutagenesis [ie: E239G]

(NN) GC
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Substitute amino acid
E239A
Screening
E239
E239G
E239R
N.B. Different from site directed
Best Mutant
Site Specific Randomization Results
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E239
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G
Plastic
Non bulky AA
Legend
20 min at pH 10.5
at 23°C (black
bars),
23°C + 0.2 mM
H2O2 (whitebars),
50°C (striped bars),
50°C + 0.2 mM
H2O2 (gray bars).
Other Site Specific Randomization
with no improvements
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V53A
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M166F vs. M166L
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No improved enzyme activity
Improved peroxide stability but no improved
thermal stability
E214(partner to E239)

No improvement in stability
Presentation Outline


Introduction
Methods and Results
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Site Directed Mutagenesis
Primary Rounds of Random Mutagenesis
Site-specific Randomization
Secondary Round of Random Mutagenesis
Primary Round of In Vivo Shuffling of Mutations
Secondary Round of In Vivo Shuffling
Discussion
Q&A
2nd Round Random Mutagenesis (R2)
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Best combination from site-directed and
random mutagenesis
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[Mutant 072(E239G, M242I, and Y272F)]
Error Prone PCR with Mutant 072
Random Mutagenesis 2 Data
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Improved mutant 072
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Increased stability at the cost of reduced activity
Presentation Outline
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Introduction
Methods and Results
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Primary Rounds of Random Mutagenesis
Site-specific Randomization
Secondary Round of Random Mutagenesis
Primary Round of In Vivo Shuffling of Mutations
Secondary Round of In Vivo Shuffling
Discussion
Q&A
In Vivo Shuffling

Separate the trade off between
activity and stability phenotypes
Shuffling out deleterious mutations

Shuffle the 10 best mutants found from 2nd round
of mutagenesis representing a spectrum of
activity and stability
High Stability
Low initial activity
High stability
High initial activity
Low stability
High initial activity
In Vivo Shuffling Process
PCR
In Vivo
Shuffling
Transform into Yeast
• Amplify 10 best mutants by PCR
• Transform into yeast
• Yeast efficiently recombines PCR
fragments
In Vivo Shuffling inside Yeast
Mutant
Fragments
3’
5’
Linearized 5’
vector with
homologous ends
for recombination
3’
Re-circularized
Autonomously Replicating
Plasmid
pJC106
Advantages of Shuffling

Shuffling generates combination of amino
acid substitutions that gave even better
enzymes than each individual mutants
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Better odds of finding best combination in
mutagenesis library from shuffling than from
manual mutagenesis
Proof of Concept of Shuffling in Yeast Cells
Different silent
or signal
sequence
substitution for
972 and 974
suggest these
mutations arose
from different
recombination
events
Non-Linked Phenotype
Results of In Vivo Shuffling

Separation activity and stability phenotypenot linked

Mutants 972 and 974 combined the residual
activity of the most stable mutant with the initial
activity of the most active mutant.
Second Round of In Vivo Shuffling
Novel
149S/T, V53A, and M166F mutant that acts
synergistically-not predicted by site specific randomization
Presentation Outline


Introduction
Methods and Results
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Site Directed Mutagenesis
Primary Rounds of Random Mutagenesis
Site-specific Randomization
Secondary Round of Random Mutagenesis
Primary Round of In Vivo Shuffling of Mutations
Secondary Round of In Vivo Shuffling
Discussion
Q&A
Discussion

3/7 including best mutants were not predicted
through random and site specific mutagenesis
requires in vivo shuffling

Found improved stability and activity of the dyetransfer inhibitor under stringent wash conditions :



High pH 10.5
High temperature 500C
High peroxide concentration 5-10mM
Future application

Shuffling a family of genes from diverse
species
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Combining protein design ideas with
techniques of random discovery
Oversights

Where did the 10 mutants for in vivo shuffling
come from?How was In Vivo Shuffling was
done? Assumed we knew the process.

Missing important mutants in Table 1 (ie: the
novel mutant and mutant with 92% activity
found in R2)
Q&A