Directed Evolution of a Fungal Peroxidase

Irene Woo

Enzong Yap

Joel R. Cherry et al.

Question

Explain the difference between initial activity and residual activity.

Presentation Outline

Introduction Methods and Results

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 2O2

- + 2H- H2O2 + O2 [SOD]

H2O2 H2O + O2 [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 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

Improved stability and activity of the dye-transfer inhibitor CiP peroxidase

• Screen with different wash conditions 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

In Vivo Shuffling

2nd round Random mutagenesis (R2)

Presentation Outline

Introduction Methods and Results

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 MutantGenerate Mutations

Enzyme inactivation step (screening/selection)

Initial activity assay [ABTS]

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”

1 POXU/ml 3 POXU/ml

Residual Activity “% max speed”

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

Introduction Methods and Results

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)

Error Prone PCR on wild type genome

Identify regions to target distinct from site-directed mutagenesis

Random Mutagenesis 1 Data

V53A increased initial activity

E239 responsible for increased residual activity Prove by inserting each individual mutation into wild type CiP

Presentation Outline

Introduction Methods and Results

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

Mutations from Site directed mutagenesis [ie: E239K] Mutations from 1st round random mutagenesis [ie: E239G] (NN) GC

E239

E239A

E239R

E239G

Substitute amino acid

Best MutantScreening

N.B. Different from site directed

Site Specific Randomization Results

E239 G Plastic Non bulky AA

Legend20 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 Randomizationwith no improvements

V53A No improved enzyme activity

M166F vs. M166L Improved peroxide stability but no improved

thermal stability E214(partner to E239)

No improvement in stability

Presentation Outline

Introduction Methods and Results

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)

Best combination from site-directed and random mutagenesis [Mutant 072(E239G, M242I, and Y272F)]

Error Prone PCR with Mutant 072

Random Mutagenesis 2 Data

Improved mutant 072

Increased stability at the cost of reduced activity

Presentation Outline

Introduction Methods and Results

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 StabilityLow initial activity

Low stabilityHigh initial activity

High stabilityHigh initial activity

In Vivo Shuffling Process

PCR

Transform into Yeast

In Vivo Shuffling

• Amplify 10 best mutants by PCR• Transform into yeast• Yeast efficiently recombines PCR

fragments

In Vivo Shuffling inside YeastMutant Fragments

Linearizedvector with homologous ends for recombination

5’

pJC106

3’

5’ 3’

Autonomously ReplicatingPlasmid

Re-circularized

Advantages of Shuffling

Shuffling generates combination of amino acid substitutions that gave even better enzymes than each individual mutants

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 phenotype- not 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 ShufflingNovel 149S/T, V53A, and M166F mutant that acts synergistically-not predicted by site specific randomization

Presentation Outline

Introduction Methods and Results

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 dye-transfer 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

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