Directed Evolution of a Fungal Peroxidase Irene Woo Enzong Yap Joel R. Cherry et al.
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Transcript of Directed Evolution of a Fungal Peroxidase Irene Woo Enzong Yap Joel R. Cherry et al.
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 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
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
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
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
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)