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

2. Question Explain the difference between initial activity and residual activity.

3. 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

4. What is Directed Evolution? Genetically alter enzymes to improve their performance under application-specific conditions

5. Coprinus cinereus (CiP) Heme Peroxidase Removes H 2 O 2 created by Superoxide dismutase 2O 2 - + 2H -  H 2 O 2 + O 2 [SOD] H 2 O 2  H 2 O + O 2 [CiP] Peroxidase catalyzes the oxidation of dyes that leach out of colored clothing in the wash rendering them colorless

6. Cyclic Redox Reaction CiP + H 2 O 2  Cpd 1 + H 2 O Cpd1 + Dye (reduced)  Cpd2 + Dye (oxidized) Cpd3 + Dye (reduced)  CiP + Dye (oxidized) + H 2 O  Dye (oxidized)= colorless

7. 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 50 0 C High peroxide concentration 5-10mM

8. How to Create Directed Mutations Mutations are accumulated in successive generations Sequential mutation coupled with random recombination. DNA shuffling

9. Experiment Flowchart Site Directed Mutagenesis (SD) 1 st round Random mutagenesis (R1) Site Specific Mutagenesis In Vivo Shuffling 2nd round Random mutagenesis (R2)

10. 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

11. Site Directed (SD) Mutagenesis http://www.web-books.com/MoBio/Free/Ch9G.htm

12. 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

13. Selection of Mutant Generate Mutations Enzyme inactivation step (screening/selection) Initial activity assay [ABTS] Residual activity assay

14. Initial Activity, %Residual Activity Mutant AMutant B Initial Activity - “mph” 10 POXU/ml5 POXU/ml Activity after screening/inactivation - “mph” 1 POXU/ml3 POXU/ml Residual Activity “% max speed” 10%60% POXU where 1 U is the amount of peroxidase required to oxidze one 1umol H 2 O 2 per min in pH7 buffer

15. SD Data Oxidizable residue sites M242I, Y272F, and M166F Destabilizing Interaction E239

16. 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

17. Random mutagenesis (R1) Error Prone PCR on wild type genome Identify regions to target distinct from site- directed mutagenesis

18. Random Mutagenesis 1 Data V53A increased initial activity E239 responsible for increased residual activity Prove by inserting each individual mutation into wild type CiP

19. 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

20. Site specific randomization Mutations from Site directed mutagenesis [ie: E239K] Mutations from 1 st round random mutagenesis [ie: E239G] (NN) GC E239 E239A E239R E239G Substitute amino acid Best Mutant Screening N.B. Different from site directed

21. Site Specific Randomization Results E239 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).

22. Other Site Specific Randomization with 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

23. 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

24. 2 nd Round Random Mutagenesis (R2) Best combination from site-directed and random mutagenesis [Mutant 072(E239G, M242I, and Y272F)] Error Prone PCR with Mutant 072

25. Random Mutagenesis 2 Data Improved mutant 072 Increased stability at the cost of reduced activity

26. 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

27. In Vivo Shuffling Separate the trade off between activity and stability phenotypes

28. Shuffling out deleterious mutations Shuffle the 10 best mutants found from 2 nd round of mutagenesis representing a spectrum of activity and stability High Stability Low initial activity Low stability High initial activity High stability High initial activity

29. 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

30. In Vivo Shuffling inside Yeast Mutant Fragments Linearized vector with homologous ends for recombination 5’ pJC106 3’ 5’ 3’ Autonomously Replicating Plasmid Re-circularized

31. 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

32. 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

33. Non-Linked Phenotype

34. 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.

35. Second Round of In Vivo Shuffling Novel 149S/T, V53A, and M166F mutant that acts synergistically-not predicted by site specific randomization

36. 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

37. 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 50 0 C High peroxide concentration 5-10mM

38. Future application Shuffling a family of genes from diverse species Combining protein design ideas with techniques of random discovery

39. 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)

40. Q&A