Directed Evolution

1. Jonathan Sun University of Illinois at Urbana Champaign BIOE 506 February 15, 2010 Directed Evolution http://www.sliceofscifi.com/wp-content/uploads/2008/02/nc_evolution_080103_ms.jpg

2. Outline • Introduction • Motivation • Methods • Applications • Conclusions University of Illinois

3. 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 University of Illinois

4. 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 University of Illinois

5. 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 University of Illinois

6. 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 University of Illinois

7. 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 University of Illinois

8. 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 University of Illinois

9. Basic Method • A parent gene is selected • Mutations/diversity are induced (mutagenesis or recombination) • Selection criteria applied • Repeat with new parent genes selected Bloom and Arnold: In the light of directed evolution: Pathways of adaptive protein evolution University of Illinois

10. Random Mutagenesis • Traditional method • Point mutation based – error prone PCR • Frequency of beneficial mutations very low • Multiple mutations virtually impossible to come out positive University of Illinois

11. 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 University of Illinois

12. RACHITT • Random Chimeragenisis on Transient Templates • Small DNA fragments hybridized on a scaffold to create a chimeric DNA fragment • Incorporates low-homology segments University of Illinois

13. 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 University of Illinois

14. 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 University of Illinois

15. MURA • Random fragmentation of parental gene • Reassembled with unidirectional primers for specific restriction site • Generates N-terminally truncated DNA shuffled libraries University of Illinois

16. 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 University of Illinois

17. 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 University of Illinois

18. 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 University of Illinois

19. 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 University of Illinois

20. The Case of a Fluorescent Protein • dsRED – parent protein evolved to have better solubility and shorter maturation time dsRed mCherry University of Illinois

21. 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 University of Illinois

22. 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 University of Illinois

23. 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 University of Illinois

24. Pharmaceuticals • Therapeutic proteins • Antibodies – natural somatic recombination • Vaccines – improved effectiveness, less side effects • Viruses – gene therapy and vaccine development University of Illinois

25. 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 insecond version • Not approved for distribution http://en.wikipedia.org/wiki/Golden_rice University of Illinois

26. 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 University of Illinois

27. Thank You! Questions? University of Illinois