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Millennium Technology Prize Winner Frances Arnold 25.5.2016
The document discusses the role of enzymes in breaking down proteins and fats, highlighting their importance in green chemistry and energy reduction. It emphasizes the evolution of enzymes through natural selection and directed evolution, which enhances their functions for various applications, including improved synthetic processes and environmental sustainability. The text also explores future possibilities for enzymatic engineering to generate novel reactions and tailored solutions in biochemistry and agriculture.
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Millennium Technology Prize Winner Frances Arnold 25.5.2016
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- 6. What do enzymesdo for us?
- 7. Enzymes break downproteins and fats— use in laundry lowers energy costs
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- 13. But the catalystsare not designed to make what we want. And they are complicated!
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- 16. MTIKEMPQPKTFGELKNL... KETSPIPQPKTFGPLGNL... KQASAIPQPKTYGPLKNL... WRRRGIPGPLGYPLVGSF... WIRKGVKGPRGLPFLGVI... FIRKGIKGPRGFPGIGML... WIRKGVKGPRGFPFFGVI... WIRKGVKGPRGFPFFGVI... WMRKGIKGPRGLPFFGII... WMRKGVKGPRGRPFVGVL... WRRRGVVGPMGFPVLGVF... REKIGLSGPEPHWFLGNL... REKIGLTGPEPHWFMGNL... RSSIGIPGPPVHWLWGNL... KVSKYPKGPLPLPFIGNI... ... Enzyme families arethe products of millions of years of mutation and natural selection. mutation and selection Ancestral enzyme Evolution makes new enzymes extremely well. Each one is well adapted to its biological role.
- 17. Humans have alteredthe biological world using evolution by artificial selection for thousands of years.
- 18. Natural mechanisms forgenerating sequence diversity are limited.
- 19. Not so inthe test tube.
- 20. Protein space: VASTLY (Verymuch more than astronomically) large Mostly empty sequences20450 20 natural amino acids EVOLUTION IS HARD! ~ 450 amino acids long
- 21. Directed evolution isa molecular optimization on a fitness landscape, where fitness is performance, defined by the user. Fitness
- 22. Evolution works because… ? …theregions that life has discovered and explored are rich in function. And, at least in some of its many dimensions, the fitness landscape is smooth. This allows adaptation, one step (one mutation) at a time.
- 23. select/screen for improvements (cells) ~10 (a few) random mutations 4 ~10~10 715 NO Parent gene (= parent protein) Directed evolution exploits smooth paths in the fitness landscape.
- 24. select/screen for improvements YES repeat (cells) ~10 (a few) random mutations 4 ~10~10 715 NO Parent gene (= parent protein) Evolved gene (= evolved protein) Directed evolution exploits smooth paths in the fitness landscape.
- 25. Performance Generation 0 10 20 30 40 50 60 70 0 1 23 4 5 6 7 0 0 Gen1 Gen2 Gen3 Gen4 Gen5 Directed evolution by iterative mutagenesis and screening can re-optimize the enzyme Evolved enzyme doing a new job Native enzyme doing its native job Native enzyme doing a new job
- 26. Diabetes affects 422million people (5.9%) of the world’s population in 2014.5
- 27. Sit Sitagliptin synthesis Solvent waste Multi-stepchemical process is expensive and generates a lot of waste. Toxic heavy metals
- 28. Sit Relativeproductivity Directed enzyme evolution Savileet al. Science, 2010, 329, 5989 Replace all that with a single enzyme step! Round of Evolution 1 10 100 1000 10000 100000 1 2 3 4 5 6 7 8 9 10 11 12 100000 10000 1000 100 10 1 Improved 70,000 fold Started here
- 29. Sit Relativeproductivity Solvent waste reduced 60% Increasedyield from starting materials Directed enzyme evolution Replace all that with a single enzyme step! Round of Evolution 1 10 100 1000 10000 100000 1 2 3 4 5 6 7 8 9 10 11 12 100000 10000 1000 100 10 1 Improved 70,000 fold Toxic heavy metals eliminated Savile et al. Science, 2010, 329, 5989
- 30. Sit Replace all thatwith a single enzyme step! FDA Approval in 2012 AWARD Thomas Edison Patent Award Toxic heavy metals eliminated Solvent waste reduced 60% Increased yield from starting materials Savile et al. Science, 2010, 329, 5989 PRESIDENTIAL
- 32. We need bettercellulases to break down biomass and generate sugars for microbial chemicals and fuel production.
- 34. MTIKEMPQPKTFGELKNL... KETSPIPQPKTFGPLGNL... KQASAIPQPKTYGPLKNL... WRRRGIPGPLGYPLVGSF... WIRKGVKGPRGLPFLGVI... FIRKGIKGPRGFPGIGML... WIRKGVKGPRGFPFFGVI... WIRKGVKGPRGFPFFGVI... WMRKGIKGPRGLPFFGII... WMRKGVKGPRGRPFVGVL... WRRRGVVGPMGFPVLGVF... REKIGLSGPEPHWFLGNL... REKIGLTGPEPHWFMGNL... RSSIGIPGPPVHWLWGNL... KVSKYPKGPLPLPFIGNI... ... We can alsouse products of natural evolution to breed new enzymes by recombination (molecular sex)
- 35. Sexual recombination populatesfitness peaks (conservative of structure and function while making large jumps in sequence) Parent proteins, found in nature
- 36. Chimeric progeny can bebetter than their parents. Sexual recombination populates fitness peaks (conservative of structure and function while making large jumps in sequence)
- 37. We used recombinationand random mutations to make thermostable cellulases that hydrolyze more biomass at elevated temperatures 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1,0 35 45 55 65 75 85 95 Cellobiose(mM) Temperature (ºC) 3C6P HJPlus HICel6 HJCel6
- 38. Enzymes (and otherproteins) are highly ‘evolvable’ Proteins can adapt via simple uphill walks New functions by changing tiny fractions of (<2 %) of the sequence Scary fact: Many beneficial mutations are far from the active site. Directed evolution can optimize enzyme function in real time, circumventing our profound ignorance of how sequence encodes function.
- 39. How can wecreate whole new enzymes to expand the chemistry of the biological world? (catalyze reactions not known in nature)
- 40. How can aninherently conservative process like this create novelty? At any given time, nature takes the most probable paths (the fewest mutational ‘steps’).
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- 42. Evolution does it:the internet of living things Crowd-sourcing problem solving for 3+ billion years: trillions of organisms at work 24/7
- 43. Atrazine • 1950–1993 non-biodegradable;accumulated in soil • From 1993 onwards, fast degradation observed
- 45. “Novelty” is oftenalready there. Access to new features relies on some (even very small) functional overlap that can be drawn out and optimized during evolution. Native reaction New reaction
- 46. Natural ‘oxene’ transfer Non-natural Fe-carbene Non-natural Fe-nitrene We evolvedheme proteins that catalyze reactions not known in nature Ser Ser O O
- 47. Heme enzyme Cyclopropane-containing serotonin-norepinephrine reuptakeinhibitor (SNRI) used to treat clinical depression Jane Wang, Hans Renata et al., ACIE (2014)
- 48. Second most abundant element inthe Earth’s crust Si 14 28.0855 Silicon ...the literature is void of examples of biologically synthesized, either in vivo or in vitro, silicon-carbon bonds. Silicon 2009, 1, 147.
- 49. N H Si OH R R catalysts / ligands SiR3 reagentsfor cross-coupling Si 18 F-acceptors for PET imaging Si(i-Pr)3 Si(i-Pr)3 materials for organic LED biological probes and drug leads O N H H2N Me3Si O OH 18F RR 3
- 50. Rhodothermus marinus cytochromec Si H MeMe N2 Me O OEt Me O OEt Si MeMeRma cyt c +
- 52. Physically possible molecules… Relevantto biology Freed from the constraints of biological function, we can explore the physically possible proteins to find new capabilities.
- 53. Bacterial enzymes thatmimic human drug metabolism so that we can test the toxicity of new drug candidates
- 54. New ‘human’ enzymesto treat cancer
- 55. Proteins to monitorbrain chemistry or enable brain stimulation with light
- 56. A palette offluorescent sensor proteins for imaging
- 57. Better agricultural practices:combat crop pests, effects of climate change…
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