This document discusses directed evolution of enzymes. It describes how Frances Arnold developed directed evolution techniques including error-prone PCR, DNA shuffling, and StEP to improve enzyme functionality for desired traits like activity, stability, selectivity. Her techniques combine random mutagenesis and recombination with selection to evolve enzymes, allowing optimization and redesigning of enzyme function. Directed evolution has led to enzymes catalyzing new reactions and expanded our understanding of what reactions enzymes can catalyze.

1. DIRECTED EVOLUTION OF ENZYMES
PRESENTED BY:
DEEPANKER BISHT
P-2145
VETERINARY BIOTECHNOLOGY DIVISION

2. outline
Introduction
Devising Strategies
Developments

3. THE NOBEL PRIZE – CHEMISTRY 2018

4. Professor of Chemical Engineering,
Bioengineering and Biochemistry
at the California Institute of
Technology (Caltech).
Mechanical Engineer
Aerospace Engineer
Finally a Protein Engineer
Conducted the first directed
evolution of enzymes
motivation
175 Men have won the Chemistry Nobel Prize……(Irony?)
Image courtesy: CalTech, Welcome Collection, Keystone/Getty images, Weizmann Institute of Science

5. Nature’s Catalytic repertoire
May be after
billions of
years…..sad
Nature- The best chemist of all time
Work by Macromolecular Protein Catalysts
Good models for Chemical and Renewable
resources harvest
Nature’s enzymes are the products of
evolution
Charles Darwin – Natural Selection

7. Directed evolution
Engineering strategy used to improve protein functionality through repeated rounds
of mutation and selection, recombining beneficial mutations as needed, and
continuing until we reach the target level of performance.
Based on natural processess but in
quicker time scale.
Recovers activity in unusual
environments (e.g. organic solvents)
Improving activity on non-native
substrates
Enhancing thermostability
Changing enantioselectivity
Arnold ,2017

8. Choice of starting state
Promiscuous activity of enzyme
under study
A starting point with no activity
for the intended reaction is useless
Uphill walk on a protein fitness
landscape
Higher elevation means
optimized proteins
Ruggedness affects the ability to
find uphill paths to fitter sequences
Romero and Arnold ,2009

9. strategic methods in practice
"Asexual" Evolution by Sequential Rounds of Random Mutagenesis
 Error prone PCR (epPCR)- introduces random point mutations in a
population of DNA products
Incorporates several parameters for
tuning mutation rate
Large mutation rates promotes greater
improvements
Can introduce undesired functionality in
the protein
Yang, Lee and Kim, 2017

10. “Sexual" Evolution by in vitro Recombination through DNA shuffling
 DNA shuffling- allow random recombination typically between parent
genes with 70% homology
Creates libraries of genes containing
combinations of mutations
Minor variations allow greater control
over the point mutagenesis
Allow rapid accumulation of beneficial
mutations identified in separate genes
Stemmer, 1994

11. “Sexual" Evolution by in vitro Recombination through StEP
 Staggered Extension Process- priming the template sequences followed by
repeated cycles of denaturation and extremely abbreviated annealing/
polymerase catalyzed extension
Prepares full length recombined genes
from a fragment pool
Explores chimeras of functional parent
enzyme sequences
Retains a high proportion of functional
progeny
Zhao et al., 1998

12. Proof-of-principle
Cytochromes P450: a very large Enzyme Superfamily whose members catalyze a
wide range of reactions
Ancestral enzyme
Mutation & Selection
Hydroxylation
Epoxidation
Sulfoxidation
Dealkylation
Decarboxlation
Nitration
Coelho et al., 2013

13. Biological Reagents +
Natural Selection

14. Can a P450 do this?
Can it evolve?
Form the Fe-Carbenoid
intermediate
Transfer the carbene to
second substrate

15. P450 BM3 268A 323 1:99 -99
Wang et al., 2014

16. applications
NEW CHEMICAL REACTIONS
Single (aqueous, room temperature) biocatalytic step replaces multiple chemical
steps with reduction in cost and waste.
Hernandez ,K. et al., 2016

17. applications
BIOFUELS- a green alternative
Suitable replacements or supplements for fossil fuels, which can be produced in a
sustainable and environmentally- friendly manner.

18. applications
Organic Synthesis and Industry
From academic settings to Industrial Applications
Bulk and Fine Chemicals
Consumer Products
Laboratory reagents and pharmaceuticals
Intermediates for pharmaceutical industry
Taste enhancers
Drugs against diabetes and vascular plaques
Lipid lowering pharmaceuticals
Lipases used in detergents

19. Summary & outlook
Highly efficient protocol for development of biocatalysts with high specificity,
limited side reactions and tolerance of diverse reaction conditions.
Versatile path towards optimised enzymes and enzymes with novel functions.
Enzymes can be tuned to catalyse new reactions and to reactions very different
from the ones catalysed by nature’s own enzymes.
Ample room for optimisation and redirection of enzyme function in terms of
reactivity, substrate specificity and chemical reactions, as well as tolerance to
various reaction conditions.
 Instead of simply asking what enzymes do in nature, we can now ask the
question, “what CAN they do?”

20. Revolution in Evolution

21. REFERENCES
 Arnold, F. (2017). Directed Evolution: Bringing New Chemistry to Life. Angewandte
Chemie International Edition, 57(16), pp.4143-4148.
Chen K, Arnold FH (1993) Tuning the activity of an enzyme for unusual environments:
sequential random mutagenesis of subtilisin E for catalysis in dimethylformamide. Proc
Natl Acad Sci U S A. 90:5618-5622.
Romero, P. and Arnold, F. (2009). Exploring protein fitness landscapes by directed
evolution. Nature Reviews Molecular Cell Biology, 10(12), pp.866-876.
 Yang, M., Lee, H. and Kim, H. (2017). Enhancement of thermostability of Bacillus subtilis
endoglucanase by error-prone PCR and DNA shuffling. Applied Biological Chemistry,
60(1), pp.73-78.
Stemmer WP (1994) DNA shuffling by random fragmentation and reassembly: in vitro
recombination for molecular evolution. Proc Natl Acad Sci U S A. 91:10747-10751.
Zhao H, Giver L, Shao Z, Affholter JA, Arnold FH (1998) Molecular evolution by
staggered extension process (StEP) in vitro recombination. Nat Biotechnol 16:258-61.
Coelho PS, Brustad EM, Kannan A, Arnold FH (2013) Olefin cyclopropanation via carbene
transfer catalyzed by engineered cytochrome P450 enzymes. Science 339:307-310.
Glieder A, Farinas ET, Arnold FH (2002) Laboratory evolution of a soluble, self-sufficient,
highly active alkane hydroxylase. Nature Biotech 20:1135–1139.
McIntosh JA, Coelho PS, Farwell CC, Wang ZJ, Lewis JC, Brown TR, Arnold FH (2013)
Enantioselective intramolecular C-H amination catalyzed by engineered cytochrome P450
enzymes in vitro and in vivo. Angew Chem Int Ed Engl 52:9309-9312.