Jonathan Sun University of Illinois at Urbana Champaign BIOE 506 February 15, 2010 .

Jonathan Sun

University of Illinois at Urbana Champaign BIOE 506

February 15, 2010

http://www.sliceofscifi.com/wp-content/uploads/2008/02/nc_evolution_080103_ms.jpg

Outline

Introduction Motivation Methods Applications Conclusions

February 15, 2010 University of Illinois 2

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

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

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


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


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


Successful Directed Evolution Desired function should be/have:

Physically feasibleBiologically or evolutionarily feasibleLibraries of mutants complex enough to

contain rare beneficial mutationsRapid screen to find desired function

Increases understanding of protein function and evolution – disconnects protein from natural context


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

Random Mutagenesis

Traditional method Point mutation based – error prone PCR Frequency of beneficial mutations very

low Multiple mutations virtually impossible to

come out positive


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


RACHITT

Random Chimeragenisis on Transient Templates

Small DNA fragments hybridized on a scaffold to create a chimeric DNA fragment

Incorporates low-homology segments


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


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


MURA

Random fragmentation of parental gene Reassembled with unidirectional primers

for specific restriction site Generates N-terminally truncated DNA

shuffled libraries


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


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


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


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


The Case of a Fluorescent Protein dsRED – parent protein evolved to have

better solubility and shorter maturation time


dsRed mCherry

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


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 detergentsCellulolytic and esterases – biofuel productionCytochrome P450 superfamily – catalyze

hydroxilation Whole metabolic pathway evolution


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 shufflingKey enzymes targetedNaturally expressed operons targetedTarget gene regulation factors


Pharmaceuticals

Therapeutic proteins Antibodies – natural somatic

recombination Vaccines – improved effectiveness, less

side effects Viruses – gene therapy and vaccine

development


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

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


Thank You!

Questions?


Jonathan Sun University of Illinois at Urbana Champaign BIOE 506 February 15, 2010 .

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