Ifrah Ishaq University Of The Punjab, Lahore.

Directed Evolution

outlineDirected EvolutionHistorical View Of Directed EvolutionProcess Of Directed EvolutionWhy Use This Approach?Types Of MutationsNaturally Evolutionary Processes

o Random Mutagenesis Methodso Gene Recombination Methods

Library SizeSelection & Screening StrategiesApplications Of Directed EvolutionAdvantages Of Directed EvolutionFuture DirectionsConclusion

Protein Engineering ApproachesThere are two approaches of protein engineering.1.Rational designUsed detailed knowledge of structure & function of the protein to make desired changes.

2. Directed evolutionRandom mutagenesis is applied to protein, a selection of mutant variant of desired characteristics.

Definition of natural selection & directed evolution?

Natural SelectionRandom genetic mutation in the genetic code from which beneficial mutations are preserved because they are beneficial for survival of organism.

Directed EvolutionA method used in protein engineering that mimics natural selection to evolve proteins or nucleic acids towards user defined goal.

Similarities Between Directed Evolution & Natural Selection

•Diversification•Selection•amplification

Directed Evolution Is first used in 70’s.

Around 0.01-1% of all random mutations estimated to be beneficial. This technique involve randomly introducing mutations at the genetic level followed by selection for the desired characteristics at the protein level.

Reason To Use The Word Evolution

Evolution is a walk from one functional protein to another in the landscape of all possible sequences.

Takes inspiration from natural process of evolution.

Historical View Of Directed Evolution 1967- in vitro Drawian experiment 1971- theory of evolution at molecular level 1980- rational mutagenesis approaches 1986- succeed in first directed evolution 1997- use directed evolution; improve

enantiselectivity of enzyme. and the progress continues in this field……..

Process Of Directed EvolutionThe progress begins by determining a target biomolecule, metabolic pathway or organism and a desired phenotypic goal.

Steps Of Directed Evolution1.Selection of gene of interest2.Generation of mutant gene library3.Expression of mutant genes4.Screening & selecting of beneficial gene5.Further rounds of mutagenesis to achieve phenotypic goal.

Diagram representation

Why use this approach?To achieve same goals as other methods of

protein engineering:Understanding protein functionImproving protein properties for industry,

medicine….To improve existing proteins functionally.Improve the working of enzymes: substrate

specificity, improve stability over a range of temperature and pH.

Requirements Of Directed Evolution

There are four pre-requistics for directed evolution:1.Availability of genes of interest2.Suitable expression system3.Method to create mutant libraries4.Screening and selection system

Genetic CodeThe genetic code is the set of rules by which

information encoded in genetic code ( DNA or RNA sequence) is translated into proteins.

Degeneracy of genetic code.Example: GAA & GAG both specify glutamic acid(redundancy). Neither of them specifies other amino acid ( no ambiguity).

Crick Wobble hypothesis.

Types Of Mutation

“ Mutation is permanent alteration of the nucleotide sequence of the genome of an organism, virus or extra chromosomal DNA or other genetic material.” _ normal sequence AGC ( serine)Silent mutation- AGT serineMissence mutation- GGC prolineNon-sense mutation- ATC terminator

Frame Shift Mutation: Deletion & Insertion

Suppressor Mutation

Second mutation cancels the effect of first mutation. May occur in same gene or in different.1.Intragenic (same gene)2.Intergenic (different gene)

Transition & Tranversion Mutation

Naturally Evolutionary Processes

There are two natural evolutionary processes which have been adapted for in-vitro evolution are:

1.Gene Recombination2.Random Mutagenesis

Gene RecombinationRecombination can be divided into four categories:-Homologous RecombinationNon- Homologous RecombinationReciprocal RecombinationSite- Specific Recombination

Random MutagenesisRandom mutagenesis can be divided into five categories:-1.Transitions2.Transversions3.Deletions4.Insertion5.Inversion

Random Mutagenesis Methods

1. Chemical MutagenesisAgents include ethyl methanesulfonate(EMS),

deaminating compounds such as nitrous acid, base analogous such as 2-aminopurine and ultra violet irradiation.

2. Mutagenic StrainsMutator strains of E.coli are deficient in one or more DNA repair genes, leading to single base substitutions at a rate of approximately 1 mutation per 1000 base pairs.

Generate mutant librariesGene of interest cloned in plasmidProcess Is simple

3. Error- prone PCRError prone PCR relies on misincorporationof nucleotides by DNA polymerase to generate point mutations.

Increased magnesium concentration Supplementation with manganese Use mutagenic dNTR analogous Role to increase mutation rate

4. Saturation Mutagenesis

Site-directed mutagenesis uses an oligonucleotide primer to introduce a single base pair substitution at specified position in gene.

Saturation mutagenesis involve substitution of all possibly a.a at pre-determined residue or continuous series of residue in protein of interest.

5. Sequence Saturation MutagenesisThis strategy is able to randomize a DNA sequence at every nucleotide position through the use of universal base. Universal base is enzymatically inserted.

6.Random Insertion/Deletion MutagenesisAllow the deletion of up to 16 bases from random sites and subsequent insertion of bases( random) at the same position.

Homologous Recombination Methods

1. DNA Shuffling

2. Gene shuffling

using endonuclease digestion at restriction sitesSequence homology still required at digested siteOverlap extension to occur

3. Family shuffling

Family of related genes with homologyCreation of chimeric libraries

4.Staggered Extension Process(step)

5. Random Chimeragenesis On Transient Templates( RACHITT)

6.Degenerate Oligonucleotides Gene Shuffling (DOGS)

Utilizes a PCR reaction with degenerate endsComplementary primers pairs to shuffle genesLimited sequence similarity & G+C content.

7.Recombination By Random Priming In Vitro Recombination (RPR)

Generate of small DNA fragmentsUtilizes elongation from random sequence primers

RPR

8.Assembly PCR or synthetic shuffling

Non-homologous Recombination Methods

1. Incremental truncation hybrid (ITCHY)

2.Non-homologous random recombination (NRR)

3. Sequence Homology-independent Protein Recombination (SHIPREC)

4.SCRATCHY

Additional diversity can also be created byshuffling of two ITCHY libraries. This method, termed SCRATCHY.

Two initial ITCHY libraries serve as starting material for DNA shuffling.

Library SizeNumber of possible variants of a protein that can be created by introducing M mutations simultaneously over N amino acids.The number of sequence variants for M substitutions in a given protein of N a.a:- 19M.N!/(N-M)!M!

Methods Of Isolating Functional Variants

There are two main categories:1.Selection2.screening

Screening & Selection Strategies

1. Phage Display

2. mRNA Display

3. Ribosome Display

4.In-vitro Compartmentalization

Applications Of Directed Evolution

Some of the examples explained below: Cephalosporins: class of antibiotic

produced via the intermediate 7-aminocephalosporanic acid (7-ACA).

Directed evolution has been used to improve the activity of cephalosporin acylases to produce these intermediates from adipyl-7-ACA or cephalosporin C.

• Atorvastatin• Is A Cholesterol Lowering Drug• Enzyme:2-deoxyribose- 5-phosphate Aldolase

(DERA)• Enzyme Target By Directed Evolution.

Atorvastatin Drug

Goal: better luciferase

Advantages of directed evolution

Frequently used in Protein Engineering:-Improving protein stabilityImproving binding affinity of antibioticsAlerting substrate specificity

Application in genetic engineering, functional genomics & gene therapy.

Applied to improve polymerases, nucleases, transposases, integrases & recombinase etc.

can modify pH or temperature dependence enzymes

vaccines – improve effectiveness; less side effects

In agriculture field, modify plants for tolerance for herbicides & toxins.

Golden rice express elevated beta-carotene.

Comparison of directed evolution and rational design

conclusion 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.

Future directions

o Directed evolution is an integral tool in the development of synthetic enzymes, ensuring they are suitable for use.

o The past success of this approach indicates that it will continue to provide many examples of safe and efficient production of chemical products.

references Sen, S., Venkata Dasu, V. and Mandal, B. (2007)

Developments in directed evolution for improving enzyme functions. Applied Biochemistry and Biotechnology, 143, 212–223.

Yuan, L., Kurek, I., English, J. and Keenan, R. (2005) Laboratory-directed protein evolution. Microbiology and Molecular Biology Reviews, 69, 373–392.

Hibbert, E.G., Baganz, F., Hailes, H.C. et al. (2005) Directed evolution of biocatalytic processes. Biomolecular Engineering, 22, 11–19.