Directed evolution

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Directed EvolutionBy

Ifrah IshaqUniversity Of The Punjab,

Lahore.

Directed evolution written by Ifrah Ishaq Page 0

Directed Evolution

OutlineDirected evolution written by Ifrah Ishaq Page 1

Directed Evolution Historical View Of Directed Evolution Process Of Directed Evolution Why Use This Approach? Types Of Mutations Naturally Evolutionary Processes

o Random Mutagenesis Methods

o Gene Recombination Methods

Library Size Selection & Screening Strategies Applications Of Directed Evolution Advantages Of Directed Evolution Future Directions Conclusion

Protein Engineering ApproachesThere are two approaches used for the protein engineering:

1. Rational Design


In rational design, the scientist used detailed knowledge of the structure and function of the protein to make desired changes. In general, this has the advantage of being inexpensive and technically easy. The major drawback of this is that the detailed structural knowledge of a protein is often unavailable and even when it is available; it can extremely difficult to predict the effects of various mutations.

2. Directed EvolutionIn directed evolution, random mutagenesis is applied to protein; a selection regime is used to pick out variants that have the desired qualities. Further rounds of mutation and selection are the applied. Directed evolution avoids this problem by creating libraries of variants processing desired properties. Definition of natural selection & directed evolution?

Natural selection

Natural selection is that, overtime random genetic mutations that occur within an organism’s genetic code from which beneficial mutations are preserved because they are beneficial for the survival of the organism.

Directed evolution

Directed evolution is the method used in protein engineering that mimics the process of natural selection to evolve proteins or nucleic acids towards a user defined goal. It operates at molecular level (i.e., no new organisms are created) and focuses on specific molecular properties.

Similarities Between Directed Evolution & Natural Selection


The similarities between the natural selection and the directed evolution are that:

Diversification: offspring’s are different from the parents Selection: survival of the fittest Amplification: procreation

Directed EvolutionDirected evolution is first used in 70’s.Around 0.01% to 1% Random mutations estimated to be beneficial. Based on natural selection processes but in much quicker timescale. This technique involves randomly introducing mutations at the genetic level followed by selection for the desired characteristics at the protein level.

Reason To Use The Word EvolutionThe reason to use the word evolution is that it takes inspiration from the natural process of evolution. The mutations that occur in the particular animal, plant or bacteria etc. then it lives in better than its competitors and survive. That animal, plant, bacteria or virus etc. would propagate. Evolution is a walk from one functional protein to another in the landscape of all possible sequences.

Historical View Of Directed EvolutionThe historical view of directed evolution described below:

1967: S.spiegelman report an in-vitro Drawian experiment using self-replicating RNA

1971: M.Eigen reports a theory of evolution at the molecular level.

1980: Rational mutagenesis approaches to engineer enzymes show only limited success.


1986: Researches at Synergen succeed in first directed evolution using an iterative rational mutagenesis approach.

1997: M.T. Reetz and K.E Jaeger et al use directed evolution to improve entantiselectvity of an enzymatic resolution.

Further research continues on with major advancements of technologies used in directed evolution approach.

Process of Directed EvolutionThe method of directed evolution involves an iterative strategy. The process begins by determining a target biomolecule, metabolic pathway, or organism and a desired phenotypic goal.The steps involved in directed evolution are:1. The first is the selection of gene of interest that formed the

desired protein.2. The gene encoding a protein of interest is mutated to generate

a library of mutant genes. A diverse library of mutants is generated in-vivo or in-vitro through methods that mirror the strategies of traditional evolution. Introduction of random mutations in the genetic material.

3. Expression of mutant genes provides the library of mutant proteins. A high through put screening or selection method is used to identify improved progeny among the library, which are subsequently used as parents in the second round of cycle.

4. The proteins are screened or selected based on the desired property.

5. The variants with modified activity are sequenced or used for further rounds of mutagenesis and selection. Rounds of these steps typically repeated, using the best variant from one round as a template for the next to achieve stepwise improvements. The process is repeated until the phenotypic goal is achieved or when no further improvement of the phenotype is observed despite repeated iterations.


Diagram Representation

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

engineering: Understanding protein function Improving protein properties for industry, medicine….

Understanding of the relationship between protein sequence, structure and function is limited.

Biotechnology- increased demand for specific properties that don’t necessarily occur naturally.

Can be used to improve existing proteins functionally.

Directed evolution provides the mean to enhance the performance of enzymes under requisite process conditions and customize the reactions they catalyze. Directed evolution tools have been used to improve synthesis yield of desired products, limit or expand substrate specificity, alter co-factor specificity and improve stability over a wide range of temperature and pH.


Requirements Of Directed Evolution

There are four pre-requisites for directed evolution represented by major steps of in-vitro evolution experiment:

i. The availability of the genes encoding the enzymes of interest.

ii. A suitable expression system.iii. An effective method to create mutant libraries.iv. And a suitable screening and selection system.Creation of diversity through the library construction methods is a crucial step in directed evolution experiments. In further steps, altered genes are cloned into plasmid for expression in a suitable host organism. The most common approaches for recombinant protein expression employ the cellular machinery of well-established organisms such as Escherichia coli, Saccharomyces cervisiae or Bacillus subtilis.To recreate evolution in laboratory, the evolution of natural selection must be accelerated such that meaningful diversity can be created and selected in much shorter time-frame, mere days to weeks favored.

Before study mutagenesis, first revise the previous knowledge of genetic code, types of mutations for understanding of approaches used in directed evolution strategy.

Genetic CodeThe genetic code is the set of rules by which information encoded in genetic code (DNA or RNA sequences) is translated into proteins (amino acid sequences) by living cells. Degeneracy is the redundancy of the genetic code. The genetic

code has redundancy but no ambiguity.


For example: although codons GAA and GAG both specify glutamic acid (redundancy) neither of them specifies any other amino acid (no ambiguity). Code is heavily redundant. That 64 codons code 20 amino

acids. Crick Wobble hypothesis: Third base makes little difference.

If first two bases have 6 H bonds, third base is irrelevant that’s why the degeneracy of the codon.

Types of Mutations “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

Silent mutations are mutations in DNA that don’t significantly alter the phenotype of the organism in which they occur. For example:Consider the normal sequence. Normal sequence AGC (serine)-Changes in the third base that shows the silent mutationAGT serineMissence mutation

This type of mutation is the change in one DNA base pair that results in the substitution of one amino acid for another in the protein made by gene. For example


Consider the normal sequence. Normal sequence AGC (serine)- Changes in the first base that shows the missence mutation.

GGC proline

Non-sense mutation A non-sense mutation is also a change in one DNA base pair. Instead of substituting one amino acid to another. The altered DNA sequence prematurely signals the cell to stop building a protein. For example:Consider the normal sequence. Normal sequence AGC (serine)-changes in the second base of the normal sequence shows the non-sense mutation.ATC terminatorDeletion (Frame shift mutation)

A deletion changes the number of bases by removing a piece of DNA. The deleted DNA may alter the function of the resulting protein.Insertion (Frame shift mutation)

An insertion changes the number of DNA bases in a gene by adding a piece of DNA. As a result, the protein made by gene may not function properly.Example of deletion and insertion:


Suppressor mutation

Second mutation cancels effect of the first mutation. May occur in same gene or in different.

o Intragenic (in same gene): +1 frame shift canceled by -1 frame shift. Improper folding compensated by other change.

o Intergenic (in different gene): usually tRNA mutation. Inserts “correct” amino acid to “wrong” codon.

Transition mutation

A mutation in which a purine/pyrimidine base pair is replaced with a base pair in the same purine/pyrimidine relationship.Transversion mutation

A mutation in which a purine/pyrimidine replaces a pyrimidine/purine base pair vice versa.Example of transition and transversion mutation:


Naturally Evolutionary ProcessesThe two natural evolutionary processes which have been adapted for in-vitro evolution are:

1. Gene Recombination2. Random Mutagenesis

Gene RecombinationGene recombination refers to the exchange of blocks of genetic material among two or more DNA strands. Recombination can be divided into four main types:1. Homologous Recombination:

Homologous recombination is that where recombination occurs between two genes with high sequence identity.

2. Non- Homologous Recombination:Non-homologous recombination is that where recombination occurs between two DNA sequences with little or no sequence identity.

3. Reciprocal Recombination:


Reciprocal recombination is that in which a symmetrical exchange of genetic material occurs between two DNA strands.

4. Site- Specific Recombination:Site-specific recombination is that in which specialized nucleotide sequence exhibiting some degree of target site specificity is moved between non-homologous sites within a genome.

Random MutagenesisRandom mutagenesis refers to changes in genome resulting from improper DNA replication or in adequate repair of DNA damage following events such as irradiation, exposure to oxidative or alkylating agent and natural deamination of cytosine. Random mutagenesis can be divided into five categories:1. Transition2. Transversion3. Deletions4. Insertions5. Inversion: which involves the 180 degree rotation of a double

stranded DNA of two base pairs or longer.In-vitro random mutagenesis methods have been developed to generate substitution, deletion or insertions. One of the simplest and most popular directed evolution tools, Error-Prone Polymerase Chain Reaction takes advantage of the fallibility of DNA polymerase to generate random pair substitution.

Random Mutagenesis Methods1.Chemical Mutagenesis


Agents include alkylating compounds such as ethyl methano sulfonate (EMS), deaminating compounds such as nitrous acid, base analogous such as 2-aminopurine and ultraviolet irradiation. Chemical mutagenesis is sufficient to deactivate genes at random for a genome-wide screen.

2.Mutagenic StrainsPropagating a gene of interest in a mutational strain represents the simplest method of random mutagenesis. Mutator strains of E.coli are deficient in one or more DNA repair genes, leading to single base substitutions at a rate of approximately 1 mutaion per 1000 base pairs and mutation cycle. To generate a mutant library, the gene of interest is cloned in plasmid or phagemid and propagated into mutator E.coli through a limited number of replications. The process is relatively easy and commercial mutator strains such as XL1-Red.

3. Error-Prone PCRError-Prone PCR relies on the mis-incorporation of nucleotides by DNA polymerase to generate point mutation in a gene sequence. The low fidelity of DNA polymerases under certain conditions generates point mutations during PCR amplification of a gene of interest. Increased magnesium concentration supplementation with manganese or the use of mutagenic dNTPs can reduce the base pairing fidelity and increase mutation rate.


4.Saturation MutagenesisSite-directed Mutagenesis uses an oligo-nucleotide primer to introduce a single base pair substitution at a specified position I a gene.Saturation Mutagenesis involves the substitution of all possible amino acids randomly at the pre-determined residue or continuous series of residues in the protein of interest.

5.Sequence Saturation MutagenesisSequence saturation mutagenesis is that in which the universal base doxyinosine is enzymatically inserted throughout the target gene. This strategy is able to randomize a DNA sequence at every nucleotide position through the use of universal base.

6.Random insertion/deletion(RID) MutagenesisIn this strategy, allows the deletion of up to 16 bases from random sites on the target gene and subsequent insertion of a random or pre-determined sequence of any number of bases at the same position.


Homologous Recombination MethodsHomologous recombination methods are explained below:

1.DNA ShufflingThis described by Stemmer. Fragments of gene through the use of DNase and then allows fragments to randomly prime one another in a PCR reaction without adding primers.

2.Gene ShufflingThis done through by using endonuclease digestion at restriction sites, rather than DNase 1 digestion, however sequence homology surrounding the digested restriction sites is still required for overlap extension to occur.

3.Family Shuffling


Family shuffling enables the creation of chimeric libraries from a family of related genes with homology.

4.Staggered Extension Process (step)This strategy utilizes primer elongation to generate small DNA fragments for recombination. In which elongation step is interrupted prematurely by heat denaturation. Subsequent annealing allows incomplete extension products to switch templates, effecting recombination of multiple DNA templates into one amplicon.

5.Random Chimeragenesis On Transient Templates(RACHITT)

In this technique, a uracil containing parent gene is made single-stranded to serve as a scaffold for the ordering of the top strand fragments of the additional, homologous parent gene and recombination occur when fragments from different parent genes hybridize to scaffold. Pfu DNA polymerase 3’- 5’ exonuclease activity removes the unhybridized 5’ or 3’ overhangs flaps created by fragment annealing and also fills gaps between the annealed fragments using transient scaffold as a template. The template strand is eliminated by treatment with uracil DNA-Directed evolution written by Ifrah Ishaq Page 16

glycosylase before applying the template chimera hybrid to PCR, resulting in amplification of double stranded, homoduplex chimmeral gene products.

6.Degenerate Oligonucleotides Gene Shuffling (DOGS)This strategy utilizes a PCR reaction with degenerate ends, complementary primer pairs to shuffle genes with limited sequence similarity and G+C content.


7.Recombination by Random Priming In Vitro Recombination (RPR)This method utilizes elongation from random sequence primers to generate a collection of small DNA fragments complementary to different areas of template sequence.

7.Assembly PCR or synthetic shufflingAlso known as assembly of designed oligonucleotides. The fragments to be shuffled are degenerate oligonucleotides that are chemically synthesized and encode all the variations in a family of homologous genes. In these reactions, overlapping primers extend one another, after multiple cycles the process yields full-length gene products in which each combination of mutation bearing oligonucleotide has been recombined.


Non-Homologous Recombination Methods

Non-Homologous recombination methods are explained below:

1.Incremental Truncation Hybrid (ITCHY)This is achieved through controlled digestion of DNA by exonuclease III to generate a collection of all possible truncated fragments of the parent genes, followed by blunt end ligation of the fragments to form hybrid proteins. Tight control of exonuclease activity is required in addition to frequent removal of digested fragments and quenching of the reaction, in order to collect a variety of fragment lengths.

2. Non-Homologous Random Recombination (NRR)Non-homologous random recombination method uses DNase fragmentation followed by blunt end ligation to generate


diverse topological rearrangements (deletions, insertions, domain reordering). Any no of DNA sequencing with little or no homology.

3.Sequence Homology-Independent Protein Recombination (SHIPREC)This strategy involves the fusion of two parent genes and creation of a library of random length fragments. Two parent genes are joined in first step. With linker between them containing a unique restriction site. The fusion product is then digested with DNase I to form a library of random fragments and fragments of length corresponding to the size of either parent gene are isolated and then treated with SI nuclease to produce blunt ends. The fragment are then circularized by blunt end ligation and relinerized by digestion at the restriction site within the linker sequence.


4.SCRATCHYDescribed by Ostermeier. Using this technique diversity can be created by shuffling of two ITCHY libraries. 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. Considering sequence variation, using only 20 amino acids. The


number of sequence variants of M substitutions in a given protein of N amino acids is 19M.N! / (N-M)!M!

Methods Of Isolating Functional VariantsTwo main categories of method exist for isolating functional variants:

1. Selection2. screening

SelectionSystems directly couple protein function to the survival of the gene.ScreeningSystems individually assay each variant and allow a quantitative threshold to be set for sorting a variant or population of variants of a desired activity.

Screening & Selection StrategiesThe screening and selection strategies are explained below:

1.Phage DisplayA technique that uses bacteriophage (virus that infect bacteria) to connect proteins with the genetic information that encodes them. In this technique a gene encoding a protein of interest is inserted into a phage coat protein gene, causing a phage to display the protein on its outside while containing the gene for the protein on its inside, resulting a connection between genotype and phenotype. These displaying phages can then be screened against other proteins, peptides or DNA sequences in order to detect interaction between displayed protein and then other molecules. In this way, large libraries of proteins can be screened and amplified in a process called in-vitro selection.


2.mRNA DisplayIt is a technique used to select proteins that bind to specific target. It allows for the identification of these selected proteins because they are covalently attached to the DNA that codes for them. The process results in translates peptides or proteins that are associated with their mRNA progenitor through a puramycin linkage. The complex then binds to an immobilized target in a selection step. The mRNA protein fusions that bind well are then reverse transcribed to cDNA and their sequence amplified through PCR. The result is a nucleotide sequence that encodes a protein with high affinity for the molecule of interest.


3.Ribosome Display Ribosome display is another display method; it uses a cell-free system for synthesis of a polypeptide chain on the mRNA template. Protein synthesis in this system is accompanied by formation of the ternary protein–ribosome–mRNA complex. This complex is then isolated from solution using capacity of the synthesized antibody fragment to bind the target antigen. Using this method it is possible to select simultaneously the highest affinity antibody fragments together with their mRNA. In this case a ribosome functions as a stabilizer of the complex. The mRNA is then subjected to reverse transcription; resulting cDNA is amplified by PCR and the resulting PCR products are used for plasmid construction for recombinant antibody fragments.


4.In-Vitro CompartmentalizationStart with the gene library that is attach with the substrate, then generate a water and oil lotion like the artificial cell that have self-machinery of transcription and translation in the compartment.so that gene transcribed into RNA and translated into enzyme. That enzyme then able to act on the substrate that attach to the gene. Usually a fluorescent product identifies that compartment. Then isolate the gene and generate a new library and then that isolated gene encoding the desired activity.


Applications Of Directed EvolutionSome of the examples explained below: Cephalosporins


Cephalosporins are a class of antibiotic produced via the intermediate 7-aminocephalosporaniacid(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. Using site-directed saturation mutagenesis and a selection system, a mutant was found that increased the catalytic efficiency toward adipyl-7-ADCA by 36-fold.

Atorvastatin Drug The cholesterol-lowering drug atorvastatin, marketed as Lipitor, is an example where biocatalysis research has been applied extensively and is in industrial use. The enzyme 2-deoxyribose- 5-phosphate aldolase (DERA) has been a target of directed evolution for the production of atorvastatin intermediates. One


variant, S238D, showed new activity towards 3-azidopropinaldehyde to form an azido pyranose which is an intermediate in atorvastatin synthesis. By screening mutants with a microplate reader or with gas chromatography, they managed to increase the synthesis of the intermediate by 10-fold.

Advantages Of Directed EvolutionDirected evolution is frequently used for protein engineering as an alternative to rational design. The advantages of directed evolution are described below:o Improving protein stability for biotechnological use at high

temperature or in harsh solvents.o Improving binding affinity of therapeutic anti bodies.o Altering substrate specificity of existing enzymes.o It has been applied to improve polymerases, nucleases,

integrases, recombinase, transposases.o Applications in genetic engineering, functional genomics and

gene therapy.o It can modify pH or temperature dependence parameters of

enzymes.o Vaccines- improve effectiveness and fewer side effects.o In agriculture field, plant produced. With increased tolerance

for herbicides or expression of toxins.o Golden rice- express elevated beta-carotene(vitamin A

precursor).

Comparison Of Directed Evolution & Rational DesignDiagrammatically explanation of directed evolution and rational design:


Future DirectionsThe complexity of today’s pharmaceutical compounds and an increasing awareness of the environmental impact of traditional chemical syntheses have opened the door to biocatalysis. Directed evolution is an integral tool in the development of synthetic enzymes, ensuring theyare suitable for use in an industrial setting. The past success of this approach indicates that it will continue to provide many examples of safe and efficient production of chemical intermediates and medical compounds.

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 crop ultimately, combining tools will lead to better understanding and applications.

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.


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Directed evolution