Directed evolution

Directed evolution written by Ifrah Ishaq Page 0
Directed Evolution
By
Ifrah Ishaq
University Of The Punjab, Lahore.

Directed Evolution

Outline
 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 Approaches
There 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 Evolution
In 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 Evolution
Directed 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 Evolution
The 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 Evolution
The 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 Evolution
The 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 Code
The 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 mutation
AGT serine
Missence 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
- 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:
-changes in the second base of the normal sequence shows the non-
sense mutation.
ATC terminator
Deletion (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 Processes
The two natural evolutionary processes which have been adapted for
in-vitro evolution are:
1. Gene Recombination
2. Random Mutagenesis
Gene Recombination
Gene 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 Mutagenesis
Random 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. Transition
2. Transversion
3. Deletions
4. Insertions
5. 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 Methods
1.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 Strains
Propagating 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 PCR
Error-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 Mutagenesis
Site-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 Mutagenesis
Sequence 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) Mutagenesis
In 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 Methods
Homologous recombination methods are explained below:
1.DNA Shuffling
This 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 Shuffling
This 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-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 shuffling
Also 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.SCRATCHY
Described 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 Size
Number 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 Variants
Two main categories of method exist for isolating functional variants:
1. Selection
2. screening
Selection
Systems directly couple protein function to the survival of the gene.
Screening
Systems 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 Strategies
The screening and selection strategies are explained below:
1.Phage Display
A 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 Display
It 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 Compartmentalization
Start 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 Evolution
Some 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 Evolution
Directed 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 Design
Diagrammatically explanation of directed evolution and rational
design:
Future Directions
The 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 they
are 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.

Directed evolution

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