Directed Evolution

Directed Evolution

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  Ifrah Ishaq University OfThe Punjab, Lahore. Directed Evolution
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  outline Directed Evolution Historical ViewOf 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
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  Protein engineering APProAches Thereare two approaches of protein engineering. 1.Rational design Used detailed knowledge of structure & function of the protein to make desired changes. 2. Directed evolution Random mutagenesis is applied to protein, a selection of mutant variant of desired characteristics.
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  Definition of naturalselection & directed evolution? Natural Selection Random genetic mutation in the genetic code from which beneficial mutations are preserved because they are beneficial for survival of organism. Directed Evolution A method used in protein engineering that mimics natural selection to evolve proteins or nucleic acids towards user defined goal.
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  Similarities Between DirectedEvolution & Natural Selection •Diversification •Selection •amplification
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  DirecteD evolution  Isfirst 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.
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  Reason To UseThe 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.
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  historicAl view ofDirecteD 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……..
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  Process of DirecteDevolution The progress begins by determining a target biomolecule, metabolic pathway or organism and a desired phenotypic goal. Steps Of Directed Evolution 1.Selection of gene of interest 2.Generation of mutant gene library 3.Expression of mutant genes 4.Screening & selecting of beneficial gene 5.Further rounds of mutagenesis to achieve phenotypic goal.
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  DiAgrAm rePresentAtion
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  why use thisAPProAch? To achieve same goals as other methods of protein engineering: Understanding protein function Improving 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.
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  requirements of DirecteD evolution Thereare four pre-requistics for directed evolution: 1.Availability of genes of interest 2.Suitable expression system 3.Method to create mutant libraries 4.Screening and selection system
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  genetic coDe The geneticcode 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.
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  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 serine Missence mutation- GGC proline Non-sense mutation- ATC terminator
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  Frame Shift Mutation:Deletion & Insertion
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  Suppressor Mutation Second mutationcancels the effect of first mutation. May occur in same gene or in different. 1.Intragenic (same gene) 2.Intergenic (different gene)
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  Transition & TranversionMutation
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  Naturally EvolutioNary ProcEssEs There aretwo natural evolutionary processes which have been adapted for in- vitro evolution are: 1.Gene Recombination 2.Random Mutagenesis
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  GENE rEcombiNatioN Recombination canbe divided into four categories:- Homologous Recombination Non- Homologous Recombination Reciprocal Recombination Site- Specific Recombination
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  raNdom mutaGENEsis Random mutagenesiscan be divided into five categories:- 1.Transitions 2.Transversions 3.Deletions 4.Insertion 5.Inversion
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  raNdom mutaGENEsis mEthods 1.Chemical Mutagenesis Agents include ethyl methanesulfonate(EMS), deaminating compounds such as nitrous acid, base analogous such as 2- aminopurine and ultra violet irradiation.
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  2. Mutagenic Strains Mutatorstrains 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 libraries Gene of interest cloned in plasmid Process Is simple
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  3. Error- pronePCR Error 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
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  4. Saturation Mutagenesis Site-directedmutagenesis 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.
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  5. Sequence SaturationMutagenesis This 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 Mutagenesis Allow the deletion of up to 16 bases from random sites and subsequent insertion of bases( random) at the same position.
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  homoloGous rEcombiNatioN mEthods 1. DNAShuffling
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  2. Gene shuffling usingendonuclease digestion at restriction sites Sequence homology still required at digested site Overlap extension to occur 3. Family shuffling Family of related genes with homology Creation of chimeric libraries
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  4.Staggered Extension Process(step)
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  5. Random ChimeragenesisOn Transient Templates( RACHITT)
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  6.Degenerate Oligonucleotides Gene Shuffling(DOGS) Utilizes a PCR reaction with degenerate ends Complementary primers pairs to shuffle genes Limited sequence similarity & G+C content. 7.Recombination By Random Priming In Vitro Recombination (RPR) Generate of small DNA fragments Utilizes elongation from random sequence primers
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  RPR
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  8.Assembly PCR orsynthetic shuffling
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  NoN-homologous RecombiNatioN methods 1. Incrementaltruncation hybrid (ITCHY)
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  2.Non-homologous random recombination (NRR)
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  3. Sequence Homology-independentProtein Recombination (SHIPREC)
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  4.SCRATCHY Additional diversity canalso be created by shuffling of two ITCHY libraries. This method, termed SCRATCHY. Two initial ITCHY libraries serve as starting material for DNA shuffling.
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  libRaRy size Number ofpossible 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!
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  methods of isolatiNg fuNctioNalVaRiaNts There are two main categories: 1.Selection 2.screening
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  scReeNiNg & selectioN stRategies 1.Phage Display
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  2. mRNA Display
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  3. Ribosome Display
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  4.In-vitro Compartmentalization
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  ApplicAtions of DirecteD evolution Someof 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.
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  • Atorvastatin • IsA Cholesterol Lowering Drug • Enzyme:2-deoxyribose- 5-phosphate Aldolase (DERA) • Enzyme Target By Directed Evolution. AtorvAstAtin Drug
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  goAl: better luciferAse
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  ADvAntAges of DirecteD evolution Frequentlyused in Protein Engineering:- Improving protein stability Improving binding affinity of antibiotics Alerting substrate specificity Application in genetic engineering, functional genomics & gene therapy. Applied to improve polymerases, nucleases, transposases, integrases & recombinase etc.
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   can modifypH 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.
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  compArison of DirecteDevolution AnD rAtionAl Design
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  conclusion  Directed evolutioncan 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.
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  future Directions o Directedevolution 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.
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  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.