1DIRECTED EVOLUTION BY in vitroRECOMBINATIONINTRODUCTION:Two distinct ways to engineer proteins have appeared during the last few decades. Theseinclude a rational protein design and a directed evolution. Here emphasis will only be on directedevolution by in vitro recombination. This technique has been used to successfully to improve oralter properties of many proteins (biocatalysts).Directed evolution appeared as a conceptually similar experimental enzyme evolution in 1970s.During the last decade, directed evolution became a key technology of molecular enzymeengineering. Directed evolution on its own, i.e., not combined with the structural data, is more orless accidental process similarly to natural evolution. During this process, successful and welladapted mutants are selected for further rounds of improvement (iterative cycle). It passes throughthe way of natural evolution and speeds up some slow steps that would normally proceed forhundreds or thousands of years.Contribution of directed evolution is substantial especially in particular cases, whenneither the three-dimensional (3-D) structures nor the catalytic mechanisms of the enzymes areknown. In comparison with rational design approach that exploits computer modeling techniquesand site-directed mutagenesis, directed evolution offers certain advantages. While rational designemphasizes the understanding of protein structure and amino acids interactions at the beginning ofthe process, directed evolution do not rely on such input data.Figure: Comparison between rational design and directed evolution
2Genetic recombination constructs libraries of hybrids by recombining fragments from twoor more parents, with the goal of discovering hybrids with beneficial properties such as improvedThermostabilityActivitySubstrate specificityDrug resistanceStability binding affinityImproved folding and solubilityNew catalytic activityPrerequisites for Directed Evolution:For directed evolution we need:1. Gene encoding protein(s) of interest2. Effective method to create mutant library3. Suitable expression (usually microbial) system4. Suitable screening and selection systemGene Diversification by in vitro Recombination:Directed evolution by in vitro recombination can be done by:1. DNA shuffling2. Random priming (RPR)3. Staggered extension process (StEP)4. Random chimeragenesis on transient templates (RACHITT)5. Incremental truncation hybrid (ITCHY)6. SCRATCHY7. Sequence Homology-Independent Protein Recombination (SHIPREC)8. Recombined Extension on Truncated Templates (RETT)9. Degenerate Oligonucleotides Gene Shuffling (DOGS)DNA shuffling:The method was first invented by W.P.C Stemmer .There are a number of techniques of invitro recombination but the main theme or principle is same which is based on simple DNAshuffling. In this technique two or more homologous genes are taken and they are fragmented byDNase I then these fragments are used to carry out a PCR like process and no external primers are
3added these fragments themselves serve as primers. Then finally full-length gene is amplified.(1).Following figure shows the simple DNA shuffling.Figure: DNA shufflingRandom Priming (RPR):It is a Simple and an efficient method. Random primers such as random hexamers areannealed to the template DNAs and then extended by a DNA polymerase at or below roomtemperature. The resulting DNA fragments are subsequently assembled into full-length genes byrepeated thermocycling in the presence of a thermostable DNA polymerase. (2). It involves Priming template with random-sequence primers . Extension to generate a pool of short DNA fragments. The fragments are reassembled during cycles of denaturation, annealing andfurther enzyme-catalyzed DNA polymerization to produce a library of full-lengthsequences.Screening or selecting the expressed gene products leads to new variants with improvedfunctions
4Figure: Schematic of random priming in vitro recombination (RPR). For simplicity, only two DNA templatesare shown. Random hexanucleotide primers are annealed to the templates and extended by Klenow fragmentto yield a pool of different sized random extension products. After the removal of the oligonucleotides and thetemplates, the homologous fragments are reassembled into full-length chimerical genes in a PCR-like process.The full-length genes will be amplified by a standard PCR and subcloned into an appropriate vector.ExampleDNA shuffling of a family of over 20 human interferon-α (Hu-IFN- α genes was used to derivevariantswith increased antiviral and antiproliferation activities in murine cells. A clone with135,000-fold improved specific activity over Hu-IFN-a2a was obtained in the first cycle ofshuffling. After a second cycle of selective shuffling, the most active clone was improved285,000-fold relative to Hu-IFN-a2a and 185-fold relative to Hu-IFN-a1. Remarkably, the threemost active clones were more active than the native murine IFNαs.These chimeras are derivedfrom up to five parental genes but contained no random point mutations. These resultsdemonstrate that diverse cytokine gene families can be used as starting material to rapidly evolvecytokines that are more active, or have superior selectivity profiles, than native cytokine genes.(3)
5Staggered Extension Process (StEP)It was introduced by Arnold and Kuchner in 1997.Staggered extension process (StEP) is simplerand less labor intensive than DNA shuffling and other PCR-based recombination techniques thatrequire fragmentation, isolation, and amplification steps. StEP recombination is based on crosshybridization of growing gene fragments during polymerase-catalyzed primer extension.Following denaturation, primers anneal and extend in a step whose brief duration and suboptimalextension temperature limit primer extension. The partially extended primers randomly reannealto different parent sequences throughout the multiple cycles, thus creating novel recombinants.(4). StEP recombination is based on template switching during polymerase catalyzed primerextension. This method uses full-length genes as templates for the synthesis of chimeric progenygenes and does not involve fragmentation. It consists of priming denatured templates followed by repeated cycles of denaturation and extremely short annealing/extension stepsRecombinogenic events occur when the partially extended primers anneal randomly to differenttemplates based on sequence complementarily and extend further.Template removal is done bypassing the mixture through Microcon 100 filter. Due to template switching, most of thepolynucleotides contain sequence information from different parental sequences.
6Figure: StEP recombination, illustrated for two gene templates. Only one primer and single strands from thetwo genes (open and solid blocks) are shown for simplicity. During priming, oligonucleotide primers anneal tothe denatured templates. Short fragments are produced by brief polymerase-catalyzed primer extension that isinterrupted by denaturation. During subsequent random annealing-abbreviated extension cycles, fragmentsrandomly prime the templates (template switching) and extend further, eventually producing full-lengthchimeric genes. The recombinant full-length gene products can be amplified in a standard PCRExampleStEP was used to convert Bacillus subtilis subtilisin E (serine protease) into an enzymefunctionally equivalent to its thermophilic homolog thermitase from Thermoactinomyces vulgaris.Five generations of random mutagenesis, recombination and screening created subtilisin E 5-3H5,whose half-life at 83°C is 3.5 min and Topt is 76°C,identical with those of thermitase. The Toptof the evolved enzyme is 17°C higher and its halflife at is 200 times greater than that of wild-typesubtilisin E(5).
7Random Chimeragenesis on Transient Templates (RACHITT):Chimeragenesis on Transient Templates (RACHITT) has been used to create librariesaveraging 12 or even 19 crossovers per gene in a single round of gene family shuffling. Theheteroduplexed top strand fragments are stabilized on the template by a single, long annealingstep, taking advantage of full length binding by each fragment, rather than the binding of smalleroverlaps, and by carrying out reactions at relatively high ionic strength. Fragments containingunannealed 5 or 3-termini are incorporated after flap trimming using the endo and exonucleolytic activities of Taq DNA polymerase and Pfu polymerase, respectively. After gap fillingand ligation, the template, which was synthesized with uracils in place of thymidine, is renderednon-amplifiable by uracil-DNA glycosylase (UDG) treatment. Other methods of DNA shufflingby gene fragmentation and reassembly can result in reconstitution of one or all of the parentalgenes at unacceptably high frequencies in the final shuffled library(6–8).Figure: RACHITT begins with production of a single stranded bottom strand “Transient Template”containing uracil and production of single stranded top strand “Donor Fragments.” The fragments areannealed to the template and joined to form a continuous chimeric top strand. Anchor oligonucleotide (Anc)protects the template 5-terminus from the nucleases used to trim unannealed fragment flaps. The template isthen degraded and the chimeric top strand amplified and cloned to result in a gene family shuffled library.
8ExampleA library of variant enzymes was created by combined shuffling of the DNA encoding thehuman Mu class glutathione transferases GST M1-1and GST M2-2. The parental GSTs are 84%sequence identical at the protein level, but their specific activities with the substratesaminochromeand 2-cyano-1, 3-dimethyl-1-nitrosoguanidine (cyanoDMNG) differ by more than100-fold. Aminochrome is of particular interest as an oxidationproduct of dopamine and ofpossible significance in the etiology of Parkinson’s disease, and cyanoDMNG is a model forgenotoxic and potentially carcinogenic nitroso compounds. GST M2-2 has at least two ordersofmagnitude higher catalytic activity with both of the substrates thanany of the other known GSTs,including GST M1-1.Variant GST sequences were expressed in E. coli, and their enzymaticactivities with aminochrome, cyanoDMNG, and 1-chloro-2, 4-dinitrobenzene (CDNB) weredetermined in bacterial lysates. Such screening of more than 70 clones demonstrated a continuousrange of activities covering at least two orders of magnitude for each of the substrates. For a givenclone, the activities with aminochrome and cyanoDMNG, in spite of their different chemistries,were clearly correlated, whereas no strong correlation was found with CDNB. This functionalcorrelation suggests a common structural basis for the enzymatic mechanisms for conjugation ofaminochrome and denitrosation of cyanoDMNG. (9).Incremental Truncation Hybrid (ITCHY)This method was introduced by Ostermeirer in 1999.The template here is a doublestranded linear DNA fragment containing 2 or more linked genes.This method results in theproduction of hybrids or chimeric genes by use of exo-nuclease III and random incorporation ofαS-dNTPs. Truncation of the targeted DNA can be achieved by two ways: Exo III can be used to digest the DNA fragment first to produce 5` overhangs andthen extending in presence of αS-dNTPs, also called as thio-itchy primer extension. Alternatively, αS-dNTPs can be incorporated during PCR amplification of theentire plasmid, also called as thio-itchy PCR amplification.o The whole plasmid is amplified in the presence of αS-dNTPs by Klenowfragment.o Then allowed to undergo Exo III cleavage. The phosphothioateinternucleotide linkages are resistant to 3′→5′ exonuclease hydrolysis,rendering the target DNA resistant to degradation in an exonuclease IIItreatment.ITCHY does not rely on the parental genes containing regions of DNA sequencehomology to create crossovers. Fusion of the truncated gene fragments by blunt end ligation thengenerates the ITCHY library.
9Figure: Schematic overview of ITCHY using both thio itchy and primer extension.SCRATCHYSCRATCHY is a combination of the incremental truncation for the creation of hybridenzymes (ITCHY) technology and DNA shuffling. It generates combinatorial libraries of hybridproteins consisting of multiple fragments from two or more parental DNA sequences with norestriction to DNA sequence identity between the original sequences. The experimentalimplementation of SCRATCHY consists of two successive steps, an initial creation of an ITCHYlibrary, followed by a homologous recombination procedure such as DNA shuffling.
10Figure:The method requires two complementary vectors, carrying the genes A and B in alternating order.Following the generation of the ITCHY library, the linearized hybrid genes are selected for parental-sizehybrid DNA constructs. After subcloning these DNA fragments into the NdeI and SpeI sites of pSALect,sequences with the correct reading frame result in the expression of a trifunctional fusion protein whichrenders the host cells resistant to ampicillin. The plasmid DNA from colonies grown under these conditions isrecovered and can be used as starting material for DNA shuffling.Sequence Homology-Independent Protein Recombination (SHIPREC)SHIPREC results in a library of chimeras in which the hybrids generatedretain propersequence alignment with the parents. When parental genes arechosen that encode homologousproteins, this type of recombination can producea library of chimeric proteins in which thecrossovers occur at structurallyrelated sites. The startingmaterial is a fusion of the two genes ofinterest, with the C-terminus of thefirst gene and the N-terminus of the second gene joinedthrough a small linkercontaining a unique restriction site (e.g., PstI). The gene fusion is thenrandomly truncated using DNase I and S1 nuclease, creating a library of fusions
11ofvarying sizefrom this library, DNA corresponding to the length of theparentalgenes is isolated and subsequently circularized. The size selection ensures that thecircularization produces chimeras which retain proper sequence alignment with the parentalgenes. The resulting chimeras are linearized by cleaving the unique restriction site in the linker(i.e., PstI), and the library is cloned into an appropriate vector for screening or selection. (13).Figure:SHIPREC overview. A gene fusion comprised of the two parental genes connected by a uniquerestriction site is constructed. (1) This fusion is randomly fragmented using DNase I and S1 nuclease toproduce a library of gene fusions exhibiting varying length and containing blunt ends. (2) Gene fusionscorresponding to the length of the parental genes are isolated using gel electrophoresis and separated from therandom digest pool. (3) Single-gene length fragments are circularized by intramolecular blunt-end ligation. (4)Circular DNA is linearized by treatment with a restriction endonuclease that cuts in the linker that separates3 and 5ends of the original parental genes. This yields a library of chimeric genes that encode for proteinswith a N-terminal region originating from Parent B and a Cterminal region originating from Parent A. (5) Thechimeras are amplified and cloned into an appropriate vector for screening/selection.ExampleSequence homology–independent protein recombination (SHIPREC) is used to createlibraries of single-crossover hybrids of unrelated or distantly related proteins. The methodmaintains the proper sequence alignment between the parents and introduces crossovers mainly at
12structurally related sites distributed over the aligned sequences. SHIPREC is used to create alibrary of interspecies hybrids of a membrane-associated human cytochrome P450 (1A2) and theheme domain of a soluble bacterial P450 (BM3). By fusing the hybrid gene library to the gene forchloramphenicol acetyl transferase (CAT), researchers were able to select for soluble and properlyfolded protein variants. Screening for 1A2 activity (diethylation of 7-ethoxyresorufin) identifiedtwo functional P450 hybrids that were more soluble in the bacterial cytoplasm than the wild-type1A2 enzyme.(13)Recombined Extension on Truncated Templates (RETT)Its starting material is RNA. Fragments can be generated by using random primers forreverse transcription or by unidirectional serial truncation of cDNA with exonuclease III. Aspecific primer is afterwards annealed to complementaryssDNA fragmentation and extended byPCR. Short fragments extended fromthis specific primer (like in StEP) are annealed to anotherssDNA fragmentand thus switch templates. This extension is repeated until full length genesaregenerated, which then is used to generate dsDNA by PCR. (14).In spite of the great importance of in vitro recombination techniques in directed geneevolution, the current techniques have still some drawbacks to be overcome for moreefficient generation of gene libraryDNA fragmentation process in DNA shuffling and RACHITT method may not berandom because DNase I hydrolyzes DNA preferentially at sites adjacent topyrimidine nucleotides, which introduces a sequence bias into recombination libraryFor the recombination of genes with low or no homology, ITCHY and SHIPREC weredeveloped.These methods have limitations that only two parental genes can be recombined andthe created hybrids are limited to one crossover.RETT does not use DNA endonucleases for generation of shuffling blocksIn RETT, unidirectional single-stranded DNA (ssDNA) fragments are created by eitherDNA polymerase in the presence of random primers or serial deletion withexonucleaseThese unidirectional ssDNA fragments only act as templates in PCR, not as primersRETT generates random recombinant gene library by template-switching ofunidirectionally growing polynucleotides from primers in the presence ofunidirectional ssDNA fragments pool used as templates
13 According to truncation pattern of unidirectional ssDNA fragments, two methods aredescribed separately. (a) Two homologous genes are presented to simplify the model. Unidirectional ssDNAfragments are prepared by reverse transcription using in vitro-transcribed target RNA astemplate in the presence of random primers. Recombinational synthesis reaction is conducted as follow: (i) Specific primer is annealed to ssDNA fragment. (ii) Specific primer is extended during one cycle of PCR.
14 (iii, iv) Short fragments extended from primer are annealed to other ssDNA fragment bytemplate-switching and extended during another cycle of PCR. (v) Steps are repeated until full-length ssDNA genes are generated. (b) Unidirectional ssDNA fragments are prepared by serial deletion with exonuclease III. Recombinational synthesis reaction is conducted by steps (i–v).Degenerate Oligonucleotides Gene Shuffling (DOGS)A method for enhancing the frequency of recombination with family shufflingIt was designed to decrease the amount of parental DNA reassembled from shufflingprocedures. For DOGS complementary degenerate primers are designed for conserved motivesfound in the candidate genes.Each of these segments is flanked by primers and individuallyamplified.For the reassembly procedure the library of fragments can be put togetherat differentratios generating many biased libraries containing no parentalgenes. (15).
15Figure:complementary degenerate primers are designed for conserved motives found in the candidate genes.Each of these segments is flanked by primers and individually amplified. For the reassembly procedure thelibrary of fragments can be put together at different ratios generating many biased libraries containing noparental genes.ExamplesThermophilic β-xylanase was made more active. It is important in paper industryfor bleaching of paper pulp. It is obtained from Dictyoglomusthermophilum strain.(15).Aharoni et al. recently reported the functional expression of a mammalianparaoxonase (PON) enzyme in Escherichia coli. PONs have gained interest due totheir role in prevention of human disease. Through DNA family shuffling of fourwild type PON1 genes derived from human, mouse, rat, and rabbit sources, a libraryof PON1 mutants was created and screened for esterase activity. Further analysis ofseveral PON1 mutants showed that improvement was largely due to increasedsolubility rather than changes in kinetic parameters. (18)
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