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Mutant Library Construction in Directed Molecular Evolution

Casting a Wider Net

Tian-Wen Wang, Hu Zhu, Xing-Yuan Ma, Ting Zhang, Yu-Shu Ma, and Dong-Zhi Wei*

Abstract
Directed molecular evolution imitates the natural selection process in the laboratory to find mutant proteins with improved properties in the expected aspects by exploring the encoding sequence space. The success of directed molecular evolution experiment depends on the quality of artificially prepared mutant libraries and the availability of convenient high-throughput screening methods. Well-prepared libraries promise the possibility of obtaining desired mutants by screening a library containing a relatively small number of mutants. This article summarizes and reviews the currently available methodologies widely used in directed evolution practices in the hope of providing a general reference for library construction. These methods include error-prone polymerase chain reaction (epPCR), oligonucleotide-based mutagenesis, and genetic recombination exemplified by DNA shuffling and its derivatives. Another designed method is also discussed, in which B-lymphocytes are fooled to mutate nonantibody foreign proteins through somatic hypermutation (SHM). Index Entries: Directed molecular evolution; error-prone PCR; oligonucleotide-based mutagenesis; DNA shuffling; mutator strain; somatic hypermutation.

1. Introduction Natural selection forces organisms to develop the ability to acclimate to harmful environmental changes in nature by continuously changing their genomes. However, it is not necessarily the case that all changes will enable organisms to survive. Actually, there are more deleterious changes that make organisms weaker and more sensitive to the changed environment than the beneficial ones that confer them better living ability. Only the variants fit to the new environment can thrive. Therefore, organisms genomes become more and more optimized, although the change is at a rather low pace in the natural selection process. Biologists have been developing strategies similar to the process taking place in the nature

to accelerate the evolution of important industrial enzymes or therapeutic proteins in laboratories. This process has been termed directed molecular evolution. It implements a simple but essentially identical Darwinian optimization algorithm. Molecular diversity is typically created by random mutagenesis, genetic recombination of a target gene (14) or a family of related genes (5). Directed evolution screens and selects a large artificially constructed library of mutants for candidates whose properties are improved in certain aspects (6). Such a strategy can thoroughly circumvent the dependence on structural information of a protein compared with the methodology of rational design, which is largely based on the linkage between structure and function (7,8). Thus,

*Author to whom all correspondence and reprint requests should be addressed. State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, P. R. China. E-mail: dzhwei@ecust.edu.cn.
Molecular Biotechnology 2006 Humana Press Inc. All rights of any nature whatsoever reserved. ISSN: 10736085/Online ISSN: 15590305/2006/34:1/055068/$30.00

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directed evolution is a powerful alternative strategy to engineer a protein, especially for proteins whose crystal structures are unavailable or whose relationships between structure and function remain to be established. Successful directed evolution experiments are the integrated results of efficient library construction (9), and robust high-throughput screening of the mutant library (10). A wellconstructed mutant library will greatly alleviate the burden in the process of screening. This is of importance when the availability of a simple and sensitive high-throughput screening method is the bottleneck in the directed evolution of a target protein. The mutant library containing less biased mutations or more crossovers is the prerequisite that one can find desirable mutants by screening a small library. In most cases, powerful screening methods are developed based on the characteristic properties of the given proteins (11,12). Knowledge on how to develop high-throughput screening methods has been reviewed by some investigators (1216). Therefore, this review will discuss the methods used for mutant library construction in directed molecular evolution.

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its simplicity and versatility (20). The rate of mutation in epPCR can be altered by modifying PCR conditions, which offers convenience in manipulation for laboratories with less expertise in doing directed evolution experiments. The epPCR methods can be classified into four groups according to how the mutation rate in amplification is enhanced.

2.1. Reducing the Fidelity of Polymerase by Adjusting PCR Reactants

Error-prone PCR uses unbalanced nucleotide concentrations to enhance the mutation rate when equimolar deoxynucleotide triphosphates (dNTP) are typically added in standard PCR reactions. Usually, the concentrations of dCTP and dTTP are increased to eliminate the mismatch preference of Taq polymerase in amplification (Taq polymerase tends to introduce AT GC mutations) and promote the incorporation of mismatched bases (2124). Very complicated mutant libraries with enriched A+T of the dihydrofolate reductase (DHFR) gene encoded by the Escherichia coli plasmid R67 were created by using biased concentrations of deoxynucleotide triphosphates (e.g., [dTTP] > [dCTP] ) in hypermutagenic epPCR, whereas another combination of unbalanced dNTP ([dTTP] > [dCTP] and [dGTP] > [dATP]) could only generate mutations at unexpectedly low frequency. The addition of divalent manganese cations could numerously change the unfavorable situation. All four transitions and a small number of transversions were produced throughout the gene when the manganese ions were present (23). The presence of manganese ions can be helpful in introducing mutations (25,26). However, its existence has deleterious effects on subsequent ligation manipulation. On the one hand, complex structures will form at the terminals of the amplified products, where restriction enzyme recognition sequences are frequently designed when manganese ions are present. Formation of complex structures will decrease the efficiency of enzymatic digestion of the PCR products. On the other hand, the existence of manganese ions

2. Error-Prone Polymerase Chain Reaction Polymerase chain reaction (PCR) can be used to specifically amplify the target DNA fragments in vitro (17,18). Commercially available polymerase tends to synthesize DNA stringently according to the base complementarity, although the fidelity in synthesis of the new strand depends on the origin of the polymerase and the commercial provider of the enzyme. Meanwhile, the fidelity of DNA synthesis by polymerase can also be greatly affected by other parameters in the PCR system, including the recipe of the reaction mixture and the thermal cycling conditions (19), making it possible to increase the frequency of mismatched incorporation of nucleotides in PCR reaction (error-prone PCR; epPCR) by adjusting the components in the PCR system and the thermal cycling parameters. The error-prone PCR method remains one of the most popular approaches to generating libraries in directed evolution experiments because of

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will adversely affect the performance of T4 DNA ligase. Therefore, it is extremely important and necessary to remove manganese ions from PCR products by performing a brief purification before subsequent manipulations. A divalent magnesium ion is the most suitable co-factor to Taq polymerase for its activity. The ion is always added to a PCR amplification system. The optimal concentration of magnesium ion closely depends on the PCR system. In epPCR, a higher concentration of magnesium ion than in standard PCR amplification is usually adopted. More magnesium ions can stabilize the uncomplementary pairing during extension (19,27). As the unique component of epPCR, manganese ions can increase the mismatch frequency in extension by lowering the recognition specificity of polymerase when biased concentrations of dNTPs are used (23,28,29).

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and transversion would take place. These two kinds of base alterations could take up the majority of the mutations. Base insertions or deletions were rarely found in the mutations caused by the participation of nucleotide analogs, because the mutations were the results of the wobbling base pairing property of the nucleotide analogs when they were substituted by the corresponding normal bases. Xu et al. (31) used two successive PCRs to randomize the encoding sequence of cutinase. The first PCR involved the carefully optimized use of manganese ions, which enhanced the rate of Taq DNA polymerase misrecognition of dNTP in a controlled manner. The introduction of dITP (2'deoxyinosine-5'-triphosphate) in the second PCR resulted in an increased rate of mispairing. The protocol was designed in such a manner that the substitution bias of the first PCR could be compensated for by the base pairing preference of dITP. Using this method, a mutation rate of one to two mutations per clone was achieved in the mutant library of the Fusarium solani pisi cutinase gene. The mutation bias was remedied by the increased frequency of GC to CG transversion.

2.2. Using Nucleotide Analogs in epPCR

Many nucleotide analogs have been designed and synthesized with the intention to take full advantage of the PCR-based methods to mutate a DNA sequence at random by using the triphosphates of mutagenic nucleosides as substrates (30). The requirements for a satisfactory nucleotide analog are as follows: (1) reasonable stability under the conditions of PCR cycling, (2) easy incorporation into the newly synthesized DNA strands, and (3) high mutagenicity. Zaccolo et al. (30) synthesized two nucleoside triphosphate analogs (5'-triphosphates of 6-(2-deoxy-b- D ribofuranosyl)-3, 4-dihydro-8H-pyrimido-[4, 5-C] [1, 2] oxazin-7-one (dP) and 8-oxo-2'deoxyguanosine (8-oxodG)) that could be efficiently incorporated into DNA strands in PCR cycling. Two separate PCRs were used to mutate the target gene with these mutagenic analogs. Analogs were added in the PCR mix in the presence of normal triphosphate nucleotides in the first PCR. The analogs worked as competitive substrate for Taq polymerase. Subsequently, only standard dNTPs were used in the second PCR to allow for the replacement of the incorporated nucleotide analogs with normal bases, in which base transition

2.3. Using Mutagenic Polymerases

Highly thermostable Taq DNA polymerase from the thermophilic bacterium Thermus aquaticus can catalyze 5' 3' synthesis of DNA strands. This enzyme has no detectable 3' 5' exonuclease activity (Taq polymerase possesses a low 5' 3' exonuclease activity), implying that Taq polymerase has no proofreading ability and cannot remove a wrong base when mismatched incorporation occurs. The mismatch rate was assumed at approx 2.7 105 in a typical amplification reaction using Taq polymerase. Thus, Taq polymerase is the first choice for epPCR (32), because epPCR expects increased misincorporation of nucleotides. Larger dosage of polymerase in epPCR reactions will increase the mismatch rate. More polymerase can also guarantee a smooth extension when mismatch happens (33). Many attempts have been made to create DNA polymerases more suitable for directed evolution

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experiments by lowering their fidelity in amplification. The wild-type thermostable DNA polymerase from Pyrococcus furiosus (Pfu-Pol) amplifies DNA with the highest accuracy. It is the best choice for PCR in which high fidelity is most desirable. Biles and Connolly (34) reengineered the polymerase by abolishing its 3' 5' proofreading exonuclease activity and changing the key amino acid in a short region that was responsible for binding the incoming dNTPs and ensuring that only the correct bases opposite to the complementary base in the template strand were inserted. By doing these steps, the investigators successfully converted the extremely accurate wild-type polymerase Pfu-Pol into a low-fidelity variant (34). This mutant polymerase shared the same specific activity, robustness, and thermal stability with the wild-type. The mutations introduced by the reengineered polymerase were random with little bias, even under exactly the same cycling conditions for high fidelity amplification where conventional mutagenic factors (unequal dNTP concentrations, base analogs, and manganese ions) were unavailable. These favorable properties were beneficial when the polymerase was applied in epPCR. The nature of all DNA polymerase, that they recognize and accept normal nucleotides with exceptionally high specificity in amplification, can explain the low mutation efficacy of mutagenic nucleotide analogs supplemented in the epPCRs mixture. The success of Ghadessy et al. (35) in evolving the wild-type Taq polymerase into an enzyme with expanded substrate spectrum offered a solution to the problem. Besides the ability to extend mismatches promiscuously, the mutant Taq polymerase also acquired a generic ability to process a diverse range of noncanonical substrates while maintained high catalytic turnover, processivity, and fidelity. Although the fidelity was not lowered in this mutant polymerase, its substrate recognition feature was compatible with the strategies commonly adopted in epPCR: supplementation of nucleotide analogs and environments favoring the formation of mismatched base pairs. The powerfulness in introducing muta-

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tions had been verified in the generation of microarray probes with up to 20-fold brighter fluorescence.

2.4. Sequence Saturated Mutagenesis Methods

Sequence saturated mutagenesis (SeSaM) is so termed because it is a very beneficial method that can explore the sequence space to the maximum extent (36,37). The procedure of directed evolution with sequence saturated mutagenesis is depicted in Fig. 1. The target gene is amplified through PCR with single-stranded DNA as template in the preparatory steps. In this PCR, 2'-deoxyadenosine5'-O-(1-thiotriphosphate) (dATPS) is supplemented in the reaction mixture as an analog of dATP to produce dATPS-containing DNA fragments that are sensitive to the cleavage by iodine. The chemically cleaved PCR product is in essence a pool of DNA fragments with a random size distribution. Then, a terminal transferase-catalyzed enzymatic elongation of these DNA fragments with various lengths is performed to tail them with universal bases. (The number of universal bases attached to the terminals is controllable by adjusting the time of the tailing reaction.) The tailed DNA fragments are extended full length by standard PCR. Mutations are introduced into the fulllength gene in this step by the pairing property of universal bases. In general, another PCR was required to eliminate the adverse effect of universal bases on subsequent cloning and expression of the mutant genes. In the last PCR, all the universal bases were replaced with standard bases. Theoretically, the random distribution of the fragment lengths in the DNA sample cleaved by iodine implies that the existence of the universe bases can be at any position along the sequence. SeSaM introduces mutations at the positions that have been universe bases added by the terminal transferase. Thus, SeSaM is a method that can randomly mutate a sequence in a manner completely independent of the mutational bias of DNA polymerases. The bias of SeSaM mainly stems from the base-pairing properties of deoxyinosine (36,38).

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synthetic DNA sequence within the encoding sequence of a protein. Therefore, there are two elements in all of these techniques. One is how the DNA sequences are randomized during the synthesis; the other is the methodology for the incorporation of the synthetic oligonucleotides. Oligonucleotide-based mutagenesis can control the DNA synthesis process, providing complete control over the level, identity and position of randomization. Currently there are many techniques available to produce the oligonucleotide mixture through controlling over the synthesis or mixing the synthetic oligonucleotides. Degenerate oligonucleotides are synthetic primer mixtures of any combination of the four natural bases at any position. This kind of synthetic DNA can be used to completely randomize a specific position within a gene. Synthetic process is susceptible to bias arising from greater incorporation of one reagent than another, because synthesis always involves a number of reagents. Careful control and utilization of optimized reagents have been proved to be a helpful way to reduce this bias in the synthetic DNA library, although this cannot ensure the reduced bias in the cloned libraries (39, 40). Another bias is caused by the mismatches in the synthesis of oligonucleotides and the degeneration of triplets when they are translated into amino acids: there are more codons for some amino acids than for others. Therefore, there will be more serine, but less tryptophan and methionine, when one completely randomized triplet is translated into amino acid. Synthesis of the oligonucleotides for each desired mutation is a simplest solution to this bias. The utilization of trinucleotide phosphoramidites in the synthesis of oligonucleotides is an alternative solution. However, whether or not adopting these two strategies to reduce the bias in oligonucleotide synthesis, in most cases, largely depends on economical consideration rather than a technical one. After the oligonucleotides are prepared, they must be incorporated into the encoding sequence of a gene to introduce mutations. Many methods based on conventional site-directed mutagenesis (SDM) techniques are available. One basic con-

Fig. 1. Schematic overview of the SeSaM method. (1) Cleaving PCR product with iodine to generate a pool of DNA fragments with a random-sized distribution. (2) Terminal transferase catalyzed tailing of DNA fragments using universal bases under controlled conditions. (3) Elongating the tailed fragments to full length by standard PCR in which mutations were introduced into the sequences as a result of the wobbling pairing property of universal bases. (4) Replacing all universal bases with standard bases to facilitate subsequent manipulations (36).

3. Utilization of Oligonucleotides DNA polymerase can catalyze the incorporation of a mismatched base at a certain rate during the synthesis of the new DNA strands. This property of polymerase makes it possible to introduce mutations. Thus, in epPCR, an even distribution of mutated sites can be expected. Unlike epPCR, oligonucleotide-based mutations can only take place in specific regions related to the synthetic oligonuceotides used in the experiment, because mutations are the direct result of the incorporated

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cern in the incorporation of the synthetic oligonucleotides is how to minimize the percentage of the wild-type sequences in the libraries. Overlap extension and megaprimer-based procedures are usually the methods of choice (4143). Stratagenes QuikChange system provides mutagenic plasmid amplification (MPA) protocol for efficient mutagenesis (44). Bias can take place in the incorporation reactions of oligonucleotide primers. Procedures involving an exponential amplification cannot evade the potential problem of amplification bias. An additional problem of oligonucleotide incorporation is that sequences with greater similarity to the wild-type DNA sequence will be incorporated at higher efficacy than those with more divergence. Furthermore, the positions of the mutated sites in the synthetic oligonucleotides can also affect the incorporation efficiency. An oligonucleotide bearing a mutation or mutations in the proximity of the 5' end can be incorporated with higher efficiency than the oligonucleotide containing uncomplementary bases near the 3' end, because the extension in PCR starts from the 3' end. Accordingly, although the synthetic oligonucleotide library is completely randomized, that is, the mutation site has an absolutely even distribution, biases will still appear in the cloned libraries. The use of carefully designed oligonucleotide and the provision of a reasonable length of fully annealing sequence at the 3' end are helpful when reduced risk of this occurrence is expected. Reduction in amplification bias is again best achieved by performing a number of separate amplifications with the smallest possible number of cycles. By assembly of appropriately prepared oligonucleotides that are designed under the guide of sequence information, full-length gene fragments can be obtained with high recombination frequency. This method has been implemented in the recombination of lipase genes of two gene families from Bacillus subtilis. From a very small library containing only 3000 lipase variants created by this method, several mutants were found with improved enantioselectivity (45).

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Oligonucleotide-based methods provide a very powerful tool for the randomization of specific chosen positions and regions within protein-encoding sequences by incorporating a synthetic DNA sequence into the full-length gene. This means that any form of randomization that is achievable in a synthetic oligonucleotide can be replicated in the full-length gene. Overlap extension and megaprimer PCR protocols are simple methods popularly used in the incorporation of these synthetic oligonucleotides.

4. Recombination Mutant library construction with epPCR or the oligonucleotide-mediated mutagenesis method starts with a single parent gene sequence. In epPCR, the majority of the changes in the mutated DNA sequences are site mutations. Although deletion or insertion of bases can also take place, there is little chance to find the replacement of consecutive bases with homogenous or completely unrelated DNA sequences. With the oligonucleotide-mediated mutagenesis method, the high dependence of the mutated sequences on the synthesized oligonucleotides severely constrains the sequence diversity. A solution to the problem was DNA shuffling introduced by Stemmer for the first time in 1994 (Fig. 2) (3) and other derivative methods developed later. The power of this sophisticated method has been proven by successes in the discovery for industrial biocatalysts and therapeutic protein agents since its establishment (4651). When the DNA shuffling method was initially developed, the encoding sequences of related genes were digested with DNase I, yielding a pool of DNA fragments with random lengths. These fragments were subsequently assembled into the full-length gene in a PCR without primers. Library diversity was generated during the reassembly process if two fragments originated from different parent sequences annealed and subsequently extended. DNA shuffling allows the spontaneous recombination of many parent genes and the generation of multiple crossovers per reassembled sequence (52). The successful assembly of these fragments

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of DNA homology (5456). However, it is usually used for the construction of mutant libraries of two parent genes with no less than 50% identity on the DNA level to ensure the percentage of functional mutants in the library. Some hybrid proteins that were fused at nonhomologous locations could also be found in the library prepared by ITCHY, but their appearance in libraries created by DNA shuffling was rarely found (56). In ITCHY method, two parent genes or gene fragments from which the resultant hybrid protein will be created are inserted into one plasmid. A suitable unique restriction enzyme recognition sequence is designed between the two encoding sequences. Digestion with the unique restriction enzyme linearizes the plasmid. Incremental truncation library is generated by digesting the parental genes with exonuclease III under controlled conditions. During the course of the truncation reaction, small aliquots are removed and quenched at such a frequency that, theoretically, every single base deletion of the two gene fragments can be collected. Fusion of the truncated gene fragments by blunt end ligation then generated the ITCHY library. Alpha-phosphothioate nucleotides can be randomly incorporated into the freshly synthesized DNA strands and formed phosphothioate internucleotide linkages resistant to 3' 5' hydrolysis by exonuclease. The occurrence of phosphothioate internucleotide linkage can block the progressive degradation of the target DNA strands by exonuclease III treatment. The utilization of alphaphosphothioate nucleotides in the fill-in reaction step (Fig. 3) or PCR amplification step (Fig. 4) will remarkably reduce the time and labor by eliminating the optimization of exonuclease hydrolysis, and the regular sampling during the truncation reaction with exonuclease III. Furthermore, if the protocol depicted in Fig. 3 is adopted, an epPCR can be carried out to enhance the random incorporation of the nucleotide analogs. The ITCHY method can be used to create recombinant proteins from distantly related sequences. But in all the recombinant proteins there is only one crossover (see Figs. 3 and 4). That is, one parent gene merely contributes the portion near

Fig. 2. DNA shuffling procedure. (1) A family of related genes were fragmented with DNase I, resulting in a pool of DNA with various lengths; (2) DNA fragments were reassembled into full-length genes in a primerless PCR reaction according to the complementarity among them. A library of mutants carrying multiple crossovers was formed (3).

to regenerate the recombinant sequences completely depends on the complementarity among the fragments when primers are absent. Hence, homology among the fragmented genes can exert a critical effect on the quality of the mutant library, because high homology among parent genes implies the ease of reassembly reaction, but it will inescapably decrease the frequency of crossover in the recombinant genes. Correspondingly, low homology promises more crossovers in the regenerated sequence, but the efficiency of reassembly will be sacrificed. Moreover, crossovers tend to aggregate in regions with high sequence identity because of the annealing-based reassembly. Accordingly, in DNA shuffling, satisfactory reassembly efficiency and crossover frequency can be achieved when one starts with genes sharing 70% sequence identity at the nucleic acid level (53). Incremental truncation for creation of hybrid enzyme (ITCHY) can create combinatorial fusion libraries between genes in a manner independent

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Fig. 3. Schematic overview of ITCHY using a-phosphothioate nucleotide (protocol 1). (1) Two genes (Gene I and Gene II) were cloned into the same plasmid. Between the two genes, a restriction enzyme recognition sequence was designed to facilitate the linearization of the recombinant plasmid with the corresponding enzyme. (2) Enzymatic treatment of the linearized plasmid with exonuclease III to produce single-stranded overhangs. (3) Polymerase catalyzed resynthesis of the complementary strand with the single-stranded target region serving as the template in the presence of small amounts of nucleotide analogs (-phosphothioate dNTPs) to allow the random, low frequency incorporation of the analog into the newly synthesized strand as indicated by stars. (4) Exonuclease III hydrolyzes the newly synthesized strands, whereas the nucleotide analogs will block enzymatic degradation. (5) Removing the single-stranded portions of the linearized plasmids with mung bean nuclease. (6) The blunt-ended constructs are recircularized by intramolecular ligation (55).

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either the 5' terminal or the 3' terminal end, although the contribution of individual parent gene to the hybrid gene differs in a given recombinant sequence. Another method, SCRATCHY, which combines the robustness of ITCHY in fusing two genes with low sequence identity and the efficacy of DNA shuffling in creating multiple crossovers, was developed by Lutz and colleagues (57). In the SCRATCHY method, two complementary ITCHY libraries that had undergone a functional screening were subsequently subjected to a DNA shuffling process to introduce multiple crossovers in the recombinant genes (note that in ITCHY, the arrangement of the two genes or gene fragments in the plasmid will determine the 5' and 3' terminal origin of the recombinant protein. The gene at the 5' terminal end in the linearized plasmid will contribute its sequence near 5' to the recombinant sequence. Correspondingly, the gene at the 3' terminal end in the linearized plasmid will provide the sequence near the 3' end of the recombinant sequence).

Fig. 4. Schematic overview of ITCHY using phosphothioate nucleotide (protocol 2). (1) Linearization of the starting plasmid by restriction digestion at the unique site between the two genes or gene fragments. (2) PCR amplification of the entire linearized vector in the presence of a mixture of dNTPs and aphosphothioate dNTPs. (3) Incubation of the plasmid with exonuclease III to hydrolyze standard dNMPs while the dNMP analogs will block enzymatic degradation. (4) The single-stranded overhangs of the plasmids are removed enzymatically with mung bean nuclease. (5) The blunt-ended constructs are recircularized by intramolecular ligation (56).

5. Other Methods 5.1. In Vitro Mutagenesis by Mutagenic Chemicals Some chemicals are mutagenic, such as nitrous acid, hydroxylamine, and bisulphate. Mutating microbes with such chemicals and screening for mutants with improved features has been practiced by microbiologists for quite a long time. All the genetic material in a microorganism is subjected to the effect of the mutagenic chemicals in these classic experiments. These chemicals have also been proved as capable agents in introducing random mutations in a specific gene in vitro other than the random regions of the whole genomic DNA (58). A research group led by Wu found that ethyl methane sulfonate (EMS) could efficiently mutate a target gene at random in vitro (59). In combination with a simple plate-based screening methodology, mutants with increased activity were obtained from a comparatively small library (about 300 mutants) when this method was applied to the Bacillus aprN18 gene, which encoded a serine protease with fibrinolytic activity. Constructing a mutant library with EMS needs nei-

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ther sophisticated equipments nor difficult manipulations. Moreover, base transition (GC AT or AT GC) and transversion (GC CG) could be found in mutant sequences with approximately comparable frequency, indicating that EMS could cause mutations with less bias when compared with the most widely adopted PCR-based methods, which tended to introduce the AT GC mutations if Taq polymerase was used.

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any genetic materials in the cells including other sequences in the plasmid besides the foreign gene of interest and their own genomic DNA. This mutator strain has been successfully used in some directed evolution experiments by researchers in different laboratories (6064). Esterase mutants capable of resolving a tough compounda sterically hindered 3-hydroxy ethyl esterwere found after seven cycles of mutation. This ester was not accepted as substrate by 20 wild-type hydrolases (65). When polyhydroxyalkanoate (PHA) synthase gene from Aeromonas punctata was subjected to mutation in XL1-RED cells, mutants harboring a single mutation in the gene gave rise to increased in vitro enzymatic activity of the overproducing mutants ranging from 1.1- to 5-fold more than that of wild-type ones, although the amount of accumulated PHA ranged from 107% to 126% of that of the wildtype (66). In addition to the application of mutator strain in the improvement of biocatalysts of industrial importance, a mutator strain has been used in the random mutagenesis in plant viral genome for generating attenuated strains or for analyzing viral gene function at the molecular level. Nucleotide sequence analysis of each virus mutant revealed that mutations were introduced randomly into the viral genome, regardless of the function of the viral gene (67). Because the mutations were generated during the amplification of the genetic material, biased mutation would be inevitable. The majority of the mutations caused by XL1-RED were single point mutations, consisting of more transitions than transversions. The occurrence frequency of single-base frame shifts resulted from base insertion or deletion was quite low. The limitation in using such a mutator strain is mainly from economic considerations. The deficiency in DNA repair makes the genome of XL1-RED quite unstable. One can only use the commercially supplied competent cell to guarantee the efficiency in the transformation manipulation. Another issue to take into account is that XL1-RED grows much slower. It has a longer doubling time than wild-type E. coli.

5.2. Using Mutator Strains

Microorganisms use four mechanisms (mismatch repair, excision repair, recombination repair, and error-prone repair) to ensure the fidelity in DNA replication. The mutation rate will increase significantly when the DNA repair function is deficient. The E. coli XL1-RED provided by Stratagene is an engineered strain deficient in the three primary DNA repair pathways: mutS (errorprone mismatch repair), mutD (deficient in 3' to 5' exonuclease of DNA polymerase III), and mutT (unable to hydrolyze 8-oxod-GTP). Thus, the mutation rate of the XL1-Red strain is approx 5000-fold higher than that of wild type E. coli (1 10 -10 to 1 10 9 mutations/generation/ cell), reaching one mutation in every 5 106 bp per generation per cell. Mutations randomly introduced by the XL1-RED mutator strain can be transitions, transversions, single-base deletions, and single-base insertions. An important advantage of mutator strain XL1RED is the convenience in manipulation. The XL1-RED bacteria will mutate the foreign genes spontaneously when the cells carrying the recombinant plasmid with the foreign gene of interest propagate. The mutated genes thus form a library. After the transformants grow for a short time, desirable mutant genes (in plasmids) can be obtained from the newly produced bacteria with a high throughput screening method. The next round of screening starts after transforming the freshly isolated plasmid harboring the mutated gene into XL1-RED competent cells. This simple procedure can be repeated several times until the expected mutants are available. What should be noted is that such an engineered deficient strain can mutate

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Methods for Mutant Library Construction 5.3. B-Lymphocytes: The Natural Evolution Accelerator
When activated by antigens, B-lymphocytes in the immune system can specifically mutate immunoglobulin (Ig) through a process called somatic hypermutation (SHM) (6872). This is the skill that B-lymphocytes have perfected, generating the myriad antibody proteins that fight off infections through the long process of natural evolution. In Tsiens elegant work, B-lymphocytes have been completely coaxed to hypermutate and produce a nonantibody protein in an inducible manner (73,74). The gene for a monomeric red fluorescent protein (mRFP) was expressed in a human B-cell line (Burkitt lymphoma Ramos) that hypermutates its Ig V genes constitutively during its proliferation. The mRFP mutants with enhanced photostability and far-red emissions were evolved through 23 rounds of iterative SHM and fluorescence-activated cell sorting (FACS) screening. In directed evolution through SHM, the evolution process initiates when the recombinant viruses carrying the mRFP gene enter into the Ramos cells with other necessary regulatory elements. The mutation is associated with the transcription. The more transcription of the mRFP gene, the more mutations will be generated. New mutations appear with each round of screening, indicating that SHM keeps exploring the sequence space. This feature of SHM significantly increases the possibility to obtain mutants with phenotypes that requires mutations that cannot be accumulated in libraries constructed through other methods. However, SHM is never a mechanism that can produce mutations free of bias. The mutation biases mostly result from SHMs known biases for particular base changes (70). The SHM-mediated protein evolution in live cells skillfully obviates labor-intensive in vitro mutagenesis and screening by sampling a large protein space and by linking the cell genotypes directly to its phenotypes. In addition, eukaryotic cells can efficiently modify the protein produced by them, making posttranslational modification no longer a problem for animal-originated pro-

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teins. This is quite valuable for some therapeutic proteins in which the posttranslational modification can affect their effect to a considerable extent.

6. Summary The attempt to enhance the performance of enzymes has been one of the core tasks of biologists for quite a long time. Before the birth of genetic engineering, microorganisms were treated with mutagenic chemicals or irradiated with high-energy rays to generate variants, from which better performers were screened and selected. The arrival of SDM, a technique that allows amino acid sequences in proteins to be altered at will, is the answer to an enzymologists prayer. Site-directed mutagenesis can be used to modify the active site of an enzyme of known structure and mechanism. For enzymes or other valuable proteins whose structures and mechanisms remain to be clarified, directed molecular evolution enables biologists to break through the limitation of insufficient understanding of the structure and the mechanism of these proteins by casting the wider netgenerating libraries containing more mutants. Many protocols have been developed to prepare protein libraries. Error-prone PCR is a derivative of standard PCR, which is performed under controlled conditions that favor mutation. It is an extensively adopted method because of its simplicity in operation and the low requirements for expertise and instruments. Oligonucleotide-based mutagenesis introduces mutations by the direct incorporation of oligonucleotides containing designed mutations at specific sites. These synthetic short sequences are designed and introduced into the target sequences by two critical steps that will influence the quality of the resultant libraries. DNA shuffling is a recombination method. Mutations generated using DNA shuffling are the results of recombination of gene fragments from different origins. DNA shuffling can only be applied to create mutant library of homologous parent genes. Incremental truncation for creation of hybrid enzyme (ITCHY) is capable of producing fusion genes independent of sequence

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homology. SCRACHYthe hybrid of DNA shuffling and ITCHYabsorbs the merits of DNA shuffling and ITCHY, making it possible to generate mutant genes carrying multiple crossovers from distantly related sequences. Commercially available engineered mutator strain E. coli XLRED with deficient DNA repairing ability is a tailor-made bacterium for directed evolution. Its in vivo mutagenesis ability immensely simplified the steps in library construction. An ingeniously designed protocol has been used to coax B-lymphocytes to falsely regard other foreign genes as the encoding sequences of immunoglobulins and to change them through somatic hypermutation. This success is especially important for therapeutic proteins for which complicated post-translational modifications are required for their full functionality.

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Acknowledgment This work was financially supported by a grant from the National High Technology Research and Development Program of China (No.2002AA2 Z345A). References
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