Directed evolution is a common technique to engineer enzymes for a diverse set of applications. Structural information and an understanding of how proteins respond to mutation and recombination are being used to develop improved directed evolution strategies by increasing the probability that mutant sequences have the desired properties. Strategies that target mutagenesis to particular regions of a protein or use recombination to introduce large sequence changes can complement full-gene random mutagenesis and pave the way to achieving ever more ambitious enzyme engineering goals. Introduction Enzymes are Nature's catalysts, tremendously accelerating the rates of a wide range of biochemical reactions, often with exquisite specificity. Harnessing enzymes for other purposes usually requires engineering them to improve their activity or stability. One approach to engineering enzymes is to make specific modifications, but this demands a detailed and frequently unattainable understanding of the relationship between sequence and function. Directed evolution bypasses this problem in much the same way as natural evolution, by combining mutation with selection or screening to identify improved variants. Because it is never possible to test more than an infinitesimal fraction of the vast number of possible protein sequences, it is essential to have a strategy for creating directed evolution sequence libraries that are rich in proteins with the desired enzymatic function. Such libraries can be designed by drawing on our knowledge of how proteins respond to mutation Directed evolution strategies Directed evolution works when the researcher can find at least one enzyme with improved properties in the sequence library. The most naïve strategy of creating a library of random protein sequences is not useful for most enzyme engineering goals. Although sequences with simple functions such as ATP binding Most directed evolution strategies involve making relatively small changes to existing enzymes. This takes advantage of the fact that enzymes often have a range of weak promiscuous activities that are quickly improved with just a few mutations Random mutagenesis The most straightforward strategy for library construction is to randomly mutate the full gene of an enzyme with a function close to the desired function. This approach requires no structural or mechanistic information, and can uncover unexpected beneficial mutations. Using sequential rounds of error-prone PCR to make an average of a few mutations per gene, followed by screening or selection for improved variants, is effective for a wide range of engineering goals. The creation of enantioselective catalysts from an enzyme whose structure is unknown is one such application. A single round of error-prone PCR produced several dozen cyclohexane monooxygenases with R or S selectivity Beneficial mutations found by random mutagenesis can be combined by DNA shuffling. A study with b-glucuronidase showed that beneficial mutations drive each other to extinction during recursive random mutagenesis, but that this problem can be eliminated by DNA shuffling Random mutagenesis can also uncover additional beneficial mutations in rationally designed enzymes. The Withers laboratory Targeted mutagenesis Some engineering goals, such as dramatically altering an enzyme's specificity or regioselectivity, may require mul- Random mutagenesis, targeted mutagenesis and recombination are three strategies for producing sequence libraries for directed evolution. (a) Random mutagenesis introduces amino acid substitutions throughout the protein and can uncover beneficial mutations distant from the active site. The red residues in the structure at top show four mutations uncovered by random mutagenesis that enhanced the activity of mammalian cytochrome P450 2B1 on several substrates Using a high-resolution crystal structure to target mutagenesis to three active site residues, Hill et al. [23] created a triple mutant of phosphotriesterase with a rate enhancement of three orders of magnitude for the degradation of organic triesters such as those used in chemical warfare agents. Crucially, two of the corresponding single mutants did not increase activity and so would not have been identified if they had been explored one at a time. The problem of inverting the enantioselectivity of a lipase offers an interesting comparison between full-gene random mutagenesis and targeted mutagenesis. Reetz and co-workers [24] used several rounds of full-gene random mutagenesis and DNA shuffling to invert the enantioselectivity of a lipase of unknown structure from S to R. Another lipase was engineered for the same goal by simultaneous mutation of four active site residues A variety of other enzymes have recently been engineered by targeted mutagenesis. Mutating three active site residues of penicillin acylase created six variants with improved activity, five of which were triple mutants [27]. Juillerat et al. [28] targeted four active site residues to engineer an O6-alkylguanine-DNA alkyltransferase for the efficient in vivo labeling of fusion proteins. They developed a selection system that allowed them to examine over 20,000 mutants and found that the best variants were triple mutants, suggesting the importance of simultaneously exploring multiple mutations. Novel DNA and RNA polymerases have also been engineered by targeted mutagenesis. Chelliserrykattil and Ellington [29] mutated four amino acids in RNA polymerase to engineer the enzyme to transcribe 2 0 -O-methyl RNA. Using a screen that selected variants that generated more RNA, they identified several mutants that incorporated nucleotides modified at the 2 0 position. Fa et al. [30] used targeted mutagenesis to engineer a DNA polymerase to specifically incorporate 2 0 -O-methyl ribonucleoside triphosphates by mutating six amino acids and selecting improved variants using phage display. Targeted mutagenesis of two active site residues was used to engineer a thioredoxin protein to replace the disulfide bond formation system in Escherichia coli Schultz and co-workers have created tRNA synthetases that charge orthogonal tRNAs with non-natural amino acids by targeting mutagenesis to five or six amino acids involved in substrate recognition. They then performed a positive selection for recognition of the non-natural amino acid and a negative selection against recognition of other amino acids The best mutants discovered by targeted mutagenesis almost always contain multiple mutations. These mutations are often beneficial as single mutants, but evidence is accumulating that at least some of them are beneficial only in combination Recombination Recombining structurally similar proteins can access larger degrees of sequence change than random mutagenesis The family shuffling protocol relies on regions of sequence identity to create crossovers that recombine the sequences of related proteins. This protocol is therefore limited to proteins with more than 70-75% identity, because libraries created from more diverged sequences tend to yield mostly parent sequences. A variety of methods have been developed to avoid this problem in the recombination of divergent sequences by using mismatched PCR primer pairs Although the studies described above demonstrate that recombining highly diverged but homologous sequences can produce libraries of diverse folded sequences, so far there has been little work to test whether it is also a useful method for discovering new functions. A tantalizing hint is that four out of fourteen chimeras of two cytochrome P450 proteins with 64% sequence identity show new product profiles Non-homologous recombination that combines fragments of unrelated proteins is another way to introduce large sequence changes. A new methodology was used to recombine the non-homologous chorismate mutase and fumarase proteins A striking application of non-homologous recombination is Ostermeier and co-workers' creation of a protein that combines the activity of a b-lactamase with the maltoseinduced conformational change of maltose-binding protein. In one experiment, they randomly inserted the lactamase sequence into the maltose-binding protein and screened for mutants with enhanced lactamase activity in the presence of maltose Conclusions Directed evolution is now an established method to engineer enzymes for a wide range of uses. Full-gene random mutagenesis continues to be a straightforward and powerful tool, and studies using this approach repeatedly illustrate that beneficial mutations can occur at unexpected sites. Targeted mutagenesis and recombination can extend directed evolution to the engineering of enzyme properties that require more than a few uncoupled changes in a protein's sequence (which are easily obtained by sequential rounds of random mutagenesis and screening). The increasing incorporation of structural and chemical knowledge will undoubtedly enhance the utility of these methods. The growing use of rational design in conjunction with directed evolution offers the exciting promise of generating libraries containing a high frequency of sequences with the desired functional properties. Update Recent work has emphasized the tendency of directed evolution to improve weak promiscuous functions by broadening specificity, as discussed i
