Directed evolution: selecting today's biocatalysts

Directed evolution has become a full-grown tool in molecular biology nowadays. The methods that are involved in creating a mutant library are extensive and can be divided into several categories according to their basic ideas. Furthermore, both screening and selection can be used to target the enzyme towards the desired direction. Nowadays, this technique is broadly used in two major applications: (industrial) biocatalysis and research. In the first field enzymes are engineered in order to produce suitable biocatalysts with high catalytic activity and stability in an industrial environment. In the latter area methods are established to quickly engineer new enzymes for every possible catalytic step, thereby creating a universal biotechnological toolbox. Furthermore, directed evolution can be used to try to understand the natural evolutionary processes. This review deals with new mutagenesis and recombination strategies published recently. A full overview of new methods for creating more specialised mutant libraries is given. The importance of selection in directed evolution strategies is being exemplified by some current successes including the beta-lactam acylases.     

Directed evolution and the creation of enantioselective biocatalysts

Directed evolution has emerged as a key technology to generate enzymes with new or improved properties that are of major importance to the biotechnology industry. A directed evolution approach starts with the identification of a target enzyme to be optimized and the cloning of the corresponding gene. An efficient expression system is needed before the target gene is subjected to random mutagenesis and/or in vitro recombination, thereby creating molecular diversity. Subsequently, improved enzyme variants are identified, preferably after being secreted into culture medium, by screening or selection for the desired property. The genes encoding the improved enzymes are then used to parent the next round of directed evolution. Enantioselectivity is a biocatalyst property of major biotechnological importance that is, however, difficult to deal with. We discuss recent examples of creating enantioselective biocatalysts by directed evolution.     

Directed polymerase evolution

Polymerases evolved in nature to synthesize DNA and RNA, and they underlie the storage and flow of genetic information in all cells. The availability of these enzymes for use at the bench has driven a revolution in biotechnology and medicinal research; however, polymerases did not evolve to function efficiently under the conditions required for some applications and their high substrate fidelity precludes their use for most applications that involve modified substrates. To circumvent these limitations, researchers have turned to directed evolution to tailor the properties and/or substrate repertoire of polymerases for different applications, and several systems have been developed for this purpose. These systems draw on different methods of creating a pool of randomly mutated polymerases and are differentiated by the process used to isolate the most fit members. A variety of polymerases have been evolved, providing new or improved functionality, as well as interesting new insight into the factors governing activity.     

Directed molecular evolution of antibodies

Antibodies play a key role in the immune system, are characterized by a homogeneous overall structure and by their ability to interact with an almost unlimited number of compounds. Encoded by a fixed number of genes, they acquire their specificity and affinity of recognition after a succession of genetic recombination and molecular mutation processes. Since the pioneer works of Kohler and Milstein in 1975 describing the possibility of producing monoclonal antibodies with pre-determined specificity, the use of antibodies in the fields of research, diagnosis and therapy has never stopped increasing. Thus, about twenty monoclonal antibodies have yet been authorized to be used in human immunotherapy. However, a majority of these molecules have been engineered to bring them into line with their clinical use: chimerization, humanization, recombinant expression of single or fused fragments. Furthermore, the recent development of in vitro molecular evolution approaches now make it possible to engineer the affinity, the specificity as well as the stability of monoclonal antibodies. The potential of in vitro molecular evolution of antibodies will be illustrated through the example of the specificity improvement of an anti-progesterone antibody.     

Directed evolution of metabolic pathways

The modification of cellular metabolism is of biotechnological and commercial significance because naturally occurring metabolic pathways are the source of diverse compounds used in fields ranging from medicine to bioremediation. Directed evolution is the experimental improvement of biocatalysts or cellular properties through iterative genetic diversification and selection procedures. The creation of novel metabolic functions without disrupting the balanced intracellular pool of metabolites is the primary challenge of pathway manipulation. The introduction of coordinated changes across multiple genetic elements, in conjunction with functional selection, presents an integrated approach for the modification of metabolism with benign physiological consequences. Directed evolution formats take advantage of the dynamic structures of genomes and genomic sub-structures and their ability to evolve in multiple directions in response to external stimuli. The elucidation, design and application of genome-restructuring mechanisms are key elements in the directed evolution of cellular metabolic pathways.     

Directed evolution of enzyme stability

Modern enzyme development relies to an increasing extent on strategies based on diversity generation followed by screening for variants with optimised properties. In principle, these directed evolution strategies might be used for optimising any enzyme property, which can be screened for in an economically feasible way, even if the molecular basis of that property is not known. Stability is an interesting property of enzymes because (1) it is of great industrial importance, (2) it is relatively easy to screen for, and (3) the molecular basis of stability relates closely to contemporary issues in protein science such as the protein folding problem and protein folding diseases. Thus, engineering enzyme stability is of both commercial and scientific interest. Here, we review how directed evolution has contributed to the development of stable enzymes and to new insight into the principles of protein stability. Several recent examples are described. These examples show that directed evolution is an effective strategy to obtain stable enzymes, especially when used in combination with rational or semi-rational engineering strategies. With respect to the principles of protein stability, some important lessons to learn from recent efforts in directed evolution are (1) that there are many structural ways to stabilize a protein, which are not always easy to rationalize, (2) that proteins may very well be stabilized by optimizing their surfaces, and (3) that high thermal stability may be obtained without forfeiture of catalytic performance at low temperatures.     

Directed evolution of biocatalytic processes

The benefits of applying biocatalysts to organic synthesis, such as their high chemo-, regio-, and enantio-specificity and selectivity, must be seriously considered, especially where chemical routes are unavailable, complex or prohibitively expensive. In cases where a potential biocatalytic route is not yet efficient enough to compete with chemical synthesis, directed evolution, and/or process engineering could be implemented for improvements. While directed evolution has demonstrated great potential to enhance enzyme properties, there will always be some aspects of biocatalytic processes that it does not address. Even where it can be successfully applied, the resources required for its implementation must currently be weighed against the feasibility of, and resources available for developing a chemical synthesis route. Here, we review the potential of combining directed evolution with process engineering, and recent developments to improve their implementation. Favourable targets for the directed evolution of new biocatalysts are the syntheses of highly complex molecules, especially where chemistry, metabolic engineering or recombineering provide a partial solution. We also review some of the recent advances in the application of these approaches alongside the directed evolution of biocatalysts.     

Directed evolution of fungal laccases

Fungal laccases are generalists biocatalysts with potential applications that range from bioremediation to novel green processes. Fuelled by molecular oxygen, these enzymes can act on dozens of molecules of different chemical nature, and with the help of redox mediators, their spectrum of oxidizable substrates is further pushed towards xenobiotic compounds (pesticides, industrial dyes, PAHs), biopolymers (lignin, starch, cellulose) and other complex molecules. In recent years, extraordinary efforts have been made to engineer fungal laccases by directed evolution and semi-rational approaches to improve their functional expression or stability. All these studies have taken advantage of Saccharomyces cerevisiae as a heterologous host, not only to secrete the enzyme but also, to emulate the introduction of genetic diversity through in vivo DNA recombination. Here, we discuss all these endeavours to convert fungal laccases into valuable biomolecular platforms on which new functions can be tailored by directed evolution.     

Directed evolution of a fungal peroxidase

The Coprinus cinereus (CiP) heme peroxidase was subjected to multiple rounds of directed evolution in an effort to produce a mutant suitable for use as a dye-transfer inhibitor in laundry detergent. The wild-type peroxidase is rapidly inactivated under laundry conditions due to the high pH (10.5), high temperature (50 degrees C), and high peroxide concentration (5-10 mM). Peroxidase mutants were initially generated using two parallel approaches: site-directed mutagenesis based on structure-function considerations, and error-prone PCR to create random mutations. Mutants were expressed in Saccharomyces cerevisiae and screened for improved stability by measuring residual activity after incubation under conditions mimicking those in a washing machine. Manually combining mutations from the site-directed and random approaches led to a mutant with 110 times the thermal stability and 2.8 times the oxidative stability of wild-type CiP. In the final two rounds, mutants were randomly recombined by using the efficient yeast homologous recombination system to shuffle point mutations among a large number of parents. This in vivo shuffling led to the most dramatic improvements in oxidative stability, yielding a mutant with 174 times the thermal stability and 100 times the oxidative stability of wild-type CiP.     

Improved biocatalysts by directed evolution and rational protein design

The efficient application of biocatalysts requires the availability of suitable enzymes with high activity and stability under process conditions, desired substrate selectivity and high enantioselectivity. However, wild-type enzymes often need to be optimized to fulfill these requirements. Two rather contradictory tools can be used on a molecular level to create tailor-made biocatalysts: directed evolution and rational protein design.     

Directed evolution for increased chitinase activity

Directed evolution through DNA shuffling and screening was used to enhance the catalytic ability of a fungal, Beauveria bassiana, chitinase, Bbchit1. The Bbchit gene was first linked to various prokaryotic signal sequences and expressed in Escherichia coli. The signal peptide, PelB, from Erwinia carotovora resulted in greatest chitinase secretion into broth. The nucleotide sequence expressing PelB signal peptide was then incorporated into an E. coli vector to express Bbchit1 variants generated by three rounds of DNA shuffling. A Bbchit1 library with 150,000 variants was constructed with a nucleotide point mutation frequency of 0.6% and screened for chitinolytic activity. Two Bbchit1 variants (SHU-1 and SHU-2) were selected that showed increased chitinolytic activity compared to the wild type. Sequence analysis of these variants revealed mutations in amino acid residues that would not normally be considered for rational design of improved chitinase activity. The amino acid substitutions occurred outside of the two putative substrate-binding sites and the catalytic region.     

Directed evolution of a thermostable l-aminoacylase biocatalyst

Enzymes from extreme environments possess highly desirable traits of activity and stability for application under process conditions. One such example is l-aminoacylase (E.C. 3.5.1.14) from Thermococcus litoralis (TliACY), which catalyzes the enantioselective amide hydrolysis of N-protected l-amino acids, useful for resolving racemic mixtures in the preparation of chiral intermediates. Variants of this enzyme with improved activity and altered substrate preference are highly desirable. We have created a structural homology model of the enzyme and applied various two different directed evolution strategies to identify improved variants. Mutants P237S and F251Y were 2.4-fold more active towards N-benzoyl valine relative to the wild type at 65 °C. F251 mutations to basic residues resulted in 4.5-11-fold shifts in the substrate preference towards N-benzoyl phenylalanine relative to N-benzoyl valine. The substrate preference of wild type decreases with increasingly branched and sterically hindered substrates. However, the mutant S100T/M106K disrupted this simple trend by selectively improving the substrate preference for N-benzoyl valine, with a >30-fold shift in the ratio of k_cat values for N-benzoyl valine and N-benzoyl phenylalanine. Mutations that favoured N-benzoyl-phenylalanine appeared at the active site entrance, whereas those improving activity towards N-benzoyl-valine occurred in the hinge region loops linking the dimerization and zinc-binding domains in each monomer. These observations support a previously proposed substrate induced conformational transition between open and closed forms of aminoacylases.