Enzymes catalyze chemical reactions with vast rate enhancements and with exquisite control. Yet naturally occurring enzymes have evolved for specific functions within the living cell, and are hardly suited for our applications. Thus, while the plethora of well-characterized enzymes found in Nature might be tempting tools for use by the organic chemist, often enzymes are not known that catalyze the desired reaction, or are not accessible due to problems with stability or expression.
Enzyme engineering serves to alter the properties of enzymes for better use, such as broadening substrate specificity, altering stereochemistry, or improving thermostability. Rational redesign involves making a small number of site-directed mutations to achieve the desired effect, usually guided by the accumulation of a large amount of structural and mechanistic data. Alternatively, evolutionary approaches known collectively as directed evolution aims to redesign enzyme properties by testing the activity of many mutants by screening or selection of large mutant libraries. Random mutagenesis and screening is repeated until the desired improvement is obtained. This evolutionary engineering algorithm does not need to rely on large volumes of structural or mechanistic data. In fact, a better understanding of the molecular determinants of substrate specificity/catalysis can often be obtained from such evolutionary experiments.
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Directed evolution requires that a linkage between genotype (DNA sequence) and phenotype (enzymatic activity) is maintained, this is usually established by growing individual mutants in wells of a microtitre plate, or by using bacterial colonies. Instead, this linkage can be established by compartmentalizing genes and translation machinery within artificial droplets (Fig 2). As many as a billion droplets can be created, allowing incredibly high-throughput screens/selections to be developed. Currently, most droplet-based screens require the use of chromo- or fluorogenic enzyme substrates for sorting ‘active droplets’. We are developing general methods for detecting the activity of bond-forming enzymes within such droplets and using this technology to evolve the substrate specificity of several enzymes.
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Riboswitches are naturally occurring short RNA sequences that control gene expression in response to binding small molecule ligands (Fig 3). The natural repertoire of riboswitch ligands is small. Nevertheless, the modular nature of riboswitches offers the opportunity to create mutant riboswitches with specificity towards non-natural ligands. Thus, the ligand binding domain of riboswitches can be replaced with laboratory selected RNA that bind any ligand of choice. The resulting non-natural riboswitch can then be optimized by directed evolution. We aim to create novel riboswitches that can be used as biosensors for various enzymatic transformations. Accordingly, such biosensors can be used for the directed evolution of almost any enzyme activity.
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Many natural products are biosynthesized via the action of large enzymatic assembly lines, whereby various enzymatic activities are organized into modules (Fig 4). Each module is responsible for the selection and incorporation of specific building block monomers into the growing enzymatic product. Thus, by engineering the specificity of these modules, the structure of the final natural product can be changed to give valuable analogues that may have improved biological activities. We are using directed evolution to alter the substrate specificity of several modules from the biosynthesis of a number of polyketide and non-ribosomal peptide based natural products.
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Protein:protein interactions are critical for the function of enzymes that bio-synthesize many natural products & small molecules. We are developing genetically encoded sensors to probe these protein interactions (Fig 5). We will use these probes to map protein:protein interfaces and to improve faulty interfaces by directed evolution. These studies will lead to the creation of new biocatalysts for the synthesis of small molecules with potentially useful biological activities.