Phage display as a tool for the directed evolution of enzymes

Since its introduction in 1985, phage display has had a tremendous impact on the discovery of peptides that bind to a variety of receptors, the generation of binding sites within predefined scaffolds, and the creation of high-affinity antibodies without immunization. Its application to enzymology has required the development of techniques that couple enzymatic activity to selection protocols based on affinity chromatography. Here, we describe both indirect methods, using transition-state analogues and suicide substrates, and direct methods, using the ability of active phage-enzymes to transform substrate into product. The methods have been applied to large libraries for mechanistic-based studies and to generate variants with new or improved properties. In addition, such techniques have been successfully used to select catalytic antibodies and improve their catalytic efficiency.     

Evolution of an organophosphate-degrading enzyme: a comparison of natural and directed evolution

Organophosphate-degrading enzyme from Agrobacterium radiobacter P230 (OPDA) is a recently discovered enzyme that degrades a broad range of organophosphates. It is very similar to OPH first isolated from Pseudomonas diminuta MG. Despite a high level of sequence identity, OPH and OPDA exhibit different substrate specificities. We report here the structure of OPDA and identify regions of the protein that are likely to give it a preference for substrates that have shorter alkyl substituents. Directed evolution was used to evolve a series of OPH mutants that had activities similar to those of OPDA. Mutants were selected for on the basis of their ability to degrade a number of substrates. The mutations tended to cluster in particular regions of the protein and in most cases, these regions were where OPH and OPDA had significant differences in their sequences.     

Non-homologous isofunctional enzymes: A systematic analysis of alternative solutions in enzyme evolution

Background  

Evolutionarily unrelated proteins that catalyze the same biochemical reactions are often referred to as analogous - as opposed to homologous - enzymes. The existence of numerous alternative, non-homologous enzyme isoforms presents an interesting evolutionary problem; it also complicates genome-based reconstruction of the metabolic pathways in a variety of organisms. In 1998, a systematic search for analogous enzymes resulted in the identification of 105 Enzyme Commission (EC) numbers that included two or more proteins without detectable sequence similarity to each other, including 34 EC nodes where proteins were known (or predicted) to have distinct structural folds, indicating independent evolutionary origins. In the past 12 years, many putative non-homologous isofunctional enzymes were identified in newly sequenced genomes. In addition, efforts in structural genomics resulted in a vastly improved structural coverage of proteomes, providing for definitive assessment of (non)homologous relationships between proteins.     

How enzymes adapt: lessons from directed evolution

Enzymes that are adapted to widely different temperature niches are being used to investigate the molecular basis of protein stability and enzyme function. However, natural evolution is complex: random noise, historical accidents and ignorance of the selection pressures at work during adaptation all cloud comparative studies. Here, we review how adaptation in the laboratory by directed evolution can complement studies of natural enzymes in the effort to understand stability and function. Laboratory evolution experiments can attempt to mimic natural evolution and identify different adaptive mechanisms. However, laboratory evolution might make its biggest contribution in explorations of nonnatural functions, by allowing us to distinguish the properties nutured by evolution from those dictated by the laws of physical chemistry.     

Saccharomyces cerevisiae in directed evolution: An efficient tool to improve enzymes

Over the past 20 years, directed evolution has been seen to be the most reliable approach to protein engineering. Emulating the natural selection algorithm, ad hoc enzymes with novel features can be tailor-made for practical purposes through iterative rounds of random mutagenesis, DNA recombination and screening. Of the heterologous hosts used in laboratory evolution experiments, the budding yeast Saccharomyces cerevisiae has become the best choice to express eukaryotic proteins with improved properties. S. cerevisiae not only allows mutant enzymes to be secreted but also, it permits a wide range of genetic manipulations to be employed, ranging from in vivo cloning to the creation of greater molecular diversity, thanks to its efficient DNA recombination apparatus. Here, we summarize some successful examples of the use of the S. cerevisiae machinery to accelerate artificial evolution, complementing the traditional in vitro methods to generate tailor-made enzymes.     

Using continuous directed evolution to improve enzymes for plant applications

Continuous directed evolution of enzymes and other proteins in microbial hosts is capable of outperforming classical directed evolution by executing hypermutation and selection concurrently in vivo, at scale, with minimal manual input. Provided that a target enzyme’s activity can be coupled to growth of the host cells, the activity can be improved simply by selecting for growth. Like all directed evolution, the continuous version requires no prior mechanistic knowledge of the target. Continuous directed evolution is thus a powerful way to modify plant or non-plant enzymes for use in plant metabolic research and engineering. Here, we first describe the basic features of the yeast (Saccharomyces cerevisiae) OrthoRep system for continuous directed evolution and compare it briefly with other systems. We then give a step-by-step account of three ways in which OrthoRep can be deployed to evolve primary metabolic enzymes, using a THI4 thiazole synthase as an example and illustrating the mutational outcomes obtained. We close by outlining applications of OrthoRep that serve growing demands (i) to change the characteristics of plant enzymes destined for return to plants, and (ii) to adapt (“plantize”) enzymes from prokaryotes—especially exotic prokaryotes—to function well in mild, plant-like conditions.

Continuous directed evolution using the yeast OrthoRep system is a powerful way to improve enzymes for use in plant engineering as illustrated by “plantizing” a bacterial thiamin synthesis enzyme.     

Structure-based stabilization of an enzyme: the case of penicillin acylase from Alcaligenes faecalis

The modeled structure of penicillin acylase from Alcaligenes faecali (AFPGA) was constructed by comparative modeling with the Modeller program. Candidate positions that could be replaced with cysteine were estimated by scanning the modeled structure of AFPGA with the program MODIP (modeling disulfide bond in protein). The mutant Q3C/P751C had a higher optimum temperature by three degrees than that of the wild type AFPGA. The half life of the double mutant Q3C/P751C at 55 degrees C was increased by 50%. To our knowledge, this was the first structure-based genetic modification of AFPGA.     

Using directed evolution to improve hydrogen production in chimeric hydrogenases from Clostridia species

Hydrogenases are enzymes that play a key role in controlling excess reducing equivalents in both photosynthetic and anaerobic organisms. This enzyme is viewed as potentially important for the industrial generation of hydrogen gas; however, insufficient hydrogen production has impeded its use in a commercial process. Here, we explore the potential to circumvent this problem by directly evolving the Fe⿿Fe hydrogenase genes from two species of Clostridia bacteria. In addition, a computational model based on these mutant sequences was developed and used as a predictive aid for the isolation of enzymes with even greater efficiency in hydrogen production. Two of the improved mutants have a logarithmic increase in hydrogen production in our in vitro assay. Furthermore, the model predicts hydrogenase sequences with hydrogen productions as high as 540-fold over the positive control. Taken together, these results demonstrate the potential of directed evolution to improve the native bacterial hydrogenases as a first step for improvement of hydrogenase activity, further in silico prediction, and finally, construction and demonstration of an improved algal hydrogenase in an in vivo assay of C. reinhardtii hydrogen production.     

Directed evolution: an approach to engineer enzymes

Directed evolution is being used increasingly in industrial and academic laboratories to modify and improve commercially important enzymes. Laboratory evolution is thought to make its biggest contribution in explorations of non-natural functions, by allowing us to distinguish the properties nurtured by evolution. In this review we report the significant advances achieved with respect to the methods of biocatalyst improvement and some critical properties and applications of the modified enzymes. The application of directed evolution has been elaborately demonstrated for protein solubility, stability and catalytic efficiency. Modification of certain enzymes for their application in enantioselective catalysis has also been elucidated. By providing a simple and reliable route to enzyme improvement, directed evolution has emerged as a key technology for enzyme engineering and biocatalysis.     

Novel methods for directed evolution of enzymes: quality, not quantity

In the past decade methods of directed molecular evolution have proven revolutionary in protein engineering. An increasing number of powerful new combinatorial techniques have joined rational design methods as effective tools for the manipulation and tailoring of biocatalysts. More and more, research in this maturing field is focusing on the quality and comprehensiveness of library construction and analysis. Additionally, in-depth studies have begun to highlight the underlying evolutionary mechanisms, limitations, and consequences of the various methodologies. Together, these investigations are creating a framework for future engineering projects.     

Sterilization as an alternative strategy to control wildlife diseases: bovine tuberculosis in European badgers as a case study

Sterilization has rarely been considered as an alternative to culling or vaccination to control wildlife diseases. Disease control by sterilization, as by culling, has most promise when the host'ss ability for compensatory growth following the removal of density-dependent inhibitions is limited, and when moderate reductions in population density cause disproportionately large reductions in disease prevalence, or even eliminate the disease. For many host/disease examples this will not be the case and vaccination may have overwhelming advantages or may be the only practical option. The impact of sterilization on host density and disease prevalence will develop relatively slowly because sterilization can prevent the recruitment of only one age-cohort at a time. Moreover, unless there is vertical transmission, this age-cohort will consist only of susceptibles. Culling, on the contrary, removes infected as well as susceptible animals. However, for certain disease/host examples, the r elative effectiveness of the different control strategies may be altered considerably if their variable effects on the probability of disease transmission are taken into account. Social perturbation or stress could render certain culling strategies ineffective or even counter-productive. Depending on how disease dynamics are influenced by the host'ss age-structure and reproductive investment, fertility control could offer epidemiological advantages that have been ignored by most disease/host models. We illustrate some of these principles by investigating the theoretical and practical feasibility of an hypothetical sterilization campaign to control bovine tuberculosis in badgers (and hence cattle) in Britain.     

Beyond directed evolution: Darwinian selection as a tool for synthetic biology

Synthetic biology is an engineering approach that seeks to design and construct new biological parts, devices and systems, as well as to re-design existing components. However, rationally designed synthetic circuits may not work as expected due to the context-dependence of biological parts. Darwinian selection, the main mechanism through which evolution works, is a major force in creating biodiversity and may be a powerful tool for synthetic biology. This article reviews selection-based techniques and proposes strict Darwinian selection as an alternative approach for the identification and characterization of parts. Additionally, a strategy for fine-tuning of relatively complex circuits by coupling them to a master standard circuit is discussed.     

Directed evolution to re-adapt a co-evolved network within an enzyme

We have previously used targeted active-site saturation mutagenesis to identify a number of transketolase single mutants that improved activity towards either glycolaldehyde (GA), or the non-natural substrate propionaldehyde (PA). Here, all attempts to recombine the singles into double mutants led to unexpected losses of specific activity towards both substrates. A typical trade-off occurred between soluble expression levels and specific activity for all single mutants, but many double mutants decreased both properties more severely suggesting a critical loss of protein stability or native folding. Statistical coupling analysis (SCA) of a large multiple sequence alignment revealed a network of nine co-evolved residues that affected all but one double mutant. Such networks maintain important functional properties such as activity, specificity, folding, stability, and solubility and may be rapidly disrupted by introducing one or more non-naturally occurring mutations. To identify variants of this network that would accept and improve upon our best D469 mutants for activity towards PA, we created a library of random single, double and triple mutants across seven of the co-evolved residues, combining our D469 variants with only naturally occurring mutations at the remaining sites. A triple mutant cluster at D469, E498 and R520 was found to behave synergistically for the specific activity towards PA. Protein expression was severely reduced by E498D and improved by R520Q, yet variants containing both mutations led to improved specific activity and enzyme expression, but with loss of solubility and the formation of inclusion bodies. D469S and R520Q combined synergistically to improve k_cat 20-fold for PA, more than for any previous transketolase mutant. R520Q also doubled the specific activity of the previously identified D469T to create our most active transketolase mutant to date. Our results show that recombining active-site mutants obtained by saturation mutagenesis can rapidly destabilise critical networks of co-evolved residues, whereas beneficial single mutants can be retained and improved upon by randomly recombining them with natural variants at other positions in the network.     

From molecular engineering to process engineering: development of high-throughput screening methods in enzyme directed evolution

With increasing concerns in sustainable development, biocatalysis has been recognized as a competitive alternative to traditional chemical routes in the past decades. As nature’s biocatalysts, enzymes are able to catalyze a broad range of chemical transformations, not only with mild reaction conditions but also with high activity and selectivity. However, the insufficient activity or enantioselectivity of natural enzymes toward non-natural substrates limits their industrial application, while directed evolution provides a potent solution to this problem, thanks to its independence on detailed knowledge about the relationship between sequence, structure, and mechanism/function of the enzymes. A proper high-throughput screening (HTS) method is the key to successful and efficient directed evolution. In recent years, huge varieties of HTS methods have been developed for rapid evaluation of mutant libraries, ranging from in vitro screening to in vivo selection, from indicator addition to multi-enzyme system construction, and from plate screening to computation- or machine-assisted screening. Recently, there is a tendency to integrate directed evolution with metabolic engineering in biosynthesis, using metabolites as HTS indicators, which implies that directed evolution has transformed from molecular engineering to process engineering. This paper aims to provide an overview of HTS methods categorized based on the reaction principles or types by summarizing related studies published in recent years including the work from our group, to discuss assay design strategies and typical examples of HTS methods, and to share our understanding on HTS method development for directed evolution of enzymes involved in specific catalytic reactions or metabolic pathways.

     

Semi-rational approaches to engineering enzyme activity: combining the benefits of directed evolution and rational design

Many research groups successfully rely on whole-gene random mutagenesis and recombination approaches for the directed evolution of enzymes. Recent advances in enzyme engineering have used a combination of these random methods of directed evolution with elements of rational enzyme modification to successfully by-pass certain limitations of both directed evolution and rational design. Semi-rational approaches that target multiple, specific residues to mutate on the basis of prior structural or functional knowledge create 'smart' libraries that are more likely to yield positive results. Efficient sampling of mutations likely to affect enzyme function has been conducted both experimentally and, on a much greater scale, computationally, with remarkable improvements in substrate selectivity and specificity and in the de novo design of enzyme activities within scaffolds of known structure.     

The enzyme as drug: application of enzymes as pharmaceuticals

Enzymes as drugs have two important features that distinguish them from all other types of drugs. First, enzymes often bind and act on their targets with great affinity and specificity. Second, enzymes are catalytic and convert multiple target molecules to the desired products. These two features make enzymes specific and potent drugs that can accomplish therapeutic biochemistry in the body that small molecules cannot. These characteristics have resulted in the development of many enzyme drugs for a wide range of disorders.     

The luminescent bacteria toxicity test: Its potential as an in vitro alternative

During the past several years, the use of animals for toxicity testing has come under critical surveillance. For ethical and economic reasons, various techniques have been developed and proposed as potential alternatives for some of the whole animal toxicity assays. One assay proposed as an alternative to animal testing is the luminescent bacteria toxicity test (LBT), provided under the trade name of Microtox®. The sensitivity and specificity of the LBT was compared with two commonly used toxicity tests–-the L-929 Minimal Eùgle's Medium (MEM) elution cytotoxicity test and the Draize test. Cytotoxicity and LBT test data from 709 medical device and biomaterial extracts were compared using a positive/negative ranking system which provided a measurement of false positive and false negative results. These data were compiled from nine separate laboratories producing or using a wide variety of biomaterials and medical device products. The LBT was more sensitive than the tissue culture assay and displayed few false negatives. LBT EC₅₀ values were compared with eye irritancy categories for a group of 34 chemicals and 27 personal care products. As with tissue culture, the LBT was more sensitive and produced minimal false negatives. The data from this study indicate the LBT has potential as a rapid, simple method to screen biomaterials and personal care products for toxicity and irritancy.     

The transition of human estrogen sulfotransferase from generalist to specialist using directed enzyme evolution

Broad specificity is believed to be a property of primordial enzymes that diverged during natural protein evolution to produce highly specific and efficient enzymes. Human estrogen sulfotransferase (SULT1E1) is a broad-specificity enzyme that detoxifies a variety of chemicals, including estrogens, by the transfer of sulfate. To study the molecular basis for the broad specificity of this enzyme and to investigate the process of SULT1E1 specialization, we have adopted a directed enzyme evolution approach. Using two iterative rounds of evolution, we generated SULT1E1 mutants with increased thermostability and narrower specificity from the broadly specific wild-type enzyme. To identify mutants with enhanced specificity, we developed an unbiased screening assay to assess sulfate transfer to three different acceptors in parallel. Such an assay enabled the isolation of SULT1E1 mutants with enhanced or wild-type activity toward an estrogen acceptor and significantly reduced activity for phenol or coumarin type of acceptors, leading to up to 3 orders of magnitude increase in specificity. We found that mutations conferring novel specificity are located in the vicinity of the active site and thus may play a direct role in reshaping the acceptor-binding site. Finally, such mutations resulted in reduced SULT1E1 thermostability, revealing a trade-off between SULT1E1 thermostability and acquisition of novel function.