Recent advances in biocatalysis by directed enzyme evolution

Naturally occurring enzymes are remarkable biocatalysts with numerous potential applications in industry and medicine. However, many of their catalyst properties often need to be further tailored to meet the specific requirements of a given application. Within this context, directed evolution has emerged over the past decade as a powerful tool for engineering enzymes with new or improved functions. This review summarizes recent advances in applying directed evolution approaches to alter various enzyme properties such as activity, selectivity (enantio- and regio-), substrate specificity, stability, and solubility. Special attention will be paid to the creation of novel enzyme activities and products by directed evolution.     

High‐throughput droplet‐based microfluidics for directed evolution of enzymes

Natural enzymes have evolved over millions of years to allow for their effective operation within specific environments. However, it is significant to note that despite their wide structural and chemical diversity, relatively few natural enzymes have been successfully applied to industrial processes. To address this limitation, directed evolution (DE) (a method that mimics the process of natural selection to evolve proteins toward a user‐defined goal) coupled with droplet‐based microfluidics allows the detailed analysis of millions of enzyme variants on ultra‐short timescales, and thus the design of novel enzymes with bespoke properties. In this review, we aim at presenting the development of DE over the last years and highlighting the most important advancements in droplet‐based microfluidics, made in this context towards the high‐throughput demands of enzyme optimization. Specifically, an overview of the range of microfluidic unit operations available for the construction of DE platforms is provided, focusing on their suitability and benefits for cell‐based assays, as in the case of directed evolution experimentations.     

酶立体选择性的定向进化及其高通量筛选方法

定向进化技术已成为开发新型生物催化剂的有力工具,特别是在对酶结构或催化机理信息缺乏的情况下。酶的立体选择性是个比较难处理的参数,其在定向进化过程中的技术瓶颈是建立快速有效的高通量筛选方法。本文概述了在酶立体选择性的定向进化方面所取得的进展,着重论述了酶立体选择性的高通量筛选方法。     

酶分子在试管内的定向进化及其在手性有机物合成中的应用

本文扼要介绍了当今改进酶分子性能的有效手段———分子定向进化技术的基本方法如易错PCR和DNA洗牌,以及派生的一些新技术的原理与进展,并列举了在手性有机物合成中一些成功的实例,为该领域的研究与应用提供了一些新的信息。     

Chromobacterium violaceum ω-transaminase variant Trp60Cys shows increased specificity for (S)-1-phenylethylamine and 4'-substituted acetophenones, and follows Swain-Lupton parameterisation

For biocatalytic production of pharmaceutically important chiral amines the ω-transaminase enzymes have proven useful. Engineering of these enzymes has to some extent been accomplished by rational design, but mostly by directed evolution. By use of a homology model a key point mutation in Chromobacterium violaceum ω-transaminase was found upon comparison with engineered variants from homologous enzymes. The variant Trp60Cys gave increased specificity for (S)-1-phenylethylamine (29-fold) and 4'-substituted acetophenones (～5-fold). To further study the effect of the mutation the reaction rates were Swain-Lupton parameterised. On comparison with the wild type, reactions of the variant showed increased resonance dependence; this observation together with changed pH optimum and cofactor dependence suggests an altered reaction mechanism.     

酶分子在试管内的定向进化及其在手性有机物合成中的应用

本文扼要介绍了当今改进酶分子性能的有效手段——分子定向进化技术的基本方法如易错PCR和DNA洗牌,以及派生的一暨新技术的原理与进展,并列举了在手性有机物合成中一些成功的实例,为该领域的研究与应用提供了一些新的信息。     

Developments in Directed Evolution for Improving Enzyme Functions

The engineering of enzymes with altered activity, specificity, and stability, using directed evolution techniques that mimic evolution on a laboratory timescale, is now well established. In vitro recombination techniques such as DNA shuffling, staggered extension process (StEP), random chimeragenesis on transient templates (RACHITT), iterative truncation for the creation of hybrid enzymes (ITCHY), recombined extension on truncated templates (RETT), and so on have been developed to mimic and accelerate nature’s recombination strategy. This review discusses gradual advances in the techniques and strategies used for the directed evolution of biocatalytic enzymes aimed at improving the quality and potential of enzyme libraries, their advantages, and disadvantages. Submitted to Applied Biochemistry and Biotechnology     

Evolutionary relationship and application of a superfamily of cyclic amidohydrolase enzymes

Cyclic amidohydrolases belong to a superfamily of enzymes that catalyze the hydrolysis of cyclic C-N bonds. They are commonly found in nucleotide metabolism of purine and pyrimidine. These enzymes share similar catalytic mechanisms and show considerable structural homologies, suggesting that they might have evolved from a common ancestral protein. Homology searches based on common mechanistic properties and three-dimensional protein structures provide clues to the evolutionary relationships of these enzymes. Among the superfamily of enzymes, hydantoinase has been highlighted by its potential for biotechnological applications in the production of unnatural amino acids. The enzymatic process for the production of optically pure amino acids consists of three enzyme steps: hydantoin racemase, hydantoinase, and N-carbamoylase. For efficient industrial application, some critical catalytic properties such as thermostability, catalytic activity, enantioselectivity, and substrate specificity require further improvement. To this end, isolation of new enzymes with desirable properties from natural sources and the optimization of enzymatic processes were attempted. A combination of directed evolution techniques and rational design approaches has made brilliant progress in the redesign of industrially important catalytic enzymes; this approach is likely to be widely applied to the creation of designer enzymes with desirable catalytic properties.     

Directed Evolution of Enzymes

On the way to a combinatorial biotechnology? The directed evolution of enzymes promises a rapid access to effective biocatalysts. New molecular biology techniques for random mutagenesis in combination with high-throughput screening might revolutionize the creation of enzymes with new and improved properties.     

Selecting Better Biocatalysts by Complementing Recoded Bacteria**

In vivo selections are powerful tools for the directed evolution of enzymes. However, the need to link enzymatic activity to cellular survival makes selections for enzymes that do not fulfill a metabolic function challenging. Here, we present an in vivo selection strategy that leverages recoded organisms addicted to non-canonical amino acids (ncAAs) to evolve biocatalysts that can provide these building blocks from synthetic precursors. We exemplify our platform by engineering carbamoylases that display catalytic efficiencies more than five orders of magnitude higher than those observed for the wild-type enzyme for ncAA-precursors. As growth rates of bacteria under selective conditions correlate with enzymatic activities, we were able to elicit improved variants from populations by performing serial passaging. By requiring minimal human intervention and no specialized equipment, we surmise that our strategy will become a versatile tool for the in vivo directed evolution of diverse biocatalysts.     

On‐Demand Production of Flow‐Reactor Cartridges by 3D Printing of Thermostable Enzymes

The compartmentalization of chemical reactions is an essential principle of life that provides a major source of innovation for the development of novel approaches in biocatalysis. To implement spatially controlled biotransformations, rapid manufacturing methods are needed for the production of biocatalysts that can be applied in flow systems. Whereas three‐dimensional (3D) printing techniques offer high‐throughput manufacturing capability, they are usually not compatible with the delicate nature of enzymes, which call for physiological processing parameters. We herein demonstrate the utility of thermostable enzymes in the generation of biocatalytic agarose‐based inks for a simple temperature‐controlled 3D printing process. As examples we utilized an esterase and an alcohol dehydrogenase from thermophilic organisms as well as a decarboxylase that was thermostabilized by directed protein evolution. We used the resulting 3D‐printed parts for a continuous, two‐step sequential biotransformation in a fluidic setup.     

Electrochemical Oxidation of Glucose Using Mutant Glucose Oxidase from Directed Protein Evolution for Biosensor and Biofuel Cell Applications

In this study, electrochemical characterisation of glucose oxidation has been carried out in solution and using enzyme polymer electrodes prepared by mutant glucose oxidase (B11-GOx) obtained from directed protein evolution and wild-type enzymes. Higher glucose oxidation currents were obtained from B11-GOx both in solution and polymer electrodes compared to wt-GOx. This demonstrates an improved electrocatalytic activity towards electrochemical oxidation of glucose from the mutant enzyme. The enzyme electrode with B11-GOx also showed a faster electron transfer indicating a better electronic interaction with the polymer mediator. These encouraging results have shown a promising application of enzymes developed by directed evolution tailored for the applications of biosensors and biofuel cells.     

高通量筛选策略在细胞色素P450单加氧酶定向进化中的应用

细胞色素P450单加氧酶具有催化活性混杂性的特点,可以催化多种氧化反应,因而在生物催化领域受到了极大的关注。然而P450单加氧酶往往存在催化活性低、稳定性差、区域和立体选择性不理想等问题,从而限制了其在生物催化领域的广泛运用。蛋白质定向进化的发展与运用为改善P450单加氧酶的催化性能提供了有效的途径,而一种高效的高通量筛选策略是保证酶蛋白定向进化成功实施的关键。本文综述了P450单加氧酶定向进化过程中高通量筛选策略的最新进展。     

Directed evolution of a gatekeeper domain in nonribosomal peptide synthesis

Modular natural products are biosynthesized by series of enzymes that activate, assemble, and process a nascent chain of building blocks. Adenylation domains are gatekeepers in nonribosomal peptide biosynthesis, providing the entry point for assembly of typical peptide-based natural products. We report the directed evolution of an adenylation domain based on a strategy of using a weak, promiscuous activity as a springboard for reprogramming the biosynthetic assembly line. Randomization of residues invoked in a "specificity-conferring code" and selection for a non-native substrate lead to mutant G2.1, favoring smaller amino acids with a specificity change of 10(5): a 170-fold improvement for L-alanine corresponds to a 10(3)-fold decrease for its original substrate (L-phenylalanine). These results establish directed evolution as a method to change gatekeeper domain specificity and suggest that adaptation of modules in combinatorial biosynthesis is achievable with few mutations during evolution.     

Evolution in microfluidic droplet

High-throughput screening (HTS) of enzymatic activity is important for directed evolution-based enzyme engineering. However, substrate and product diffusion can severely compromise these HTS assays. In this issue of Chemistry & Biology, Kintses and coworkers describe a microfluidic platform for the directed evolution of enzymes in droplets that allows for the screening of 10(7) mutants per round of evolution.     

Laboratory Evolution of Stereoselective Enzymes: A Prolific Source of Catalysts for Asymmetric Reactions

Asymmetric catalysis plays a key role in modern synthetic organic chemistry, with synthetic catalysts and enzymes being the two available options. During the latter part of the last century the use of enzymes in organic chemistry and biotechnology experienced a period of rapid growth. However, these biocatalysts have traditionally suffered from several limitations, including in many cases limited substrate scope, poor enantioselectivity, insufficient stability, and sometimes product inhibition. During the last 15 years, the genetic technique of directed evolution has been developed to such an extent that all of these long‐standing problems can be addressed and solved. It is based on repeated cycles of gene mutagenesis, expression, and screening (or selection). This Review focuses on the directed evolution of enantioselective enzymes, which constitutes a fundamentally new approach to asymmetric catalysis. Emphasis is placed on the development of methods to make laboratory evolution faster and more efficient, thus providing chemists and biotechnologists with a rich and non‐ending source of robust and selective catalysts for a variety of useful applications.     

Engineering of Therapeutic and Detoxifying Enzymes

Therapeutic enzymes present excellent opportunities for the treatment of human disease, modulation of metabolic pathways and system detoxification. However, current use of enzyme therapy in the clinic is limited as naturally occurring enzymes are seldom optimal for such applications and require substantial improvement by protein engineering. Engineering strategies such as design and directed evolution that have been successfully implemented for industrial biocatalysis can significantly advance the field of therapeutic enzymes, leading to biocatalysts with new-to-nature therapeutic activities, high selectivity, and suitability for medical applications. This minireview highlights case studies of how state-of-the-art and emerging methods in protein engineering are explored for the generation of therapeutic enzymes and discusses gaps and future opportunities in the field of enzyme therapy.     

A practical high-throughput screening system for enantioselectivity by using FTIR spectroscopy

For the first time FTIR spectroscopy has been applied to the measurement of enantiomeric purity. The underlying concept is based on the use of pseudo-enantiomers that are (13)C-labeled at appropriate positions. Upon applying Lambert-Beer's law in the determination of the concentrations of both enantiomers, the ee values are accessible, accuracy to within +/-5 % of the true values being possible. The application of a commercially available high-throughput FTIR system results in a slightly decreased accuracy (+/-7% for the ee values), but this allows a throughput of up to 10000 samples per day. The method is of interest in the area of combinatorial symmetric catalysis and directed evolution of enantioselective enzymes.     

Computational Enzyme Design

Recent developments in computational chemistry and biology have come together in the “inside‐out” approach to enzyme engineering. Proteins have been designed to catalyze reactions not previously accelerated in nature. Some of these proteins fold and act as catalysts, but the success rate is still low. The achievements and limitations of the current technology are highlighted and contrasted to other protein engineering techniques. On its own, computational “inside‐out” design can lead to the production of catalytically active and selective proteins, but their kinetic performances fall short of natural enzymes. When combined with directed evolution, molecular dynamics simulations, and crowd‐sourced structure‐prediction approaches, however, computational designs can be significantly improved in terms of binding, turnover, and thermal stability.