人工合成多样性突变文库研究进展*

突变文库的构建是定向进化研究过程中一个关键步骤,主要利用天然存在的系统或者人工合成的分子技术来产生多样性核酸分子文库,为制备和筛选具有一定特性的蛋白酶、多肽、人工抗体等提供庞大的遗传基因库,也可用于合成生物学中相关基因元件的研究与筛选,为目标生物制品的高效工业化生产提供动力。随着对突变文库构建技术研究的日益深入,各种文库构建策略相继被开发出来,并在生物能源、生物化工、生物医药、生物试剂和食品工业等方面得到了广泛的应用。然而,定向进化中的文库构建策略多有不同,各种突变文库构建技术的核心方法也在不断创新。主要介绍近年来实验室中人工合成多样性文库的前沿技术,并对文库构建技术在自动化和智能化方向的发展进行了展望。     

Steering directed protein evolution: strategies to manage combinatorial complexity of mutant libraries

How to explore protein sequence space efficiently and how to generate high-quality mutant libraries that allow to identify improved variants with current screening technologies are key questions for any directed protein evolution experiment. High-quality mutant libraries can be generated through improved random mutagenesis methodologies and by restricting diversity generation through computational methods to residues which have high success probabilities. Advances in mutant library design and computational tools to focus diversity generation are summarized in this minireview and discussed from an experimentalist point of view in the context of directed protein evolution.     

筛选在非生长条件下突变体酶的新方法

利用双层平板筛选由定向进化方法产生的瑞氏木霉内切葡聚糖酶Ⅲ（EGⅢ）突变体库,根据水解圈在平权上形成的速度判断酶活高低。采用这种方法在不同的筛选条件下分别筛选得到了几株在低温或碱性条件下高活性的EGⅢ突变体。比色法测定的酶活性与平板筛选结果一致。该方法的建议将使通过定向进货方法改造现有蛋白质,获得具有新的优良性状的工业用酶的途径得到更加广泛的应用。     

To the Final Goal: Can We Predict and Suggest Mutations for Protein to Develop Desired Phenotype?

Directed evolution of proteins is a good approach to develop desired phenotypes from existing proteins. Fully experimental protein evolution usually utilizes randomization of a given protein sequence by error-prone PCR or gene shuffling followed by high-throughput selection or timeconsuming screening method. However, these random methods create mutant library full of deleterious mutations. In addition, they need high-throughput screening or selection method to search for positive clones from an enormous size of mutant library. Construction of a mutant library while retaining the original function is important for efficient protein evolution because it greatly reduces time and effort for the identification of positive mutants. Therefore, researchers have tried to reduce the size of mutant library by minimizing the occurrence of deleterious mutants. Such efforts have led to the creation of a concept of ‘small but smart library’. For this goal, neutral drift theory has been applied. Although smart library greatly reduces the library size, it is still the beyond the capacity of low-throughput assay. In parallel, computational analysis of protein structure and efforts to discriminate mutatable residues from all residues of a given protein have been consistently pursued. Accumulated knowledge of protein evolution through random mutation and selection has improved our understanding of functions of amino acids in protein structure. Protein evolution by rational design is being developed based on such understanding. In this review, we describe how the use of semi-rationally designed library rather than completely random one has impacted the overall procedure of directed evolution. We also describe efforts made to evaluate the effect of single mutation. Such efforts will bring lazy boys to the final goal - computational mutation suggestion system.     

Sensitive assay for laboratory evolution of hydroxylases toward aromatic and heterocyclic compounds

Powerful directed evolution methods have been developed for tailoring proteins to our needs in industrial applications. Here, the authors report a medium-throughput assay system designed for screening mutant libraries of oxygenases capable of inserting a hydroxyl group into a C-H bond of aromatic or O-heterocyclic compounds and for exploring the substrate profile of oxygenases. The assay system is based on 4-aminoantipyrine (4-AAP), a colorimetric phenol detection reagent. By using 2 detection wavelengths (509 nm and 600 nm), the authors achieved a linear response from 50 to 800 microM phenol and standard deviations below 11% in 96-well plate assays. The monooxygenase P450 BM-3 and its F87A mutant were used as a model system for medium-throughput assay development, identification of novel substrates (e.g., phenoxytoluene, phenylallyether, and coumarone), and discovery of P450 BM-3 F87A mutants with 8-fold improvement in 3-phenoxytoluene hydroxylation activity. This activity increase was achieved by screening a saturation mutagenesis library of amino acid position Y51 using the 4-AAP protocol in the 96-well format.     

高通量筛选工具的设计与应用

定向进化方法作为新兴的高效蛋白质工程手段,其内容包括蛋白质突变体文库的构建和有效突变体的快速筛选。高通量筛选方法是定向进化方法的重要组成部分,是成功获得有效突变体的关键。筛选的突变体数量越多,获得有效突变体的几率越大。以下介绍了目前已经成功应用于或有潜力应用于定向进化改造蛋白质的几种高通量筛选工具。高通量筛选工具的不断设计与开发将推动蛋白质工程领域的技术革新。     

Advances in ultrahigh-throughput screening technologies for protein evolution

Inspired by natural evolution, directed evolution randomly mutates the gene of interest through artificial evolution conditions with variants being screened for the required properties. Directed evolution is vital to the enhancement of protein properties and comprises the construction of libraries with considerable diversity as well as screening methods with sufficient efficiency as key steps. Owing to the various characteristics of proteins, specific methods are urgently needed for library screening, which is one of the main limiting factors in accelerating evolution. This review initially organizes the principles of ultrahigh-throughput screening from the perspective of protein properties. It then provides a comprehensive introduction to the latest progress and future trends in ultrahigh-throughput screening technologies for directed evolution.     

High-throughput screening of enzyme libraries

Directed evolution is becoming a widely used technique for modifying or enhancing protein performance. Ultimately, the success of directed protein evolution experiments hinges on the efficiency of the methods used to screen libraries for mutants with properties of interest. Although there is still a paucity of general methods for enzyme library screening, in recent years a number of promising strategies have emerged and are increasingly being used to explore challenging issues in protein engineering.     

Chemical and biochemical strategies for the randomization of protein encoding DNA sequences: library construction methods for directed evolution

Directed molecular evolution and combinatorial methodologies are playing an increasingly important role in the field of protein engineering. The general approach of generating a library of partially randomized genes, expressing the gene library to generate the proteins the library encodes and then screening the proteins for improved or modified characteristics has successfully been applied in the areas of protein–ligand binding, improving protein stability and modifying enzyme selectivity. A wide range of techniques are now available for generating gene libraries with different characteristics. This review will discuss these different methodologies, their accessibility and applicability to non-expert laboratories and the characteristics of the libraries they produce. The aim is to provide an up to date resource to allow groups interested in using directed evolution to identify the most appropriate methods for their purposes and to guide those moving on from initial experiments to more ambitious targets in the selection of library construction techniques. References are provided to original methodology papers and other recent examples from the primary literature that provide details of experimental methods.     

随机突变文库构建与筛选研究进展

定向进化是一个循环过程,在构建多样化基因序列和筛选功能基因变体之间交替进行.该技术目前已被广泛应用于DNA序列、基因功能和蛋白质结构的优化和分析.定向进化包括随机基因文库的生成、基因在合适宿主中的表达和突变文库的筛选.构建基因文库的关键是库容量和突变多样性,而筛选变体的关键是高灵敏度和高通量.文中讨论了定向进化技术的最...     

Affinity Maturation of Cry1Aa Toxin to the Bombyx mori Cadherin-Like Receptor by Directed Evolution

Improvement of the activity and insecticidal spectrum of cloned Cry toxins of Bacillus thuringiensis should allow for their wider application as biopesticides and a gene source for gene-modified crops. The insecticidal activity of Cry toxins depends on their binding to the receptor. Therefore, as a model, we aimed to generate improved binding affinity mutant toxins against Bombyx mori cadherin-like receptor (BtR175) using methods of directed evolution with the expectation of insecticidal activity improved mutants. Four serial amino acid residues of ⁴³⁹QAAG⁴⁴² or ⁴⁴³AVYT⁴⁴⁶ of Cry1Aa were replaced with random amino acids and were displayed on the T7 phage for library construction. Through five cycles of panning of the phage libraries using BtR175, 11 mutant phage clones were concentrated, and mutant toxin sequences were confirmed. The binding affinities of the three mutants were 42-, 15-, and 13-fold higher than that of the wild type, indicating that mutants with improved binding affinity to cadherin can be easily selected from randomly replaced loop 3 mutant libraries using directed evolution. We discuss the development of a genetic engineering method based on directed evolution to improve the binding affinity of Cry toxin to receptors.     

Construction of diverse adeno-associated viral libraries for directed evolution of enhanced gene delivery vehicles

Rational design of improved gene delivery vehicles is a challenging and potentially time-consuming process. As an alternative approach, directed evolution can provide a rapid and efficient means for identifying novel proteins with improved function. Here we describe a methodology for generating very large, random adeno-associated viral (AAV) libraries that can be selected for a desired function. First, the AAV2 cap gene is amplified in an error-prone PCR reaction and further diversified through a staggered extension process. The resulting PCR product is then cloned into pSub2 to generate a diverse (>10(6)) AAV2 plasmid library. Finally, the AAV2 plasmid library is used to package a diverse pool of mutant AAV2 virions, such that particles are composed of a mutant AAV genome surrounded by the capsid proteins encoded in that genome, which can be used for functional screening and evolution. This procedure can be performed in approximately 2 weeks.     

mRNA display for the selection and evolution of enzymes from in vitro-translated protein libraries

The mRNA display technology enables the in vitro selection and directed evolution of functional proteins from libraries of more than 10(12) different mutants in a single test tube. The size of these libraries is well beyond the limit of screening technologies and of most in vivo and in vitro selection methods. The mRNA display technology has been used to select peptides and proteins that bind to a specific ligand, as well as novel enzymes. This protocol details the procedure to produce mRNA-displayed proteins (3 d) and to subject them to a selection and evolution of enzymes for bond-forming reactions (4-10 weeks). This method is demonstrated by the generation of new RNA ligase enzymes.     

High-throughput assays for lipases and esterases

In the past few years a considerable number of high-throughput screening (HTS) systems have been developed, especially for lipases and esterases. In this review, a range of HTS methods for the directed evolution of these hydrolases are covered. This includes spectrophotometric and fluorimetric formats as well as other approaches to allow for fast, efficient and reliable identification of desired enzyme variants within large mutant libraries. In addition, methods for library creation and application of lipases and esterases are briefly covered.     

Advances in laboratory evolution of enzymes

We address recent developments in the area of laboratory, or directed evolution, with a focus on enzymes and on new methodologies of generic potential. We survey three main areas: (i) library making techniques, including the application of computational and rational methods for library design; (ii) screening and selection techniques, including recent applications of enzyme screening by FACS (fluorescence activated cell sorter); (iii) new approaches for performing directed evolution, and in particular, the application of 'neutral drifts' (libraries generated by rounds of mutation and selection for the enzyme's original function) and of consensus mutations to generate highly evolvable starting points for directed evolution.     

Laboratory-Directed Protein Evolution

Systematic approaches to directed evolution of proteins have been documented since the 1970s. The ability to recruit new protein functions arises from the considerable substrate ambiguity of many proteins. The substrate ambiguity of a protein can be interpreted as the evolutionary potential that allows a protein to acquire new specificities through mutation or to regain function via mutations that differ from the original protein sequence. All organisms have evolutionarily exploited this substrate ambiguity. When exploited in a laboratory under controlled mutagenesis and selection, it enables a protein to “evolve” in desired directions. One of the most effective strategies in directed protein evolution is to gradually accumulate mutations, either sequentially or by recombination, while applying selective pressure. This is typically achieved by the generation of libraries of mutants followed by efficient screening of these libraries for targeted functions and subsequent repetition of the process using improved mutants from the previous screening. Here we review some of the successful strategies in creating protein diversity and the more recent progress in directed protein evolution in a wide range of scientific disciplines and its impacts in chemical, pharmaceutical, and agricultural sciences.     

Unbiased libraries in protein directed evolution

Directed evolution is a powerful approach to study the molecular basis of protein evolution and to engineer proteins for a wide range of applications in synthetic organic chemistry and biotechnology. There are many methods based on random or focused mutagenesis to engineer successfully any protein trait. Focused approaches such as site-directed and saturation mutagenesis have become methods of choice for improving protein activity, selectivity, stability and many other traits because the screening step can be practically handled (bottleneck in directed evolution). Although novel mutagenesis methods based on CRISPR or solid-phase gene synthesis can eliminate bias when creating protein libraries, traditional PCR approaches, although imperfect, remain widely used due to their ease and low cost. One of the most common approaches in focused mutagenesis relies on NNK mutagenesis, however, the primer-based 22c-trick and small-intelligent methods have emerged as key tools for constructing less biased and unbiased libraries when all 20 canonical amino acids are needed for various reasons. In this minireview, we assess studies employing such methods for library creation and their areas of application. We also discuss the advantages and disadvantages of both methods and provide a perspective for creating smarter libraries.     

体内连续定向进化研究进展

定向进化为合成生物学的发展提供了一种简单高效的工具,尤其在化学品合成和医药开发方面发挥着重要的作用.但是传统的定向进化技术存在操作繁琐、耗时和效率低的问题,不能满足大量突变文库的构建和筛选.近几年,一项将突变、翻译(进化非基因)、筛选和复制过程进行无缝连接的体内连续定向进化技术开始出现,该技术在噬菌体、细菌和真核细胞中...     

Quantitative screening of yeast surface-displayed polypeptide libraries by magnetic bead capture

Magnetic bead capture is demonstrated here to be a feasible alternative for quantitative screening of favorable mutants from a cell-displayed polypeptide library. Flow cytometric sorting with fluorescent probes has been employed previously for high throughput screening for either novel binders or improved mutants. However, many laboratories do not have ready access to this technology as a result of the limited availability and high cost of cytometers, restricting the use of cell-displayed libraries. Using streptavidin-coated magnetic beads and biotinylated ligands, an alternative approach to cell-based library screening for improved mutants was developed. Magnetic bead capture probability of labeled cells is shown to be closely correlated with the surface ligand density. A single-pass enrichment ratio of 9400 +/- 1800-fold, at the expense of 85 +/- 6% binder losses, is achieved from screening a library that contains one antibody-displaying cell (binder) in 1.1 x 10(5) nondisplaying cells. Additionally, kinetic screening for an initial high affinity to low affinity (7.7-fold lower) mutant ratio of 1:95,000, the magnetic bead capture method attains a single-pass enrichment ratio of 600 +/- 200-fold with a 75 +/- 24% probability of loss for the higher affinity mutant. The observed high loss probabilities can be straightforwardly compensated for by library oversampling, given the inherently parallel nature of the screen. Overall, these results demonstrate that magnetic beads are capable of quantitatively screening for novel binders and improved mutants. The described methods are directly analogous to procedures in common use for phage display and should lower the barriers to entry for use of cell surface display libraries.     

几种定向进化技术的比较及文库构建策略

蛋白质定向进化在蛋白质工程中取得了令人瞩目的成就,其核心技术---随机突变库构建技术已成为近年来体外定向进化研究的热点。在概述定向进化基本原理基础上,对几种随机突变技术进行了介绍、分类和比较,并对突变库的特征及构建策略予以分析和描述 。