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。     

酶分子体外定向进化的研究进展

分子体外定向进化是改造酶分子的新策略,它主要通过体外模拟自然进化机制,利用基因随机突变、重组和定向筛选技术,使进化过程朝着人们需要的方向发展。简要介绍了酶分子体外定向进化的发展历史,详细介绍了突变文库构建和筛选方法的最新研究进展及应用情况。     

几种高通量定向筛选技术及其比较

在定向进化过程中,多样性的突变文库一旦构建好后,实验成败与否的关键就在于建立针对目的改造特征的高通量筛选方案。从突变文库中筛选需要的、功能进化的目的基因,关键在于是否有一种或几种高通量筛选技术。介绍了几种目前常用高通量定向筛选技术的基本原理,并对其特性进行了比较。     

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、细菌和真核细胞中均取得了突破性进展,极大地促进了定向进化技术的革新和应用。随着体内连续定向进化技术的不断发展,筛选方法和设备也不断改善。对体内连续定向进化技术、筛选方法和设备最新研究进展作一综述,并讨论当前面临的挑战和机遇。     

酶的定向进化策略

定向进化技术已成为改造酶分子的一种有效策略,是在体外模拟自然进化进程,通过随机突变和（或）体外重组产生基因的多样性．再经筛选（或选择）获得所需性能大幅提高的酶。该文介绍近几年来酶定向进化技术研究的最新进展以及一些成功实例。     

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 。     

蛋白质定向进化技术的研究进展

蛋白质定向进化技术是蛋白质分子改造的一个重要策略.重点介绍了易错PCR、DNA改组等对编码蛋白质的基因进行随机突变和重组的技术,以及构建突变体库和高通量筛选的方法,并探讨了定向进化技术在蛋白质工程中的应用及前景.     

基于荧光激活细胞分选技术的超高通量酶活性筛选方法及其应用

基于荧光激活细胞分选(FACS)技术的超高通量酶活性筛选方法是新出现的一类高通量筛选技术.它利用流式细胞仪高灵敏度、高通量的特点,能以极高的速度(108/天)对大容量酶基因文库进行筛选.FACS筛选技术的出现突破了常规筛选方法低效、耗时、费力等瓶颈问题,极大地提升了人类对大容量基因文库的探索能力,因此在新酶基因筛选、酶活性检测、酶定向进化等领域有广泛的应用潜力.综述了FACS超高通量酶活性筛选方法的最新研究进展,着重介绍了其在酶定向进化中的应用.     

通过原位易错PCR一步构建基因突变文库

主要介绍一种通过原位易错PCR构建随机突变文库的新技术。本实验室最近发表的一项国际专利中,利用来源于海栖热孢菌的极耐热性DNA连接酶,在传统PCR循环中加入一个连接步骤,即变性—退火—延伸—连接的四步循环法PCR,从而实现环状质粒的PCR指数扩增(PPCP)。原位易错PCR中所用引物为一段线性双链DNA,它含有与模板质粒不同的筛选标记,产物转化宿主菌后,模板质粒在筛选平板上被直接剔除。筛选到的阳性突变子可用作模板直接进入突变文库的再次构建,通过筛选获得二级或多级累加的正突变。利用这种方法构建了一个木聚糖酶基因和一个纤维素酶基因的随机突变文库,并筛选出具有正向突变的蛋白,证明以PPCP为基础的原位易错PCR技术,为基因定向进化提供了一种快速有效的随机突变文库构建的新方法。     

斑马鱼反转录病毒插入突变技术的发展及其在基因饱和突变与筛选中的应用

用于大规模基因突变与筛选的主要策略有化学诱变、插入突变、基因诱捕。插入突变是一种通过外源DNA整合的方式来获得突变体,并克隆得到对应突变基因的方法。运用反转录病毒介导的插入突变技术,在脊椎动物斑马鱼中已经获得了许多影响胚胎发育和细胞生长过程的突变体,并找到了对应的基因。基因诱捕技术也被运用于反转录病毒载体的构建。这套系统的建立使斑马鱼成为第一个有可能达到基因饱和突变和筛选的脊椎动物。     

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.     

Mutant library construction in directed molecular evolution

Directed molecular evolution imitates the natural selection process in the laboratory to find mutant proteins with improved properties in the expected aspects by exploring the encoding sequence space. The success of directed molecular evolution experiment depends on the quality of artificially prepared mutant libraries and the availability of convenient high-throughput screening methods. Well-prepared libraries promise the possibility of obtaining desired mutants by screening a library containing a relatively small number of mutants. This article summarizes and reviews the currently available methodologies widely used in directed evolution practices in the hope of providing a general reference for library construction. These methods include error-prone polymerase chain reaction (epPCR), oligonucleotide-based mutagenesis, and genetic recombination exemplified by DNA shuffling and its derivatives. Another designed method is also discussed, in which B-lymphocytes are fooled to mutate nonantibody foreign proteins through somatic hypermutation (SHM).     

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。     

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.     

High-throughput screens and selections of enzyme-encoding genes

The availability of vast gene repertoires from both natural sources (genomic and cDNA libraries) and artificial sources (gene libraries) demands the development and application of novel technologies that enable the screening or selection of large libraries for a variety of enzymatic activities. We describe recent developments in the selection of enzyme-coding genes for directed evolution and functional genomics. We focus on HTS approaches that enable selection from large libraries (>10(6) gene variants) with relatively humble means (i.e. non-robotic systems), and on in vitro compartmentalization in particular.     

蛋白质定向进化的研究进展

在工业生物催化过程和生物细胞工厂构建方面,蛋白质定向进化被广泛地应用于酶的分子改造.蛋白质定向进化不仅可以针对某一目的蛋白进行改造,还可以改善代谢途径、优化代谢网络、获得期望表型细胞.为了获得更高效的突变效率,快捷、高通量的筛选方法,提高蛋白质定向进化的效果,研究者不断开发蛋白质体内、体外进化方法,取得了新的进展和应用.本文介绍了最近发展的蛋白质定向进化技术的原理、方法及特点,总结了突变文库的筛选方法和蛋白质定向进化的最新应用,最后讨论了蛋白质定向进化存在的挑战和未来发展方向.     

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.