蛋白质体外进化建库技术研究

蛋白质体外进化技术是蛋白质工程发展的一个里程碑,也是改造蛋白质的一种有效工具。它不仅具有重要的应用价值,而且有助于蛋白质结构与功能的研究。通过蛋白质体外进化技术已成功地改造了许多蛋白质,有些已应用于工农业生产。体外进化技术分为两步:建库和筛选。本文主要对蛋白质体外进化策略及对体外随机突变技术、DNA重组技术、利用活细胞自身修复系统构建突变文库等几种定向进化突变文库建立技术进行了介绍与论述,同时还对蛋白质体外进化技术的应用及与其它学科结合的研究前景进行了分析,为获得具有改进功能或全新功能的蛋白质提供理论基础。     

体外分子定向进化研究进展

体外定向进化作为近几年发展起来的一种蛋白质改造新策略,可以在未知目标蛋白三维结构信息和作用机制的情况下,通过对编码基因的随机突变、重组和定向筛选,获得具有改进功能或全新功能的蛋白质,使几百万年的自然进化过程在短期内得以实现,因而是发现新的生物活性分子和反应途径的重要方法,已在短短几年内取得了令人瞩目的成就.     

蛋白质定向进化及其在微生物代谢调控中的应用

蛋白质定向进化是非理性改造蛋白质的一种有效方法。利用蛋白质定向进化技术可以改变代谢流,扩展或构建新的代谢途径,弱化或消除不必要或有害的代谢途径,从而达到提高某种代谢产物产率或降解有害物质的目的。蛋白质定向进化技术在代谢调控中的应用有效拓宽了代谢工程的应用范围。本文介绍了主要的蛋白质定向进化技术如易错PCR技术、DNA改组技术、交错延伸技术和临时模板随机嵌合技术等,评述了蛋白质定向进化技术对微生物细胞代谢中的关键蛋白进行定向改造,从而改善其代谢能力,调控微生物代谢等的应用。     

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.     

蛋白质新功能定向进化研究策略

利用定向进化策略改造蛋白质功能已经在农业、工业和医药等领域得到了广泛的应用.蛋白质工程的最新进展是利用定向进化策略对自然界蛋白质引入新功能,但由于其决定因素比较复杂,是研究者面临的一个重大挑战.详细介绍了国外近年发展的蛋白质新功能定向进化研究策略:对传统突变体库构建策略进行改进以及非同源重组改造技术的开发,是早期引入蛋白质新功能的常用手段,利用计算/理性设计与定向进化相结合引入蛋白质新功能是近年定向进化研究的一个重大突破,而噬菌体展示技术是蛋白质新功能筛选的主要策略.蛋白质新功能的分子进化模型已逐渐成为蛋白质工程改造的新思路.     

蛋白质定向进化的研究进展及其应用前景

定向进化是改造蛋白质分子的有效新策略.它不需要了解蛋白质的空间结构,主要通过在实验室里模拟自然进化过程,采用错误倾向PCR等方法对编码蛋白质的基因进行随机突变,经DNA改组、交错延伸等技术进行体外重组,设计高通量筛选方法来选出需要的突变体.本综述了定向进化技术的发展及应用.     

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新进展,这些新技术极大地加速和简化了传统的定向进化过程,为定向进化的发展注入了强劲动力。     

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。     

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。     

蛋白质体外定向进化策略研究进展

定向进化已广泛应用于蛋白质的分子改造,是改善蛋白质特性的最有效策略之一。为了获得更高的突变效率,构建含有更多突变体的文库,不断地发明创新各种高效的蛋白质体外定向进化方法。对近年来出现的几种定向进化方法三核苷酸突变(TriNex)、序列饱和突变(SeSaM)、随机链交换突变法(RAISE)、重叠延伸蛋白域文库法(PDLGO)和迭代饱和突变法(ISM)的原理、特点及应用进行综述。     

高通量筛选系统在定向改造中的新进展

酶和细胞工厂是工业生物技术的核心,在医药、化工、食品、农业、能源等诸多领域发挥重要作用。一般天然酶和细胞均需通过分子改造提高其催化效率、稳定性及立体选择性等。定向改造为快速改善酶和细胞工厂的性能提供了可能性,其中灵敏可靠的高通量筛选方法是决定酶和细胞工厂成功高效定向改造的关键。文中阐述并分析讨论了各种筛选方法的优缺点、适用范围以及信号产生策略,并总结了近3年超高通量筛选技术在酶和细胞工厂定向改造中的最新研究进展。在此基础上,讨论了高通量筛选系统目前面临的限制性因素,并对高通量筛选方法未来的发展趋势作出了展望。希望生物技术和仪器开发等各领域的研究者能够紧密合作,实现协同发展,进一步提升高通量筛选技术的可靠性和适用性。     

Optimization of protein-based volumetric optical memories and associative processors by using directed evolution

The potential use of proteins in device applications has advanced in large part due to significant advances in the methods and procedures of protein engineering, most notably, directed evolution. Directed evolution has been used to tailor a broad range of enzymatic proteins for pharmaceutical and industrial applications. Thermal stability, chemical stability, and substrate specificity are among the most common phenotypes targeted for optimization. However, in vivo screening systems for photoactive proteins have been slow in development. A high-throughput screening system for the photokinetic optimization of photoactive proteins would promote the development of protein-based field-effect transistors, artificial retinas, spatial light modulators, photovoltaic fuel cells, three-dimensional volumetric memories, and optical holographic processors. This investigation seeks to optimize the photoactive protein bacteriorhodopsin (BR) for volumetric optical and holographic memories. Semi-random mutagenesis and in vitro screening were used to create and analyze nearly 800 mutants spanning the entire length of the bacterio-opsin (bop) gene. To fully realize the potential of BR in optoelectronic environments, future investigations will utilize global mutagenesis and in vivo screening systems. The architecture for a potential in vivo screening system is explored in this study. We demonstrate the ability to measure the formation and decay of the red-shifted O-state within in vivo colonies of Halobacterium salinarum, and discuss the implications of this screening method to directed evolution. These authors contributed equally to this work.     

Advances in directed molecular evolution of reporter genes

Many reporter genes, such as gfp, gusA, and lacZ, are widely used for research into plants, animals, and microorganisms. Reporter genes, which offer high levels of sensitivity and convenience of detection, have been utilized in transgenic technology, promoter analysis, drug screening, and other areas. Directed molecular evolution is a powerful molecular tool for the creation of designer proteins for industrial and research applications, including studies of protein structure and function. Directed molecular evolution is based mainly on in vitro recombination methods, such as error-prone PCR and DNA shuffling. The strategies of directed evolution of enzyme biocatalysts have been the subject of several recent reviews. Here, we briefly summarize successes in the field of directed molecular evolution of reporter genes and discuss some of the applications.     

Enzyme-mediated hydrogel encapsulation of single cells for high-throughput screening and directed evolution of oxidoreductases

Directed evolution of oxidoreductases to improve their catalytic properties is being ardently pursued in the industrial, biotechnological, and biopharma sectors. Hampering this pursuit are current enzyme screening methods that are limited in terms of throughput, cost, time, and complexity. We present a directed evolution strategy that allows for large-scale one-pot screening of glucose oxidase (GOx) enzyme libraries in well-mixed homogeneous solution. We used GOx variants displayed on the outer cell wall of yeasts to initiate a cascade reaction with horseradish peroxidase (HRP), resulting in peroxidase-mediated phenol cross-coupling and encapsulation of individual cells in well-defined fluorescent alginate hydrogel shells within ~10 min in mixed cell suspensions. Following application of denaturing stress to whole-cell GOx libraries, only cells displaying GOx variants with enhanced stability or catalytic activity were able to carry out the hydrogel encapsulation reaction. Fluorescence-activated cell sorting was then used to isolate the enhanced variants. We characterized three of the newly evolved Aspergillus niger GOx enzyme sequences and found up to ~5-fold higher specific activity, enhanced thermal stability, and differentiable glycosylation patterns. By coupling intracellular gene expression with the rapid formation of an extracellular hydrogel capsule, our system improves high-throughput screening for directed evolution of H ₂O ₂-producing enzymes many folds.     

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.

     

Automated assay for screening the enzymatic release of reducing sugars from micronized biomass

Background  

To reduce the production cost of bioethanol obtained from fermentation of the sugars provided by degradation of lignocellulosic biomass (i.e., second generation bioethanol), it is necessary to screen for new enzymes endowed with more efficient biomass degrading properties. This demands the set-up of high-throughput screening methods. Several methods have been devised all using microplates in the industrial SBS format. Although this size reduction and standardization has greatly improved the screening process, the published methods comprise one or more manual steps that seriously decrease throughput. Therefore, we worked to devise a screening method devoid of any manual steps.     

Directed evolution of an endoinulinase from Talaromyces purpureogenus toward efficient production of inulooligosaccharides

Inulinases are fructofuranosyl hydrolases that target the β‐2,1 linkage of inulin and hydrolyze it into fructose, glucose and inulooligosaccharides (IOS), the latter are of growing interest as dietary fibers. Inulinases from various microorganisms have been purified, characterized and produced for industrial applications. However, there remains a need for inulinases with increased catalytic activity and better production yields to improve the hydrolysis process and fulfill the growing industrial demands for specific fibers. In this study, we used directed enzyme evolution to increase the yield and activity of an endoinulinase enzyme originated from the filamentous fungus Talaromyces purpureogenus (Penicillium purpureogenum ATCC4713). Our directed evolution approach yielded variants showing up to fivefold improvements in soluble enzyme production compared to the starting point which enabled high‐yield production of highly purified recombinant enzyme. The distribution of the enzymatic reaction products demonstrated that after 24 h of incubation, the main product (57%) had a degree of polymerization of 3 (DP3). To the best of our knowledge, this is the first application of directed enzyme evolution to improve inulooligosaccharide production. The approach enabled the screening of large genetic libraries within short time frames and facilitated screening for improved enzymatic activities and properties, such as substrate specificity, product range, thermostability and pH optimum. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:868–877, 2018     

Colorimetric inorganic pyrophosphate assay using a double cycling enzymatic method

The biosynthesis of aminocoumarin antibiotics involves the action of amide synthetases which construct amide bonds between aminocoumarins and various acyl moieties. Libraries of aminocoumarin analogues have been generated by in vivo fermentation, via feeding known amide synthetase substrates into producing microbial strains. Critically, such feeding studies rely on the inherent or engineered substrate promiscuity of each amide synthetase. We have initiated a program of directed evolution in order to create mutant amide synthetases for the synthesis of new nonnatural amino coumarin analogues. We used the clorobiocin enzyme CloL as a model amide synthetase to design and validate a fluorimetric high-throughput screen, which can be used to report the activity of mutant amide synthetases toward a broad range of coumarin and acyl donor substrates. Our assay monitors the decrease in fluorescence of aminocoumarins on acylation. The utility of the assay was illustrated by screening a library of amide synthetase mutants created by error-prone PCR. The substrate specificity of an amide synthetase was also rapidly probed using this assay, affording several newly identified substrates. It is anticipated that this high-throughput screen will accelerate the creation of amide synthetase mutants with new specificities by directed evolution.