植物分子系统分类学的新技术—VNTR及DNA指纹图

随着分子生物学技术日新月异的发展,许多先进的实验方法与较成熟的分析技术已开始进入植物系统分类学领域,使得这门学科逐步往分子水平迈进,可以直接对遗传变异和进化的分子基础DNA进行分析,以研究并揭示植物系统分类和进化的分子规律。继RFLP(Restriction Fragment Length Polymorphism,限制性酶切片段长度多态性)技术被植物系统分类学采纳并应用以来,最近,分子生物学     

微生物酶分子改造研究进展

近年来,越来越多的酶蛋白已经采用重组微生物反应器进行高效生产。为了改善酶蛋白的催化性能,提高其环境适应性,同时提高酶蛋白的表达量,降低生产成本,各种针对酶蛋白分子改造的基因工程技术已经得到大量的应用。综述了用于酶分子改造和进化的主要分子生物学方法,如定点突变、易错PCR、基因改组、密码子优化等技术及其应用成就。     

核酸技术在真菌系统分类与鉴定中的应用

近年来以核酸操作为主要内容的分子生物学技术的崛起,为从核酸分子水平上研究真菌的遗传本质,为真菌的进化研究、系统分类和鉴定开辟了新的途径。本文对近几年在真菌系统分类与鉴定研究中应用较多的ＤＮＡ—ＤＮＡ杂交、限制性片段长度多态性分析、多聚酶链式反应技术和核酸测序等几种技术进行简要评述。     

酶的分子设计、改造与工程应用

酶工程的研究已经发展到分子水平 ,在体外通过基因工程、化学、物理等手段改造酶分子结构与功能 ,大幅提高了酶分子的进化效率和催化效率 ,生产有价值的非天然酶。对酶工程学若干“热点”和前沿课题的研究、应用进行了概述 ,分析了国际上酶工程研究及应用技术、手段、方法 ,包括体外分子进化、核酶和抗体酶的设计、酶分子的定向固定化技术、酶蛋白分子的化学修饰、融合酶、人工合成及模拟酶等技术 ,并展望了酶工程的技术进步和应用的新进展。     

分子生物学技术在蕨类植物研究中的应用

近年来,分子标记、基因克隆、RNA干扰和基因芯片等分子生物学技术已应用于蕨类植物的系统进化、遗传多样性、孢子萌发以及生理代谢分子机制的研究。本文介绍近年来分子生物学技术在蕨类植物研究中的应用进展。     

大数据时代杂合酶的设计及其新趋势

酶分子的高效性和稳定性是工业广泛应用的物质基础。利用分子生物学技术可以将不同酶分子通过串联、插入、翻译后融合等方式构建成符合工业需求的杂合酶,但应用中多结构域杂合酶在表达量与酶活等方面仍存在弊端,而基于特定蛋白质结构域的多功能设计成为新趋势。高通量测序技术的发展,使得生物学家正面临着爆炸式增长的大数据集。近年来"蛋白质功能区"概念的提出,拓宽了人们对蛋白质结构与功能组织层次的认知,功能区残基聚簇的协同演化可导致同一家族不同蛋白质功能的差异。基于海量大数据分析可以快速定位特定功能区以及协同进化的关键位点,再利用合成生物学技术就可实现多种功能残基在同一蛋白质中的精准嫁接,完成天然酶分子的再设计。这将是杂合酶技术发展的新阶段,也会成为生物大数据时代下蛋白质设计的新趋势。     

启动子和细胞全局转录机制的定向进化在微生物代谢工程中的应用

通过随机突变和定向选择而进行的定向进化(又称分子进化或人工进化)在改造酶的催化特性和稳定性、扩展酶的底物范围等方面具有广泛的应用。近年来,定向进化也开始应用在对结构基因的启动子区域和具有调节功能的蛋白如转录因子等进行代谢工程改造,并成功选育了对环境胁迫因素具有较强耐受性,以及发酵效率提高的微生物菌种。以下着重介绍近年来启动子的定向进化,包括启动子的强度和调节功能的分子进化,以及细胞全局转录工程等技术在微生物代谢工程中的应用,这些定向进化技术使人们可以更精细地调节基因表达水平,并可同时改变细胞内多个基因的转录水平,是代谢工程研究新的有力工具。     

分子生物学方法在鲸类学研究中的应用

80年代以来,随着分子生物学的迅猛发展,涌现出一批分子进化和种群遗传分析方法和技术。       

纤毛虫分子系统发育学的研究进展

在回顾纤毛虫分子系统发育学产生发展历史的基础上,介绍了随着20年中RFLP、RAPD和DNA序列分析等分子生物学技术作为该学科的主要研究方法在种群遗传多样性与进化、种上阶元系统发育学两方面取得的研究成果和近期研究进展,最后在探讨纤毛虫分子系统发育学存在的一些问题和解决方法的同时,预测了纤毛虫分子系统发育学今后将极大地推动真核生物的起源与进化,内共生等重要生物进化问题的研究。     

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。     

Evolution of enzymes in metabolism: a network perspective

Several models have been proposed to explain the origin and evolution of enzymes in metabolic pathways. Initially, the retro-evolution model proposed that, as enzymes at the end of pathways depleted their substrates in the primordial soup, there was a pressure for earlier enzymes in pathways to be created, using the later ones as initial template, in order to replenish the pools of depleted metabolites. Later, the recruitment model proposed that initial templates from other pathways could be used as long as those enzymes were similar in chemistry or substrate specificity. These two models have dominated recent studies of enzyme evolution. These studies are constrained by either the small scale of the study or the artificial restrictions imposed by pathway definitions. Here, a network approach is used to study enzyme evolution in fully sequenced genomes, thus removing both constraints. We find that homologous pairs of enzymes are roughly twice as likely to have evolved from enzymes that are less than three steps away from each other in the reaction network than pairs of non-homologous enzymes. These results, together with the conservation of the type of chemical reaction catalyzed by evolutionarily related enzymes, suggest that functional blocks of similar chemistry have evolved within metabolic networks. One possible explanation for these observations is that this local evolution phenomenon is likely to cause less global physiological disruptions in metabolism than evolution of enzymes from other enzymes that are distant from them in the metabolic network.     

蛋白质工程：从定向进化到计算设计

定向进化通过建立突变体文库与高通量筛选方法,快速提升蛋白的特定性质,是目前蛋白质工程最为常用的蛋白质设计改造策略。近十年随着计算机运算能力大幅提升以及先进算法不断涌现,计算机辅助蛋白质设计改造得到了极大的重视和发展,成为蛋白质工程新开辟的重要方向。以结构模拟与能量计算为基础的蛋白质计算设计不但能改造酶的底物特异性与热稳定性,还可从头设计具有特定功能的人工酶。近年来机器学习等人工智能技术也被应用于计算机辅助蛋白质设计改造,并取得瞩目的成绩。文中介绍了蛋白质工程的发展历程,重点评述当前计算机辅助蛋白质设计改造方面的进展与应用,并展望其未来发展方向。     

Artificial enzymes with protein scaffolds: Structural design and modification

Recent development in biochemical experiment techniques and bioinformatics has enabled us to create a variety of artificial biocatalysts with protein scaffolds (namely ‘artificial enzymes’). The construction methods of these catalysts include genetic mutation, chemical modification using synthetic molecules and/or a combination of these methods. Designed evolution strategy based on the structural information of host proteins has become more and more popular as an effective approach to construct artificial protein-based biocatalysts with desired reactivities. From the viewpoint of application of artificial enzymes for organic synthesis, recently constructed artificial enzymes mediating oxidation, reduction and C–C bond formation/cleavage are introduced in this review article.     

Nature-inspired engineering of an artificial ligase enzyme by domain fusion

The function of most proteins is accomplished through the interplay of two or more protein domains and fine-tuned by natural evolution. In contrast, artificial enzymes have often been engineered from a single domain scaffold and frequently have lower catalytic activity than natural enzymes. We previously generated an artificial enzyme that catalyzed an RNA ligation by >2 million-fold but was likely limited in its activity by low substrate affinity. Inspired by nature''s concept of domain fusion, we fused the artificial enzyme to a series of protein domains known to bind nucleic acids with the goal of improving its catalytic activity. The effect of the fused domains on catalytic activity varied greatly, yielding severalfold increases but also reductions caused by domains that previously enhanced nucleic acid binding in other protein engineering projects. The combination of the two better performing binding domains improved the activity of the parental ligase by more than an order of magnitude. These results demonstrate for the first time that nature''s successful evolutionary mechanism of domain fusion can also improve an unevolved primordial-like protein whose structure and function had just been created in the test tube. The generation of multi-domain proteins might therefore be an ancient evolutionary process.     

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.     

多氯联苯降解菌Burkholderia xenovorans LB400研究进展

Burkholderia xenovorans LB400是一株多氯联苯(polychlorinated biphenyls,PCBs)降解菌,可以氧化含有1?6个氯取代基的多氯联苯。近年来,由于其广泛的底物谱和优异的降解性能,菌株LB400已成为研究原核生物降解多氯联苯的生物化学和分子生物学方面的模式生物。目前关于PCBs的微生物降解研究已不再局限于对微生物资源的挖掘,而是更多地聚焦在LB400等降解菌的PCBs降解基因、降解酶的酶学特性以及酶的人工分子进化等方面。同时,LB400作为早期发现的降解菌,其对多氯联苯的降解途径、底物范围及相关机制也被广泛探讨;但是对于PCBs降解相关基因的调控研究较少。因此,本文以Burkholderia xenovorans LB400对多氯联苯降解为核心,通过综述其代谢途径、代谢相关基因和酶系以及降解应用等方面的研究进展,以期为深入探讨Burkholderia xenovorans LB400的应用以及进一步在遗传、分子和生化水平研究其他多氯联苯降解菌株提供借鉴。     

Synthetic biology with RNA motifs

Structural motifs in naturally occurring RNAs and RNPs can be employed as new molecular parts for synthetic biology to facilitate the development of novel devices and systems that modulate cellular functions. In this review, we focus on the following: (i) experimental evolution techniques of RNA molecules in vitro and (ii) their applications for regulating gene expression systems in vivo. For experimental evolution, new artificial RNA aptamers and RNA enzymes (ribozymes) have been selected in vitro. These functional RNA molecules are likely to be applicable in the reprogramming of existing gene regulatory systems. Furthermore, they may be used for designing hypothetical RNA-based living systems in the so-called RNA world. For the regulation of gene expressions in living cells, the development of new riboswitches allows us to modulate the target gene expression in a tailor-made manner. Moreover, recently RNA-based synthetic genetic circuits have been reported by employing functional RNA molecules, expanding the repertory of synthetic biology with RNA motifs.     

The use of molecular modelling in the understanding of configurational specificity (R or S) in asymmetric reactions catalyzed by Saccharomyces cerevisiae or isolated dehydrogenases

This method gives a general ideal how to use crystallographic information of enzymes to understand reactions catalyzed by these biocatalysts, commonly used by biochemists to produce chiral products. The interactions of three acetoacetic esters with the enzymes L-lactate dehydrogenase and alcohol dehydrogenase were studied through molecular modelling computer program. These artificial substrates have been widely used to produce chiral synthons. Through this methodology it was possible to understand the conformational specificity of these enzymes with respect to the products and how these enzymes can be inhibited by modifying the structures of the artificial substrates. Also, it was possible to predict whether some type of artificial substrate will suffer reduction by cells that contain these dehydrogenases and what kind of configuration (R or S) the final product will have.     

蛋白质人为进化的研究进展

蛋白质人为进化是目前蛋白质工程研究的热点之一.易错PCR、DNA shuffling及高突变菌株的应用,使许多蛋白质在功能上大幅度改善.     

Determinants of thermostability in the cytochrome P450 fold

Cytochromes P450 are found throughout the biosphere in a wide range of environments, serving a multitude of physiological functions. The ubiquity of the P450 fold suggests that it has been co-opted by evolution many times, and likely presents a useful compromise between structural stability and conformational flexibility. The diversity of substrates metabolized and reactions catalyzed by P450s makes them attractive starting materials for use as biocatalysts of commercially useful reactions. However, process conditions impose different requirements on enzymes to those in which they have evolved naturally. Most natural environments are relatively mild, and therefore most P450s have not been selected in Nature for the ability to withstand temperatures above ~ 40 °C, yet industrial processes frequently require extended incubations at much higher temperatures. Thus, there has been considerable interest and effort invested in finding or engineering thermostable P450 systems. Numerous P450s have now been identified in thermophilic organisms and analysis of their structures provides information as to mechanisms by which the P450 fold can be stabilized. In addition, protein engineering, particularly by directed or artificial evolution, has revealed mutations that serve to stabilize particular mesophilic enzymes of interest. Here we review the current understanding of thermostability as it applies to the P450 fold, gleaned from the analysis of P450s characterized from thermophilic organisms and the parallel engineering of mesophilic forms for greater thermostability. We then present a perspective on how this information might be used to design stable P450 enzymes for industrial application. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.