DNA重排及体外分子进化

ＤＮＡ重排是目前为止最简便、最有效的体外定向进化技术,可以对单一基因、质粒、代谢途径、部分甚至整个基因组进行改造。本综述了ＤＮＡ重排的基本原理、特点、与其它体外进化技术的不同,着重介绍了其在体外分子进化上的广泛应用,并对应用前景进行了展望。     

Molecular evolution of adomet synthetase by DNA recombination with a novel Separate-Mixing method

We describe a new approach to in vitro DNA recombination termed the Separate-Mixing method in this study. The reaction process of this method consists of two stages: at the first stage the reaction was implemented in two parallel teams, which generated random recombination by template-switching of growing poly-nucleotides from primers in the presence of unidirectional single-stranded DNA fragments used as templates, and then both teams were mixed together for further extension and recombination of DNA sequences at the second stage. Due to this particular strategy, the reaction process was also accompanied by two other processes of DNA shuffling and StEP simultaneously. Two AdoMet synthetase genes, sam2 from Saccharomyces cerevisiae and metK from Escherichia coli, which have only 56% homology on the DNA level, were used for recombination with the Separate-Mixing method. DNA recombination was available after a single round of reaction. When 10 randomly selected recombinants were sequenced, an unshuffled parental clone was not found, nor was unexpected insertion, deletion, or rearrangement detected. An evolved gene, sam’, was obtained after screening and selection, which could obviously increase the accumulation of AdoMet in S. cerevisiae. Published in Russian in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 3, pp. 546–553. This article was submitted by the authors in English.     

一个简便的DNA改组(DNA shuffling)操作程序

介绍了一个简便的DNA改组操作程序.首先利用PCR扩增了两段具有高度序列同源性的1 700 bp左右的基因片段,两者相比较同源性大于93%.然后将其等量混合后,在Mg²⁺存在的条件下,用DNaseⅠ切割成10～50 bp的小片段.这些小片段在不外加引物的前提下,利用PCR反应进行重聚,再将重聚物经过两轮正常的PCR扩增,获得了与原来片段大小相当的基因片段.这一技术有利于从一组序列同源性程度较高的基因库构建随机嵌合基因.     

Analysis of shuffled gene libraries

In vitro recombination of homologous genes (family shuffling) has been proposed as an effective search strategy for laboratory evolution of genes and proteins. Few data are available, however, on the composition of shuffled gene libraries, from which one could assess the efficiency of recombination and optimize protocols. Here, probe hybridization is used in a macroarray format to analyze chimeric DNA libraries created by DNA shuffling. Characterization of hundreds of shuffled genes encoding dioxygenases has elucidated important biases in the shuffling reaction. As expected, crossovers are favored in regions of high sequence identity. A sequence-based model of homologous recombination that captures this observed bias was formulated using the experimental results. The chimeric genes were found to show biases in the incorporation of sequences from certain parents, even before selection. Statistically different patterns of parental incorporation in genes expressing functional proteins can help to identify key sequence-function relationships.     

DNA改组技术在水蛭素实验进化中的应用

蛋白质的改造是生物工程的重大研究课题.由于结构和功能预测的不精确性,而使按照三维结构信息进行定位诱变往往达不到预期的目的.近年来,另一条改造蛋白质的途径有较大的发展,即在实验室条件下模拟生物分子的自然进化,通过变异和靶功能的选择来获得改进性能的蛋白质[1],此过程称为生物分子实验定向进化.DNA改组(DNAshuffling)是一种改造基因和蛋白质的有效实验进化技术[2].它是在体外进行基因随机片段的重组,从而增加基因的多样性,促使有利变异与不利变异分离,通过选择使有利变异得到优化组合[3].DNA改组包含3个步骤:基因的随机片段化,自身引发PCR和重组合PCR.经过DNA改组的突变体库有可能选择到性能更优的突变体.为进行亲和淘选,需将突变体展示在噬菌体的表面[4].     

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.     

DNA改组(DNA shuffling)及其研究进展

自1994年首次提出至今,DNA改组已经成为定向进化最为有效的方法之一.无论DNA、蛋白质还是生物体的进化,DNA改组都有十分突出的表现.通过对十余年来DNA改组的发展作以简要综述,为日后相关研究的开展提供理论基础.     

Increasing the thermal stability of an oligomeric protein, beta-glucuronidase.

The reporter enzyme beta-glucuronidase was mutagenized and evolved for thermostability. After four cycles of screening the best variant was more active than the wild-type enzyme, and retained function at 70 degrees C, whereas the wild-type enzyme lost function at 65 degrees C. Variants derived from sequential mutagenesis were shuffled together, and re-screened for thermostability. The best variants retained activities at even higher temperatures (80 degrees C), but had specific activities that were now less than that of the wild-type enzyme. The mutations clustered near the tetramer interface of the enzyme, and many of the evolved variants showed much greater resistance to quaternary structure disruption at high temperatures, which is also a characteristic of naturally thermostable enzymes. Together, these results suggest a pathway for the evolution of thermostability in which enzymes initially become stable at high temperatures without loss of activity at low temperatures, while further evolution leads to enzymes that have kinetic parameters that are optimized for high temperatures.     

酶的定向进化研究及其在工业生物催化中的应用

综合概括了各种蛋白质改造与微生物代谢途径改造的非理性定向进化技术的原理、特点,介绍了这些技术在高性能工业生物催化剂改造中的应用。     

Engineered enzymes for chemical production

In order to enable competitive manufacturing routes, most biocatalysts must be tailor-made for their processes. Enzymes from nature rarely have the combined properties necessary for industrial chemical production such as high activity and selectivity on non-natural substrates and toleration of high concentrations of organic media over the wide range of conditions (decreasing substrate, increasing product concentrations, solvents, etc.,) that will be present over the course of a manufacturing process. With the advances in protein engineering technologies, a variety of enzyme properties can be altered simultaneously, if the appropriate screening parameters are employed. Here we discuss the process of directed evolution for the generation of commercially viable biocatalysts for the production of fine chemicals, and how novel approaches have helped to overcome some of the challenges.     

分段DNA shuffling: 一种大分子海藻糖合酶有效的定向进化方法

[目的]红色亚栖热菌(Meiothermus ruber)海藻糖合酶(Trehalose synthase,M-TreS)将麦芽糖转化生成海藻糖只需一步反应,且具有很好的热稳定性及pH耐受性,是潜在的工业生产海藻糖的酶源.为了提高该酶的性能,有必要对其进行定向进化.[方法]M-TreS基因(M-treS)大小为2 889bp.该蛋白质分子本身具有很大的进化空间,但是却不宜进行全长基因Shuffling.分段DNA shuffling是为大分子蛋白质(基因≥2 000 bp)的进化而设计的一种方法.该方法分为三步:(1)用两对引物分别扩增目的基因的上游片段和下游片段;(2)上下游片段各自进行Shuffling; (3)利用重叠延伸PCR连接上下游突变群,建立完整基因的突变文库.[结果]结合易错PCR,通过该方法经一轮进化获得一株酶活力是野生型1.6倍、催化效率是野生型2倍的突变株.序列分析表明,该突变株共有6个位点发生了氨基酸的替代,其中一个来自易错突变,2个来自同源重组,3个为随机突变.[结论]分段DNA shuffling是进化大分子蛋白质的有效方法.     

Delineation of an evolutionary salvage pathway by compensatory mutations of a defective lysozyme.

Model-free approaches (random mutagenesis, DNA shuffling) in combination with more "rational," three-dimensional information-guided randomization have been used for directed evolution of lysozyme activity in a defective T4 lysozyme mutant. A specialized lysozyme cloning vector phage, derived from phage lambda, depends upon T4 lysozyme function for its ability to form plaques. The substitution W138P in T4 lysozyme totally abolishes its plaque-forming ability. Compensating mutations in W138P T4 lysozyme after sequential random mutagenesis of the whole gene as well as after targeted randomization of residues in the vicinity of Trp138 were selected. In a second stage, these mutations were randomly recombined by the recombinatorial PCR method of DNA shuffling. Shuffled and selected W138P T4 lysozyme variants provide the hybrid lambda phage with sufficient lysozyme activity to produce normal-size plaques, even at elevated temperature (42 degrees C). The individual mutations with the highest compensatory information for W138P repair are the substitutions A146F and A146M, selected after targeted randomization of three residues in the neighborhood of Trp138 by combinatorial mutagenesis. The best evolved W138P T4 lysozymes, however, accumulated mutations originating from both randomly mutagenized as well as target-randomized variants.     

Efficient site-directed saturation mutagenesis using degenerate oligonucleotides.

We describe a reliable protocol for constructing single-site saturation mutagenesis libraries consisting of all 20 naturally occurring amino acids at a specific site within a protein. Such libraries are useful for structure-function studies and directed evolution. This protocol extends the utility of Stratagene's QuikChange Site-Directed Mutagenesis Kit, which is primarily recommended for single amino acid substitutions. Two complementary primers are synthesized, containing a degenerate mixture of the four bases at the three positions of the selected codon. These primers are added to starting plasmid template and thermal cycled to produce mutant DNA molecules, which are subsequently transformed into competent bacteria. The protocol does not require purification of mutagenic oligonucleotides or PCR products. This reduces both the cost and turnaround time in high-throughput directed evolution applications. We have utilized this protocol to generate over 200 site-saturation libraries in a DNA polymerase, with a success rate of greater than 95%.     

改进酶稳定性的定向进化技术

酶作为一种生物催化剂,以其独特的优良特性,在绿色化学和清洁生产中得到了广泛的应用。随着酶定向进化技术的建立和发展,通过定向进化改进酶稳定性的研究越来越多。详细综述了各种定向进化方法的特点及在提高酶稳定性方面的应用,并从结构和功能的角度进一步解释了相关机理。     

Simultaneous optimization of enzyme activity and quaternary structure by directed evolution

Natural evolution has produced efficient enzymes of enormous structural diversity. We imitated this natural process in the laboratory to augment the efficiency of an engineered chorismate mutase with low activity and an unusual hexameric topology. By applying two rounds of DNA shuffling and genetic selection, we obtained a 400-fold more efficient enzyme, containing three non-active-site mutations. Detailed biophysical characterization of the evolved variant suggests that it exists predominantly as a trimer in solution, but is otherwise similarly stable as the parent hexamer. The dramatic structural and functional effects achieved by a small number of seemingly innocuous substitutions highlights the utility of directed evolution for modifying protein-protein interactions to produce novel quaternary states with optimized activities.     

A comparison of directed evolution approaches using the beta-glucuronidase model system

Protein engineers can alter the properties of enzymes by directing their evolution in vitro. Many methods to generate molecular diversity and to identify improved clones have been developed, but experimental evolution remains as much an art as a science. We previously used DNA shuffling (sexual recombination) and a histochemical screen to direct the evolution of Escherichia coli beta-glucuronidase (GUS) variants with improved beta-galactosidase (BGAL) activity. Here, we employ the same model evolutionary system to test the efficiencies of several other techniques: recursive random mutagenesis (asexual), combinatorial cassette mutagenesis (high-frequency recombination) and a versatile high-throughput microplate screen. GUS variants with altered specificity evolved in each trial, but different combinations of mutagenesis and screening techniques effected the fixation of different beneficial mutations. The new microplate screen identified a broader set of mutations than the previously employed X-gal colony screen. Recursive random mutagenesis produced essentially asexual populations, within which beneficial mutations drove each other into extinction (clonal interference); DNA shuffling and combinatorial cassette mutagenesis led instead to the accumulation of beneficial mutations within a single allele. These results explain why recombinational approaches generally increase the efficiency of laboratory evolution.     

In vitro evolution of thermostable p53 variants

The tumor suppressor p53 is conformationally unstable at physiological temperature. Even the activated p53delta30 variant, which lacks the self-inhibiting carboxy terminal domain, has a half-life of only 8 min at 37 degrees C in vitro. We have developed a genetic approach to identify p53 variants that stabilize the active conformation. The human p53delta30 gene was randomly mutated, and the resulting library was expressed in Escherichia coli under conditions that apparently denatured the parental protein. Stable p53 variants were identified based on their ability to specifically bind a p53 consensus site. The initial thermostable variants were randomly recombined by DNA shuffling, and substitutions that were functionally additive or synergistic were identified in a second more stringent round of screening. The DNA binding activity of N239Y/N268D/E336V p53delta30 variant has a half-life of 100 min at 37 degrees C, 12 times longer than that of the parental protein. The thermostable variants should be more amenable to crystallographic studies and more effective in gene therapies than the wild-type protein.     

Quality control of DNA break metabolism: in the 'end', it's a good thing

DNA ends pose specific problems in the control of genetic information quality. Ends of broken DNA need to be rejoined to avoid genome rearrangements, whereas natural DNA ends of linear chromosomes, telomeres, need to be stable and hidden from the DNA damage response. Efficient DNA end metabolism, either at induced DNA breaks or telomeres, does not result from the machine-like precision of molecular reactions, but rather from messier, more stochastic processes. The necessary molecular interactions are dynamically unstable, with constructive and destructive processes occurring in competition. In the end, quality control comes from the constant building up and tearing down of inappropriate, but also appropriate reaction steps in combination with factors that only slightly shift the equilibrium to eventually favour appropriate events. Thus, paradoxically, enzymes antagonizing DNA end metabolism help to ensure that genome maintenance becomes a robust process.