Directed evolution of beta-galactosidase from Escherichia coli into beta-glucuronidase

In vitro directed evolution through DNA shuffling is a powerful molecular tool for creation of new biological phenotypes. E. coli beta-galactosidase and beta-glucuronidase are widely used, and their biological function, catalytic mechanism, and molecular structures are well characterized. We applied an in vitro directed evolution strategy through DNA shuffling and obtained five mutants named YG6764, YG6768, YG6769, YG6770 and YG6771 after two rounds of DNA shuffling and screening, which exhibited more beta-glucuronidase activity than wild-type beta-galactosidase. These variants had mutations at fourteen nucleic acid sites, resulting in changes in ten amino acids: S193N, T266A, Q267R, V411A, D448G, G466A, L527I, M543I, Q626R and Q951R. We expressed and purified those mutant proteins. Compared to the wild-type protein, five mutant proteins exhibited high beta-glucuronidase activity. The comparison of molecular models of the mutated and wildtype enzymes revealed the relationship between protein function and structural modification.     

Directed evolution of a beta-galactosidase from <Emphasis Type="Italic">Pyrococcus woesei</Emphasis> resulting in increased thermostable beta-glucuronidase activity

We performed directed evolution on a chemically synthesized 1,533-bp recombinant beta-galactosidase gene from Pyrococcus woesei. More than 200,000 variant colonies in each round of directed evolution were screened using the pYPX251 vector and host strain Rosetta-Blue (DE3). One shifted beta-galactosidase to beta-glucuronidase mutant, named YG6762, was obtained after four rounds of directed evolution and screening. This mutant had eight mutated amino acid residues. T29A, V213I, L217M, N277H, I387V, R491C, and N496D were key mutations for high beta-glucuronidase activity, while E414D was not essential because the mutation did not lead to a change in beta-glucuronidase activity. The amino acid site 277 was the most essential because mutating H back to N resulted in a 50% decrease in beta-glucuronidase activity at 37°C. We also demonstrated that amino acid 277 was the most essential site, as the mutation from N to H resulted in a 1.5-fold increase in beta-glucuronidase activity at 37°C. Although most single amino acid changes lead to less than a 20% increase in beta-glucuronidase activity, the YG6762 variant, which was mutated at all eight amino acid sites, had a beta-glucuronidase activity that was about five and seven times greater than the wild-type enzyme at 37 and 25°C, respectively. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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).     

过氧化氢酶基因定向进化文库的构建及其评价

采用易错聚合酶链反应和DNA改组技术构建野生型梅花鹿过氧化氢酶(CAT)基因的突变体文库,并随机对两种方法所得产物各5个样品做序列测定。序列分析结果表明突变率分别为0.329%和27.58%,易错聚合酶链反应体系的错配率可以比普通PCR体系提高约10倍,DNA改组的突变率则更高,但是难以避免由于突变率太高造成的目的基因无法正确翻译这一情况。另外,应用邻接法(neighbor-joining, NJ)对随机选择的过氧化氢酶基因突变体序列和野生型序列做核酸和蛋白质序列的NJ进化树,进化关系与突变率分析基本一致。     

Significantly enhanced stability of glucose dehydrogenase by directed evolution

An NaCl-independent stability-enhanced mutant of glucose dehydrogenase (GlcDH) was obtained by using in vitro directed evolution. The family shuffling method was applied for in vitro directed evolution to construct a mutant library of GlcDH genes. Three GlcDH-coding genes from Bacillus licheniformis IFO 12200, Bacillus megaterium IFO 15308 and Bacillus subtilis IFO 13719 were each cloned by direct PCR amplification into the p Trc99A expression vector and expressed in the host, Escherichia coli. In addition to these three GlcDH genes, a gene encoding a previously obtained GlcDH mutant, F20 (Q252L), derived from B. megaterium IWG3, was also subjected to directed evolution by the family shuffling method. A highly thermostable mutant, GlcDH DN-46, was isolated in the presence or absence of NaCl after the second round of family shuffling and filter-based screening of the mutant libraries. This mutant had only one novel additional amino acid residue exchange (E170K) compared to F20, even though DN-46 was obtained by family shuffling of four different GlcDH genes. The effect of temperature and pH on the stability of the GlcDH mutants F20 and DN46 was investigated with purified enzymes in the presence or absence of NaCl. In the absence of NaCl, F20 showed very poor thermostability (half-life =1.3 min at 66 degrees C), while the half-life of isolated mutant DN-46 was 540 min at 66 degrees C, i.e., 415-fold more thermostable than mutant F20. The activity of the wild-type and F20 enzymes dropped critically when the pH value was changed to the alkaline range in the absence of NaCl, but no such decrease was apparent with the DN-46 enzyme in the absence of NaCl.     

DNA shuffling技术及其在基因工程疫苗中的应用

DNA shuffling技术是一项全新的体外人工进化模式,它通过基因在分子水平上的重组,再定向筛选具有预期性状的突变体,获得同时具有多个亲本基因的特征的突变基因。该文介绍DNA shuffling技术的基本原理,并列举了由该技术发展而来的新技术及其在基因工程疫苗领域的应用,展望了DNAshuffling技术的发展方向。     

Guided selection of an anti-gamma-seminoprotein human Fab for antibody directed enzyme prodrug therapy of prostate cancer

Background The HAMA response is a major challenge when murine antibodies are repeatedly administered for antibody directed enzyme prodrug therapy in vivo. In this study we have achieved humanization of the anti-γ-seminoprotein E₄B₇ murine mAb by guided selection. Methods Using optimal Ig Fab primers, human Fd and CL gene repertoires were amplified by RT-PCR from PBMCs of prostate cancer patients. The human Lc gene repertoire was first paired with the murine Fd gene of E₄B₇ mAb to construct a pComb3X hybrid Fab display library. This hybrid library was screened with purified γ-seminoprotein antigen. The human Fd gene repertoire was then paired with the selected human Lc to construct a fully human Fab library. After four more rounds of panning, completely human Fab antibodies specific for γ-seminoprotein were selected and further identified. Results First, using the E₄B₇ Fd gene as a template, light chain shuffling was achieved by panning the hybrid library. Then, using the selected Lc as a template, a human Fab antibody against γ-seminoprotein was produced through heavy chain Fd shuffling. Western blotting, ELISA, and flow cytometry results demonstrated that the resulting human Fab antibody resembled the parental E₄B₇ mAb in that they both recognized the same epitope with similar affinities. Fluorescent cell staining and immunohistochemistry analysis further confirmed that this newly constructed human anti-γ-seminoprotein Fab antibody indeed specifically bound prostate cancer cells and tissue. Conclusions Through guided-selection, we successfully produced a human anti-γ-seminoprotein Fab antibody. This work lays the foundation for optimal antibody-directed enzyme prodrug therapy of prostate cancer using a fully human Fab antibody. Zhang Qing and Zhang Si-He are co-first authors on the publication.     

DNA shuffling: Modifying the hand that nature dealt

Summary DNA shuffling is a technique being utilized for in vitro recombination of a single gene or pools of homologous genes. The genes are fragmented into randomly sized pieces, and polymerase chain reaction (PCR) reassembly of full-length genes from the fragments, via self-priming, yields recombination due to PCR template switching. After these PCR products are screened and the interesting products sequenced, improved clones are reshuffled to recombine useful mutations in additive or synergistic ways, in effect mimicking the process of natural sexual recombination. Proteins can be ‘bred’ with the appropriate individual properties and then their ‘progeny’ screened for the desired combination of traits. DNA shuffling is a powerful tool enabling rapid and directed evolution of new genes, operons and whole viral genomes.     

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.     

Directed evolution of operon of trehalose-6-phosphate synthase/phosphatase from Escherichia coli

Trehalose is a nonspecific protective agent for biomacromolecules. Trehalose-6-phosphate synthase (OtsA)/phosphatase (OtsB), which is encoded by the gene operon otsBA located at -42 of the Escherichia coli genome, is the main enzyme system that catalyzes the synthesis of trehalose in E. coli. We cloned the operon and modified it by directed evolution. Unlike in the previously reported work, we modified the whole operon and screened the positive mutant simultaneously. Thus we believe that the gene complex solves the negative effects between two enzymes if one of them diversifies its structure or functions and finds the form most suitable for trehalose synthesis. It thus mimics the natural process, in which the functional improvement of organisms is related to alterations in coordinated enzymes. The evolution procedure was carried out in a sequence of error-prone PCR, shuffling PCR, and then strict screening of the mutants. After screening of a library of more than 4000 colonies, about 15 positive colonies were analyzed, resulting in a higher concentration of trehalose than control. One of them, E. coli TS7, shows 12.3-fold higher trehalose synthesis ability than E. coli DH5alpha. In contrast, we introduced the cDNA sequence of the tps1 gene from Saccharomyces cerevisiae, which has 54% identity with the gene otsA, as one of the templates in shuffling PCR. By hybrid evolution and screening, we obtained 10 positive colonies with higher concentrations of trehalose than control. E. coli TS22 appears to have 5.3-fold higher trehalose synthesis ability than E. coli DH5alpha and 1.6-fold more than E. coli DEF3(pOTS11). This result demonstrated that coevolution and hybrid evolution, as powerful protocols in protein engineering, are effective in modifying enzyme. It indicates that repeating the process of genomic evolution in nature is feasible.     

从噬菌体抗体库中淘选血纤维蛋白特异的单链抗体

为构建小鼠噬菌体抗体库 ,以获得对人血纤维蛋白特异的抗体 ,由小鼠脾脏提取 m RNA,经反转录 PCR扩增出抗体重链、轻链可变区基因片段 ,将二者和一段编码十五肽 (Gly4 Ser) 3的 DNA接头借助重组 PCR组装成为单链抗体 (single- chain antibody,Sc Ab)基因 .将单链抗体基因插入噬菌体展示载体 p CANTAB- 5E,通过电击法转化大肠杆菌 TG1细胞 ,用辅助噬菌体 M1 3K0 7超感染 ,构建了库容量在 1 0 8以上的噬菌体单链抗体库 .利用亲和选择方法 (淘选 ) ,从噬菌体抗体库中选得血纤维蛋白特异的单链抗体 .模拟抗体成熟过程 ,用 DNA改组 (DNA shuffling)技术使抗体基因重新组合 ,构建新的改组抗体库 ,并从中选择到提高了亲和力的噬菌体单链抗体 .抗体基因在大肠杆菌中表达 ,表达蛋白经 Sephadex G- 75柱层析分离 ,得到初步纯化的单链抗体蛋白 .     

利用雨生红球藻表达系统高通量筛选活力提高的阿特拉津氯水解酶突变子

阿特拉津氯水解酶定向改造的关键是开发一种廉价的、表型改变明显的高通量筛选方法。利用高错误倾向PCR和DNA洗牌相结合的突变方法,对来源于假单胞菌ADP和节杆菌AD1的阿特拉津氯水解酶基因进行随机突变,以雨生红球藻为受体、以阿特拉津为选择压力对突变文库进行高通量筛选。筛选到的12个突变子序列分析显示,突变均为点替换,位点分散在全基因上,是在高错误倾向PCR及DNA洗牌过程中逐渐累积形成的。酶活力分析显示,突变子的酶活力均高于野生株,在添加1.0 mg/L阿特拉津培养液中的活力是野生株的1.9~3.6倍,在添     

Reducing mutational bias in random protein libraries

The success of protein optimization through directed molecular evolution depends to a large extent on the size and quality of the displayed library. Current low-fidelity DNA polymerases that are commonly used during random mutagenesis and recombination in vitro display strong mutational preferences, favoring the substitution of certain nucleotides over others. The result is a biased and reduced functional diversity in the library under selection. In an effort to reduce mutational bias, we combined two different low-fidelity DNA polymerases, Taq and Mutazyme, which have opposite mutational spectra. As a first step, random mutants of the Bacillus thuringiensis cry9Ca1 gene were generated by separate error-prone polymerase chain reactions (PCRs) with each of the two polymerases. Subsequent shuffling by staggered extension process (StEP) of the PCR products resulted in intermediate numbers of AT and GC substitutions, compared to the Taq or Mutazyme error-prone PCR libraries. This strategy should allow generating unbiased libraries or libraries with a specific degree of mutational bias by applying optimal mutagenesis frequencies during error-prone PCR and controlling the concentration of template in the shuffling reaction while taking into account the GC content of the target gene.     

Directed evolution improves the fibrinolytic activity of nattokinase from Bacillus natto

Nattokinase (subtilisin NAT, NK) is a relatively effective microbial fibrinolytic enzyme that has been identified and characterized from Bacillus natto. In the current report, DNA family shuffling was used to improve the fibrinolytic activity of nattokinase. Three homologous genes from B. natto AS 1.107, Bacillus amyloliquefaciens CICC 20164 and Bacillus licheniformis CICC 10092 were shuffled to generate a mutant library. A plate-based method was used to screen the mutant libraries for improved activity. After three rounds of DNA shuffling, one desirable mutant with 16 amino acid substitutions was obtained. The mutant enzyme was purified and characterized. The kinetic measurements showed that the catalytic efficiency of the mutant NK was approximately 2.3 times higher than that of the wild-type nattokinase. In addition, the molecular modeling analysis suggested that the mutations affect the enzymatic function by changing the surface conformation of the substrate-binding pocket. The current study shows that the evolution of nattokinase with improved fibrinolytic activity by DNA family shuffling is feasible and provides useful references to facilitate the application of nattokinase in thrombolytic therapy.     

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.     

Improved catalytic efficiency of Endo-β-1,4-glucanase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> BME-15 by directed evolution

Bacillus subtilis endo-β-1,4-glucanase (Cel5A) hydrolyzes cellulose by cleavage of the internal bonds in the glucose chains, producing new ends randomly. Using directed evolution techniques of error-prone polymerase chain reaction (PCR) and DNA shuffling, several Cel5A variants with improved catalytic activity had been screened from the mutant library, which contained 71,000 colonies. Compared with the wild-type enzyme, the variants (M44-11, S75 and S78) showed 2.03 to 2.68-fold increased activities toward sodium carboxymethyl cellulose (CMC), while the M44-11 also exhibited a wider pH tolerance and higher thermostability. Structural models of M44-11, S75, S78, and WT proteins revealed that most of the substitutions were not located in the strictly conserved regions, except the mutation V255A of S75, which was closed to the nucleophile Glu257 in the catalytic center of the enzyme. Moreover, V74A and D272G of M44-11, which were not located in the substrate binding sites and the catalytic center, might result in improved stability and catalytic activity. These results provided useful references for directed evolution of the enzymes that belonged to the glycoside hydrolase family 5 (GH5).     

Compensation of the enantioselectivity-activity trade-off in the directed evolution of an esterase from Rhodobacter sphaeroides by site-directed saturation mutagenesis

Despite directed evolution being a practical and efficient method of improving the properties of enzymes, a trade-off between the targeted property and other essential properties often exists which hinders the efficiency of directed evolution. In our previous work, mutant CVH of an esterase from Rhodobacter sphaeroides with high enantioselectivity was obtained by directed evolution, unfortunately its activity cannot catch another mutant YH. To compensate the trade-off of mutant CVH, site-directed saturation mutagenesis was conducted on four residues, three (Asn62, Met121, and Leu145) were hot spots determined from directed evolution, and one (Tyr27) was introduced to make up the large distance between a mutation (Asn62) and the substrate. A new mutant (HMVY) with high enantioselectivity and comparable activity to YH was obtained. According to the kinetic analysis and molecular dynamics simulations, it was understood that the high enantioselectivity and poor activity of mutant CVH was caused by different decrement of efficiency constants to two isomers, (R)-, (S)-methyl mandelate, and the high enantioselectivity and activity of mutant HWVY was caused by improved activity towards the preferred substrate ((S)-methyl mandelate), which provided the interpretation of the trade-off compensation. This work could provide a way to compensate the trade-off of enantioselectivity and activity in the process of enzyme evolution.     

Development of a selection system for the detection of L-ribose isomerase expressing mutants of <Emphasis Type="Italic">Escherichia coli</Emphasis>

L-Arabinose isomerase (E.C. 5.3.1.14) catalyzes the reversible isomerization between L-arabinose and L-ribulose and is highly selective towards L-arabinose. By using a directed evolution approach, enzyme variants with altered substrate specificity were created and screened in this research. More specifically, the screening was directed towards the identification of isomerase mutants with L-ribose isomerizing activity. Random mutagenesis was performed on the Escherichia coli L-arabinose isomerase gene (araA) by error-prone polymerase chain reaction to construct a mutant library. To enable screening of this library, a selection host was first constructed in which the mutant genes were transformed. In this selection host, the genes encoding for L-ribulokinase and L-ribulose-5-phosphate-4-epimerase were brought to constitutive expression and the gene encoding for the native L-arabinose isomerase was knocked out. L-Ribulokinase and L-ribulose-5-phosphate-4-epimerase are necessary to ensure the channeling of the formed product, L-ribulose, to the pentose phosphate pathway. Hence, the mutant clones could be screened on a minimal medium with L-ribose as the sole carbon source. Through the screening, two first-generation mutants were isolated, which expressed a small amount of L-ribose isomerase activity.     

通过DNA改组技术获得高活性β-葡萄糖苷酸酶

β 葡萄糖苷酸酶是在植物转基因中广泛应用的报告基因 .以质粒pBI12 1中的GUS基因为基础 ,利用DNA改组方法 ,经DNaseⅠ降解 ,PrimerlessPCR ,PrimerPCR对GUS基因进行了突变和改组 ,然后将改组的GUS基因连接到原核表达载体pG2 5 1中 ,构建了库容为 10 8的突变体库 .经过活性的筛选 ,得到活性提高的克隆 ,再以此为基础 ,经过新的改组、筛选得到活性大幅度提高的克隆GUS2 4 .基因测序显示 ,GUS2 4与GUS基因之间的同源性为 99 7% ,共有 6个核苷酸位点发生了改变 ,分别是 :379位的A突变为G ,396位的T突变为C ,711位的G突变为A ,95 8位T突变为C ,990位的T突变为C ,1649位的A突变为G .核苷酸序列推导的氨基酸序列显示 ,3个氨基酸发生了突变 ,12 7位的Ser突变为Gly ,32 0位的Trp突变为Arg ,5 5 0位的Asn突变为Ser.X gluc染色检测和荧光测活结果显示GUS2 4基因表达的 β 葡萄糖苷酸酶基较GUS基因表达产物活性提高 3倍