一种高效的多点组合突变克隆构建方法（英文）

高质量的突变方法和高效的筛选方法相结合可以提高酶定向进化的效率。文中开发了一种高效的多点组合突变(Multi-points combinatorial mutagenesis,MCM)的克隆方法。MCM方法通过引入DNA组装、融合PCR和杂交技术,实现高效多点组合突变。应用优化后的方法定向进化改造苯甲酰甲酸脱羧酶(Benzoylformate decarboxylase,BFD)来测试MCM方法的效率。通过电转至大肠杆菌感受态Escherichia coli TreliefTM 5α所获得的单菌落数量(Colony-formingunits,CFUs)超过106 CFUs/μgDNA。经验证90/100单菌落精确组装;5个位点L109、L110、H281、Q282和A460同时组合突变的效率达到88%。最后,筛选到一种kcat/Km提高10倍的突变酶(L109Y、L110D、H281G、Q282V和A460M)。因此,应用该方法可以有效地创建突变体库,促进酶的定向进化技术的快速发展。     

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.     

基于多重计算设计策略提高枯草芽孢杆菌脂肪酶的热稳定性

提高酶的热稳定性是生物催化领域的热点和难点,计算机辅助的理性设计相比于传统的定向进化更加高效,在酶工程领域中的应用越来越广泛和深入。文中以枯草芽孢杆菌脂肪酶A为模式蛋白,首先,利用Rosetta-VIP计算设计对酶的结构空腔进行分析,选择了16个有利于结构空腔填充(ΔΔE<0)的单点突变,并以突变位点的溶剂可及表面积和进化保守性为二次筛选依据,测定了其热稳定性与酶活性。有6个单点突变体(F17A、V74I、L114P、I135V、M137A、I157L)的热稳定性得到了提高,其中Tm值最大提高3.18℃。结果表明,单点突变体满足ΔΔE越低、蛋白溶剂可及表面积减少且符合序列保守性,则得到保留原有酶活力的正向突变的可能性越大。此外,将热稳定性提高的6个单点突变进行迭代组合突变,两点组合突变体的Tm最大提高4.04℃,三点组合突变体的Tm最大提高5.13℃,四点组合突变体的Tm提高了7.30℃,六点组合突变体的Tm提高了7.43℃。因此,基于酶的分子结构的空腔分析、溶剂可及表面积及氨基酸序列保守性计算的多重虚拟筛选方法,可有效提高酶的热稳定性。     

枯草芽孢杆菌β-糖苷酶的克隆表达、体外定向进化及结构模拟

摘要:【目的】从枯草芽孢杆菌基因组DNA中扩增出bglC基因并在大肠杆菌中表达,分析表达产物的酶学性质并进行结构模拟,为进一步研究其生理功能及结构解析奠定基础。【方法】将bglC基因克隆到大肠杆菌(Escherichia coli)BL21(DE3)中表达,通过定向进化获取水解效率提高的突变株,经Ni-NTA镍离子层析柱纯化后,测定野生枯草芽孢杆菌β-糖苷酶与突变酶的性质。利用CD光谱,非变性聚丙烯酰胺凝胶电泳及三维结构建模,分析野生酶与突变酶的高级结构。【结果】野生酶的比活力为9.7 U/mg,最适催化温度为60℃,最适pH值是7.0。经过突变和筛选,我们得到一个突变体BS-GLY_M1(A242T/T385A/S425L),其比活力达到17.1 U/mg,最适温度为55 ℃,最适pH值是7.0,在55 ℃下的半衰期为3.5 h,比野生酶增加2 h。突变酶对4-硝基苯基-β-半乳糖苷、乳糖和熊果苷的催化效率(Km/Kcat)有所提高。酶在天然条件下以二聚体、四聚体状态存在,推测它以二聚体为基本功能单位。结构模拟结果表明突变后酶的三维结构有轻微地变化,这可能是酶热稳定性和催化效率提高的原因。【结论】枯草芽孢杆菌β-糖苷酶可以在大肠杆菌中高效表达并可以通过定向进化提高其水解效率。     

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...     

基于单细胞超高通量筛选的α-1,2-岩藻糖基转移酶定向进化

2’-岩藻糖基乳糖在婴幼儿配方奶粉、保健品和医药等产品开发方面极具应用价值。幽门螺杆菌(Helicobacter pylori NCTC11639)来源的α-1,2-岩藻糖基转移酶(FutC)是目前合成2’-岩藻糖基乳糖的重要生物催化剂,但是天然酶存在异源表达量低、催化活性差等缺陷。针对α-1,2-岩藻糖基转移酶定向进化过程中缺少高通量筛选方法的问题,发展了基于荧光激活细胞分选(fluorescence-activated cell sorting,FACS)的FutC超高通量筛选方法。对FutC进行了定向进化实验,通过对FutC的随机突变库进行了3轮筛选,从库容量为5.4×10⁵突变文库中成功筛选出活力提高2.6倍、2.7倍、3倍的3种突变体(K282E,K102E,R105C),证明了此筛选方法的有效性。本研究为α-1,2-岩藻糖基转移酶的分子活性改造奠定了良好基础。     

基于单细胞超高通量筛选的α-1,2-岩藻糖基转移酶定向进化

2’-岩藻糖基乳糖在婴幼儿配方奶粉、保健品和医药等产品开发方面极具应用价值。幽门螺杆菌(Helicobacter pylori NCTC11639)来源的α-1,2-岩藻糖基转移酶(FutC)是目前合成2’-岩藻糖基乳糖的重要生物催化剂,但是天然酶存在异源表达量低、催化活性差等缺陷。针对α-1,2-岩藻糖基转移酶定向进化过程中缺少高通量筛选方法的问题,发展了基于荧光激活细胞分选(fluorescence-activated cell sorting,FACS)的FutC超高通量筛选方法。对FutC进行了定向进化实验,通过对FutC的随机突变库进行了3轮筛选,从库容量为5.4×10⁵突变文库中成功筛选出活力提高2.6倍、2.7倍、3倍的3种突变体(K282E,K102E,R105C),证明了此筛选方法的有效性。本研究为α-1,2-岩藻糖基转移酶的分子活性改造奠定了良好基础。     

定向进化技术的最新进展

筛选是制约酶定向进化改造的瓶颈。为解决这一难题,近年来一系列基于组合活性中心饱和突变(Combinatorial active-site saturation test,CAST)及迭代饱和突变(Iterative saturation mutagenesis,ISM)的半理性设计新方法被开发出来,包括单密码子饱和突变(Single code saturation mutagenesis,SCSM)、双密码子饱和突变(Double code saturation mutagenesis,DCSM)和三密码子饱和突变(Triple code saturation mutagenesis,TCSM)。通过构建"小而精"的高质量突变体文库,对特定靶点进行组合突变,并成功应用于多种生物催化剂的立体/区域选择性及催化活力等多参数的改造。文中综述了近年来定向进化技术的最新进展及其在生物催化剂定向改造中的应用。     

基于分子结构与生物信息学等多维度特征的定向进化改造甲基对硫磷水解酶

甲基对硫磷水解酶作为环境友好绿色催化剂,可降解甲基对硫磷等在土壤中残留的有机磷农药,这使得如何提高甲基对硫磷水解酶的水解效率成为酶工程中备受关注的问题。基于分子结构与生物信息学等多维度特征,确定了催化活性中心周围的9个关键位点。构建了一个含1 500个突变体的筛选文库,进行定向进化。经过两轮筛选,最终确定2株催化效率提高的突变体,L258V和L273M。其催化效率常数kcat/Km值分别为134.1(mmol/L)-1·s-1和166.5(mmol/L)-1·s-1,与野生酶相比较分别提高了27.3%和58.1%。这种通过半理性设计构建定向进化文库的方法,不仅有效的提高了甲基对硫磷水解酶的筛选效率,更为酶的改造提供了新的思路。     

生理pH条件下精氨酸脱亚胺酶的分子改造研究

精氨酸脱亚胺酶（arginine deiminase,EC 3.5.3.6,ADI）因其可作为精氨酸营养缺陷型肿瘤细胞的靶向治疗药物而受到广泛关注. 目前,支原体来源的重组ADI处于肝癌和黑素瘤的三期临床研究阶段. 作为药用酶,当前报道的ADI在体内生理条件下普遍存在酶活低、半衰期短、底物亲和性弱等局限性.本研究结合随机突变及基于理性设计的定点突变两种方法,对研究室前期自主筛选得到的变形假单胞菌Pseudomonas plecoglossicida来源的ADI经一轮定向进化后所获优势突变株M314(A128T/H404R/I410L)进行分子改造.通过对随机突变法获得的1480个突变株进行96孔板高通量筛选,得到优良突变株M173(A128T/H404R/I410L/K272R);同时,基于同源序列比对及ADI蛋白三维结构同源建模,采用PyMOL软件理性预测和分析其活性中心及附近保守区域氨基酸位点对蛋白功能的影响,选择了6个位点D78E、L223I、P230I、S245D、A275N、R400M分别在M314的基础上进行定点突变,最终获得优势突变株M04(A128T/H404R/I410L/S245D). 通过对突变株的酶学性质以及动力学参数分析发现：生理pH值下,突变株M173的酶比活（12.32 U/mg）在M314（9.02 U/mg）的基础上提升3659%,Kcat/Km提高5236%;而突变株M04的最适pH由6.5升高至7.0,更接近体内生理pH,其比酶活（14.66 U/mg）较M314提升62.53 %,Kcat/Km提高了37.12%. 综上结果,本研究结合两种分子改造方法成功地对该ADI在生理pH条件下的酶活和酶学性质进行了改良,并为蛋白质的分子改造策略提供了理论基础和实验依据.     

Teaching TetR to recognize a new inducer

Tet Repressor (TetR) recognizes the inducer tetracycline (tc) with high affinity. The tc analog 4-de(dimethylamino)-6-deoxy-6-demethyl-tetracycline (cmt3) is not an inducer for TetR. Induction specificity for cmt3 was generated by employing a directed evolution approach to screen appropriate TetR mutants in four successive steps. The specificity of the best TetR mutant is more than 20,000-fold increased for cmt3 over tc as judged by the ratio of their respective binding constants. Two rounds of directed evolution via DNA shuffling revealed His64 as a key residue for inducer specificity. The best TetR mutant with cmt3 specificity contains the H64K exchange, leading to a 300-fold decreased tc and a 20-fold increased cmt3 affinity. Another round of directed evolution made use of randomized oligonucleotides to mutate selected residues close to the tc-binding pocket of TetR and yielded TetR S135L with a 250-fold increased cmt3 affinity. The double mutant TetR H64K S135L was constructed and again subjected to directed evolution using randomized oligonucleotides to alter residues in the "secondary shell" of the tc-binding pocket. The resulting best mutants TetR H64K E114Q S135L, TetR A61V H64K Q109E Q116E S135L and TetR H64K T112K S135L are fully inducible by cmt3 and not by tc. Thus, their inducer specificity has been redesigned. The molecular mechanism of changed inducer recognition is discussed, based on binding constants with several tc analogs and in light of the TetR crystal structure.     

Directed evolution of N-carbamyl-D-amino acid amidohydrolase for simultaneous improvement of oxidative and thermal stability

Directed evolution of N-carbamyl-D-amino acid amidohydrolase from Agrobacterium tumefaciens NRRL B11291 was attempted in order to simultaneously improve oxidative and thermal stability. A mutant library was generated by DNA shuffling, and positive clones with improved oxidative and thermal stability were screened on the basis of the activity staining method on a solid agar plate containing pH indicator (phenol red) and substrate (N-carbamyl-D-p-hydroxyphenylglycine). Two rounds of directed evolution resulted in the best mutant 2S3 with a significantly improved stability. Oxidative stability of the evolved enzyme 2S3 was about 18-fold higher than that of the wild type, and it also showed an 8-fold increased thermostability. The K(m) value of 2S3 was comparable to that of wild-type enzyme, but k(cat) was slightly decreased. DNA sequence analysis revealed that six amino acid residues (Q23L, V40A, H58Y, G75S, M184L, and T262A) were substituted in 2S3. From the mutational analysis, four mutations (Q23L, H58Y, M184L, and T262A) were found to lead to an improvement of both oxidative and thermal stability. Of them, T262A had the most significant effect, and V40A and G75S only increased the oxidative stability.     

Improvement of oxidative and thermostability of N-carbamyl-d-amino Acid amidohydrolase by directed evolution

N-Carbamyl-D-amino acid amidohydrolase (N-carbamoylase), which is currently employed in the industrial production of unnatural D-amino acid in conjunction with D-hydantoinase, has low oxidative and thermostability. We attempted the simultaneous improvement of the oxidative and thermostability of N-carbamoylase from Agrobacterium tumefaciens NRRL B11291 by directed evolution using DNA shuffling. In a second generation of evolution, the best mutant 2S3 with improved oxidative and thermostability was selected, purified and characterized. The temperature at which 50% of the initial activity remains after incubation for 30 min was 73 degrees C for 2S3, whereas it was 61 degrees C for wild-type enzyme. Treatment of wild-type enzyme with 0.2 mM hydrogen peroxide for 30 min at 25 degrees C resulted in a complete loss of activity, but 2S3 retained about 79% of the initial activity under the same conditions. The K(m) value of 2S3 was estimated to be similar to that of wild-type enzyme; however k(cat) was decreased, leading to a slightly reduced value of k(cat)/K(m), compared with wild-type enzyme. DNA sequence analysis revealed that six amino acid residues were changed in 2S3 and substitutions included Q23L, V40A, H58Y, G75S, M184L and T262A. The stabilizing effects of each amino acid residue were investigated by incorporating mutations individually into wild-type enzyme. Q23L, H58Y, M184L and T262A were found to enhance both oxidative and thermostability of the enzyme and of them, T262A showed the most significant effect. V40A and G75S gave rise to an increase only in oxidative stability. The positions of the mutated amino acid residues were identified in the structure of N-carbamoylase from Agrobacterium sp. KNK 712 and structural analysis of the stabilizing effects of each amino acid substitution was also carried out.     

Distinct functional consequences of MUTYH variants associated with colorectal cancer: Damaged DNA affinity,glycosylase activity and interaction with PCNA and Hus1

MUTYH is a base excision repair (BER) enzyme that prevents mutations in DNA associated with 8-oxoguanine (OG) by catalyzing the removal of adenine from inappropriately formed OG:A base-pairs. Germline mutations in the MUTYH gene are linked to colorectal polyposis and a high risk of colorectal cancer, a syndrome referred to as MUTYH-associated polyposis (MAP). There are over 300 different MUTYH mutations associated with MAP and a large fraction of these gene changes code for missense MUTYH variants. Herein, the adenine glycosylase activity, mismatch recognition properties, and interaction with relevant protein partners of human MUTYH and five MAP variants (R295C, P281L, Q324H, P502L, and R520Q) were examined. P281L MUTYH was found to be severely compromised both in DNA binding and base excision activity, consistent with the location of this variation in the iron-sulfur cluster (FCL) DNA binding motif of MUTYH. Both R295C and R520Q MUTYH were found to have low fractions of active enzyme, compromised affinity for damaged DNA, and reduced rates for adenine excision. In contrast, both Q324H and P502L MUTYH function relatively similarly to WT MUTYH in both binding and glycosylase assays. However, P502L and R520Q exhibited reduced affinity for PCNA (proliferation cell nuclear antigen), consistent with their location in the PCNA-binding motif of MUTYH. Whereas, only Q324H, and not R295C, was found to have reduced affinity for Hus1 of the Rad9–Hus1–Rad1 complex, despite both being localized to the same region implicated for interaction with Hus1. These results underscore the diversity of functional consequences due to MUTYH variants that may impact the progression of MAP.     

High-Activity Barley \bold\ralpha-Amylase by Directed Evolution

Barley -amylase isozyme 2 was cloned into and constitutively secreted by Saccharomyces cervisiae. The gene coding for the wild-type enzyme was subjected to directed evolution. Libraries of mutants were screened by halo formation on starch agar plates, followed by high-throughput liquid assay using dye-labeled starch as the substrate. The concentration of recombinant enzyme in the culture supernatant was determined by immunodetection, and used for the calculation of specific activity. After three rounds of directed evolution, one mutant (Mu322) showed 1000 times the total activity and 20 times the specific activity of the wild-type enzyme produced by the same yeast expression system. Comparison of the amino acid sequence of this mutant with the wild type revealed five substitutions: Q44H, R303K and F325Y in domain A, and T94A and R128Q in domain B. Two of these mutations, Q44H and R303K, result in amino acids highly conserved in cereal -amylases. R303K and F325Y are located in the raw starch-binding fragment of the enzyme molecule.     

Characterization of mutations in oligomerization domain of Lac repressor protein

A series of mutant lac repressor proteins at positions 281 or 282 was isolated for detailed characterization. Although Cys281 modification by sulfhydryl reagents abrogates pH effects on inducer binding and diminishes operator binding (Daly, T. J., Olson, J. S., and Matthews, K. S. (1986) Biochemistry 25, 5468-5474), substitution at this site by alanine, serine, phenylalanine, isoleucine, or methionine did not abolish completely the pH shift nor affect operator affinity. Thus, ionization of the sulfhydryl residue does not account fully for the alterations in inducer affinity and cooperativity of binding observed with elevated pH. Substitution for Cys281 did, however, alter the kinetic parameters for inducer association with the protein. The polarity of the side chain at 281 influenced the rates of sugar binding, presumably by altering the rate of opening/closing of the binding site. Furthermore, the presence of the branched side chain of isoleucine at position 281 disrupted oligomerization of the repressor. In contrast to the tolerance for substitution at 281, the only amino acid side chain exchanges for Tyr282 which yielded tetrameric protein with near normal operator binding characteristics were phenylalanine and leucine; this result is consistent with studies of suppressed nonsense mutations at position 282 which indicated repression occurred only for the corresponding substitutions (Kleina, L. G., and Miller, J. H. (1990) J. Mol. Biol. 221, 295-318). Despite the tetrameric character of the Y282F mutant protein, the pH dependence and cooperativity of inducer binding for this mutant protein were altered. All amino acid substitutions other than phenylalanine and leucine at this position resulted in either monomeric protein or no detectable repressor in the cell. Thus, the hydrophobic character of the side chain at position 282 is essential for tetramer formation, and the phenyl ring alone alters inducer binding parameters. The monomeric mutant proteins with substitutions for Tyr282 exhibited lower stability than their tetrameric counterparts, and the absence of dimer formation suggests alterations at this site affect both dimer and tetramer interfaces. Based on previous genetic studies and our detailed mutant characterization, the region encompassing 281 and 282, indicated by secondary structure prediction to be a turn or coil, is essential for oligomer formation and additionally exerts a strong influence on the dynamic properties of the protein, presumably mediated by interactions at the subunit interface which regulate the rate of opening and closing of the inducer binding cleft.     

Directed evolution of a Baeyer–Villiger monooxygenase to enhance enantioselectivity

The Baeyer-Villiger monooxygenase (BVMO) BmoF1 from Pseudomonas fluorescens DSM 50106 was shown before to enantioselectively oxidize different 4-hydroxy-2-ketones to the corresponding hydroxyalkyl acetates, being the first example of a BVMO-catalyzed kinetic resolution of aliphatic acyclic ketones. However, the wild-type enzyme exhibited only moderate E values (E approximately 55). Thus, the enantioselectivity was enhanced by means of directed evolution and optimization of reaction conditions since it was found that higher E values (E approximately 70 for wild-type BmoF1) could already be obtained when performing biotransformations in shake flasks rather than small tubes. In a first step, random mutations were introduced by error-prone polymerase chain reaction, and BmoF1 mutants (>3,500 clones) were screened for improved activity and enantioselectivity using a microtiter-plate-based screening method. Mutations S136L and L252Q were found to increase conversion compared to wild type, while several mutations (H51L, F225Y, S305C, and E308V) were identified enhancing the enantioselectivity to a varying extent (E approximately 75-90). In a second step, beneficial mutations were recombined by consecutive cycles of QuikChange site-directed mutagenesis resulting in a double mutant (H51L/S136L) showing both improved conversion and enantioselectivity (E approximately 86).     

The zinc finger domain of the archaeal minichromosome maintenance protein is required for helicase activity

The minichromosome maintenance (MCM) proteins, a family of six conserved polypeptides found in all eukaryotes, are essential for DNA replication. The archaeon Methanobacterium thermoautotrophicum Delta H contains a single homologue of MCM with biochemical properties similar to those of the eukaryotic enzyme. The amino acid sequence of the archaeal protein contains a putative zinc-binding domain of the CX(2)CX(n)CX(2)C (C(4)) type. In this study, the roles of the zinc finger domain in MCM function were examined using recombinant wild-type and mutant proteins expressed and purified from Escherichia coli. The protein with a mutation in the zinc motif forms a dodecameric complex similar to the wild-type enzyme. The mutant enzyme, however, is impaired in DNA-dependent ATPase activity and single-stranded DNA binding, and it does not possess helicase activity. These results illustrate the importance of the zinc-binding domain for archaeal MCM function and suggest a role for zinc binding in the eukaryotic MCM complex as well, since four out of the six eukaryotic MCM proteins contain a similar zinc-binding motif.     

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.