利用SpyTag/SpyCatcher构建胞内自组装多酶复合体实现高效生物合成

SpyTagr和SpyCatche可通过自发反应形成共价键,产生稳定的分子自组装体。酶分子自组装体因具有高效有序的催化特性在合成生物学和纳米技术领域具有重要的应用价值。为探索SpyTag/SpyCatcher在大肠杆菌胞内多酶复合体系形成有序自组装分子能力,将SpyTagr和SpyCatche分别与P450BM3m单加氧酶和葡萄糖脱氢酶GDH进行融合表达,以期产生具有辅酶再生循环系统、高效生物合成靛蓝分子的SpyTag/SpyCatcher双酶自组装复合体。首先,通过电泳及质谱对重组工程菌表达蛋白进行分析,证实SpyCatcher-P450BM3m与SpyTag-GDH在胞内成功形成了自组装多酶复合体;然后,系统分析不同培养条件下组装体合成靛蓝的能力。结果发现,经0.5mmol/L IPTG诱导后,菌体在16℃继续培养18h后,工程菌对吲哚(2mmol/L)与葡萄糖(4mmol/L)的全细胞催化能力最强,靛蓝产量最高达258mg/L,是未组装多酶系统的1.9倍,比P450BM3m单酶表达系统高约2.4倍;反应70min后达到反应平衡,转化率为52%。成功实现了SpyTag/SpyCatcher介导的多酶体系在大肠杆菌细胞中的自组装和高效转化体系,为胞内多酶复合物组装体的设计提供了新思路。     

降解纤维素的“超分子机器”研究进展

综述了目前关于纤维小体组装模式、纤维小体结构多样性及人工设计纤维小体等方面的研究进展.纤维小体是某些厌氧菌产生的由多个亚基共同组装而成的大分子机器,是致力于组织、协调多种酶组分协同高效催化降解木质纤维素的胞外蛋白质复合体.纤维小体是厌氧微生物水解纤维素的主体,具有非常高效的打破结晶纤维素的结晶结构和降解纤维素链的作用.纤维小体对木质纤维素降解的高效性来自于其自发组装而成的复杂的高级结构,其结构的复杂性因不同的厌氧微生物而有所不同.     

微丝骨架蛋白分子重组与细胞转化

本文介绍正常细胞微丝骨架组装特点和与细胞贴壁及运动的关系;细胞转化后,应力纤维和粘着斑破坏,微丝骨架蛋白分子重组装,肌动蛋白小体形成等变化与转化细胞恶性行为的相关性。本文并提出今后有关本领域的研究方向。     

解纤维梭菌纤维小体黏附域与锚定域相互作用的差异性

黏附域(cohesin)与锚定域(dockerin)的相互作用是纤维素酶复合体-纤维小体(cellulosome)组装的基础,该作用是自然界已知最强的相互作用力之一。为解析纤维小体的装配机制,本研究以解纤维梭菌(Ruminiclostridium cellulolyticum)纤维小体为研究对象,通过Pull-down和等温滴定量热(ITC)的方法,分析并比较不同簇的3个黏附域与7个锚定域之间的相互作用。结果表明,不同簇黏附域与锚定域的相互作用具有显著差异。其中,Coh1与Doc-0729的相互作用最强,K_a为10⁸ M^-1,Coh7与Doc-0729、0931作用力强,K_a为10⁷ M^-1,而Coh8与Doc-0931、1656、0752作用强,K_a也达到10⁷ M^-1。总之,Doc-0729、0931、1656与3个Coh的结合力均较高。本研究揭示了纤维小体黏附域与锚定域的组装具有偏爱性...     

纤维小体在燃料乙醇中的应用

纤维小体在木质纤维素的降解中起着重要作用。它不仅含有降解纤维素所需的各种纤维素酶系,而且组装成具有高效催化活性的多酶复合体形式。介绍了纤维小体基本结构与功能,重点概述了其在生物燃料乙醇中的应用并对纤维小体的研究提出了展望。     

蛋白质自组装机器在人工多酶复合体系中的研究与应用进展

随着合成生物学的研究与发展,人们利用微生物细胞或无细胞体系对代谢途径中的多酶体系进行编程和重组,成功合成了大量的功能化合物。但由于多酶体系分散度高,造成体系代谢流速和流量不平衡,代谢效率和产量降低。生物体内存在多种天然的多酶自组装复合体,如纤维素小体机器、细胞信号转导中的激酶级联通路等。研究表明,这些体系中存在的底物通道效应和协同作用机制是多酶复合体具有高催化效率的原因。模拟和借鉴天然多酶体系,并结合生物体中蛋白质与DNA、RNA等相互作用设计和构建人工自组装多酶体系,是提高代谢效率的重要途径。现对蛋白质自组装机器在人工多酶体系中的研究进展进行综述。     

利用分子环化技术提高瘤胃微生物木聚糖酶热稳定性

在高温下保持催化活性是工业酶的重要性质。近年来,采用基因工程、蛋白质工程技术提高野生酶进行催化活性或耐热等性质取得了重要进展。文中利用新近建立起来的异肽键介导的SpyTag/SpyCatcher系统对瘤胃微生物来源的木聚糖酶XYN11-6进行分子环化,获得稳定的环化酶C-XYN11-6。在60℃、70℃和80℃下处理10 min,C-XYN11-6的残余活性为81.53%、73.98%和64.41%,分别是相同条件下线性蛋白L-XYN11-6残余活性的1.48、2.92、3.98倍。经60–90℃热处理10 min后,C-XYN11-6仍保持可溶状态,而L-XYN11-6几乎完全聚沉。内源荧光和8-苯胺-1-萘磺酸(8-anilino-1-naphthalenesulfonic acid,ANS)结合荧光光谱分析显示,较之L-XYN11-6,热处理环境中C-XYN11-6更能够维持其构象稳定。值得注意的是,分子环化提高了C-XYN11-6对0.1–50 mmol/L Ca~(2+)或0.1 mmol/L Cu~(2+)的耐受能力。综上所述,文中利用SpyTag/SpyCatcher系统获得热稳定性和离子稳定性提高的环化酶,为工业酶的分子改良及扩大其在工业领域的应用建立了良好基础。     

基于双工程菌的“锁扣”生物活材料构建及其体内肠道滞留应用

生物活材料的研究主要集中在利用单一细菌生产生物膜、水塑料等体外应用。由于菌株尺寸较小，当其应用于体内时，容易发生逃逸，导致滞留效果较差。为解决这一难题，本研究借助大肠杆菌(Escherichia coli)表面展示系统(Neae)，在两个菌株表面分别展示SpyTag和SpyCatcher，构建一种双菌“锁扣”型生物活材料生产系统。两菌株之间通过SpyTag和SpyCatcher的结合，发生原位交联，从而长时间滞留在肠道部位。体外实验表明两菌株混合几分钟后，会发生明显的沉降。此外，利用共聚焦成像和微流控平台进一步证明了该系统在流动状态下的粘附效果。最后，为了验证该系统在体内应用的可行性，小鼠连续3d口服A菌(p15A-Neae-SpyTag/sfGFP)和B菌(p15A-Neae-SpyCatcher/mCherry)，收集肠道组织进行冷冻切片染色。结果表明，相较于未结合菌株，该双菌系统能更多滞留在小鼠肠道，为生物活材料进一步的体内应用奠定了基础。     

多酶共固定化的研究进展

固定化酶技术是现代生物催化的核心技术。过去几十年里,固定化酶技术的研究主要集中在单酶固定化。近年来,多酶共固定化由于具有可增加反应的局部浓度、提高反应收率等优点而得到研究者的广泛关注。本文根据国内外研究现状并结合本实验研究从多酶非特异性共价共固定化、非特异性非共价共固定化、非共价包埋固定化以及位点特异性固定化四个方面阐述多酶固定化方法的研究进展,并分析和展望了其在工业上的应用前景。     

基于肌红蛋白的氧激活蛋白的理性设计

金属酶催化了生命活动及工业催化中的很多重要反应。天然金属酶的研究往往受限于蛋白质本身结构复杂、表达纯化困难等问题。通过理性设计,可以在分子量小、结构简单、易于表达的骨架蛋白中模拟天然金属酶的结构、光谱和功能,构建人工金属酶。人工酶可以为天然酶的机制研究提供新的平台。本文中,笔者探讨以肌红蛋白为骨架蛋白,通过结构特征的模拟、非天然氨基酸的引入等手段,模拟氧激活蛋白,尤其是氧化酶。综述以对理性设计得到的人工氧化酶的反应中间体进行研究的进展,发现金属酶活性中心的酪氨酸作为催化反应的关键残基,对于氧化反应的调控非常重要;而在小分子量骨架蛋白中模拟天然金属酶是一种人工金属酶分子设计的方法,可以用于研究血红素酶外的其他金属酶。     

Programmable assembly of 2D crystalline protein arrays into covalently stacked 3D bionanomaterials

Rational embellishment of self-assembling two-dimensional (2D) proteins make it possible to build 3D nanomaterials with novel catalytic, optoelectronic and mechanical properties. However, introducing multiple sites of embellishment into 2D protein arrays without affecting the self-assembly is challenging, limiting the ability to program in additional functionality and new 3D configurations. Here we introduce two orthogonal covalent linkages at multiple sites in a 2D crystalline-forming protein without affecting its self-assembly. We first engineered the surface-layer protein SbsB from Geobacillus stearothermophilus pV72/p2 to display isopeptide bond-forming protein conjugation pairs, SpyTag or SnoopTag, at four positions spaced 5.7-10.5 nm apart laterally and 3 nm axially. The C-terminal and two newly-identified locations within SbsB monomer accommodated the short SpyTag or SnoopTag peptide tags without affecting the 2D lattice structure. Introducing tags at distinct locations enabled orthogonal and covalent binding of SpyCatcher- or SnoopCatcher-protein fusions to micron-sized 2D nanosheets. By introducing different types of bifunctional cross-linkers, the dual-functionalized nanosheets were programmed to self-assemble into different 3D stacks, all of which retain their nanoscale order. Thus, our work creates a modular protein platform that is easy to program to create dual-functionalized 2D and lamellar 3D nanomaterials with new catalytic, optoelectronic, and mechanical properties.     

Stoichiometric Assembly of the Cellulosome Generates Maximum Synergy for the Degradation of Crystalline Cellulose,as Revealed by In Vitro Reconstitution of the Clostridium thermocellum Cellulosome

The cellulosome is a supramolecular multienzyme complex formed by species-specific interactions between the cohesin modules of scaffoldin proteins and the dockerin modules of a wide variety of polysaccharide-degrading enzymes. Cellulosomal enzymes bound to the scaffoldin protein act synergistically to degrade crystalline cellulose. However, there have been few attempts to reconstitute intact cellulosomes due to the difficulty of heterologously expressing full-length scaffoldin proteins. We describe the synthesis of a full-length scaffoldin protein containing nine cohesin modules, CipA; its deletion derivative containing two cohesin modules, ΔCipA; and three major cellulosomal cellulases, Cel48S, Cel8A, and Cel9K, of the Clostridium thermocellum cellulosome. The proteins were synthesized using a wheat germ cell-free protein synthesis system, and the purified proteins were used to reconstitute cellulosomes. Analysis of the cellulosome assembly using size exclusion chromatography suggested that the dockerin module of the enzymes stoichiometrically bound to the cohesin modules of the scaffoldin protein. The activity profile of the reconstituted cellulosomes indicated that cellulosomes assembled at a CipA/enzyme molar ratio of 1/9 (cohesin/dockerin = 1/1) and showed maximum synergy (4-fold synergy) for the degradation of crystalline substrate and ∼2.4-fold-higher synergy for its degradation than minicellulosomes assembled at a ΔCipA/enzyme molar ratio of 1/2 (cohesin/dockerin = 1/1). These results suggest that the binding of more enzyme molecules on a single scaffoldin protein results in higher synergy for the degradation of crystalline cellulose and that the stoichiometric assembly of the cellulosome, without excess or insufficient enzyme, is crucial for generating maximum synergy for the degradation of crystalline cellulose.     

Modeling the self-assembly of the cellulosome enzyme complex

Most bacteria use free enzymes to degrade plant cell walls in nature. However, some bacteria have adopted a different strategy wherein enzymes can either be free or tethered on a protein scaffold forming a complex called a cellulosome. The study of the structure and mechanism of these large macromolecular complexes is an active and ongoing research topic, with the goal of finding ways to improve biomass conversion using cellulosomes. Several mechanisms involved in cellulosome formation remain unknown, including how cellulosomal enzymes assemble on the scaffoldin and what governs the population of cellulosomes created during self-assembly. Here, we present a coarse-grained model to study the self-assembly of cellulosomes. The model captures most of the physical characteristics of three cellulosomal enzymes (Cel5B, CelS, and CbhA) and the scaffoldin (CipA) from Clostridium thermocellum. The protein structures are represented by beads connected by restraints to mimic the flexibility and shapes of these proteins. From a large simulation set, the assembly of cellulosomal enzyme complexes is shown to be dominated by their shape and modularity. The multimodular enzyme, CbhA, binds statistically more frequently to the scaffoldin than CelS or Cel5B. The enhanced binding is attributed to the flexible nature and multimodularity of this enzyme, providing a longer residence time around the scaffoldin. The characterization of the factors influencing the cellulosome assembly process may enable new strategies to create designers cellulosomes.     

Cell-surface binding domains from Clostridium cellulovorans can be used for surface display of cellulosomal scaffoldins in Lactococcus lactis

Engineering microbial strains combining efficient lignocellulose metabolization and high-value chemical production is a cutting-edge strategy towards cost-sustainable 2^nd generation biorefining. Here, protein components of the Clostridium cellulovorans cellulosome were introduced in Lactococcus lactis IL1403, one of the most efficient lactic acid producers but unable to directly ferment cellulose. Cellulosomes are protein complexes with high cellulose depolymerization activity whose synergistic action is supported by scaffolding protein(s) (i.e., scaffoldins). Scaffoldins are involved in bringing enzymes close to each other and often anchor the cellulosome to the cell surface. In this study, three synthetic scaffoldins were engineered by using domains derived from the main scaffoldin CbpA and the Endoglucanase E (EngE) of the C. cellulovorans cellulosome. Special focus was on CbpA X2 and EngE S-layer homology (SLH) domains possibly involved in cell-surface anchoring. The recombinant scaffoldins were successfully introduced in and secreted by L. lactis. Among them, only that carrying the three EngE SLH modules was able to bind to the L. lactis surface although these domains lack the conserved TRAE motif thought to mediate binding with secondary cell wall polysaccharides. The synthetic scaffoldins engineered in this study could serve for assembly of secreted or surface-displayed designer cellulosomes in L. lactis.     

A synthetic biology approach for evaluating the functional contribution of designer cellulosome components to deconstruction of cellulosic substrates

Background

Select cellulolytic bacteria produce multi-enzymatic cellulosome complexes that bind to the plant cell wall and catalyze its efficient degradation. The multi-modular interconnecting cellulosomal subunits comprise dockerin-containing enzymes that bind cohesively to cohesin-containing scaffoldins. The organization of the modules into functional polypeptides is achieved by intermodular linkers of different lengths and composition, which provide flexibility to the complex and determine its overall architecture.

Results

Using a synthetic biology approach, we systematically investigated the spatial organization of the scaffoldin subunit and its effect on cellulose hydrolysis by designing a combinatorial library of recombinant trivalent designer scaffoldins, which contain a carbohydrate-binding module (CBM) and 3 divergent cohesin modules. The positions of the individual modules were shuffled into 24 different arrangements of chimaeric scaffoldins. This basic set was further extended into three sub-sets for each arrangement with intermodular linkers ranging from zero (no linkers), 5 (short linkers) and native linkers of 27–35 amino acids (long linkers). Of the 72 possible scaffoldins, 56 were successfully cloned and 45 of them expressed, representing 14 full sets of chimaeric scaffoldins. The resultant 42-component scaffoldin library was used to assemble designer cellulosomes, comprising three model C. thermocellum cellulases. Activities were examined using Avicel as a pure microcrystalline cellulose substrate and pretreated cellulose-enriched wheat straw as a model substrate derived from a native source. All scaffoldin combinations yielded active trivalent designer cellulosome assemblies on both substrates that exceeded the levels of the free enzyme systems. A preferred modular arrangement for the trivalent designer scaffoldin was not observed for the three enzymes used in this study, indicating that they could be integrated at any position in the designer cellulosome without significant effect on cellulose-degrading activity. Designer cellulosomes assembled with the long-linker scaffoldins achieved higher levels of activity, compared to those assembled with short-and no-linker scaffoldins.

Conclusions

The results demonstrate the robustness of the cellulosome system. Long intermodular scaffoldin linkers are preferable, thus leading to enhanced degradation of cellulosic substrates, presumably due to the increased flexibility and spatial positioning of the attached enzymes in the complex. These findings provide a general basis for improved designer cellulosome systems as a platform for bioethanol production.

     

Resonance assignments of cohesin and dockerin domains from Clostridium acetobutylicum ATCC824

Cohesin and dockerin domains are critical assembling components of cellulosome, a large extracellular multienzyme complex which is used by anaerobic cellulolytic bacteria to efficiently degrade lignocellulose. According to sequence homology, cohesins can be divided into three major groups, whereas cohesins from Clostridium acetobutylicum are beyond these groups and emanate from a branching point between the type I and type III cohesins. Cohesins and dockerins from C. acetobutylicum show low sequence homology to those from other cellulolytic bacteria, and their interactions are specific in corresponding species. Therefore the interactions between cohesins and dockerins from C. acetobutylicum are meaningful to the studies of both cellulosome assembling mechanism and the construction of designer cellulosome. Here we report the NMR resonance assignments of one cohesin from cellulosome scaffoldin cipA and one dockerin from a cellulosomal glycoside hydrolase (family 9) of C. acetobutylicum for further structural determination and functional studies.     

Design and production of active cellulosome chimeras. Selective incorporation of dockerin-containing enzymes into defined functional complexes

Defined chimeric cellulosomes were produced in which selected enzymes were incorporated in specific locations within a multicomponent complex. The molecular building blocks of this approach are based on complementary protein modules from the cellulosomes of two clostridia, Clostridium thermocellum and Clostridium cellulolyticum, wherein cellulolytic enzymes are incorporated into the complexes by means of high-affinity species-specific cohesin-dockerin interactions. To construct the desired complexes, a series of chimeric scaffoldins was prepared by recombinant means. The scaffoldin chimeras were designed to include two cohesin modules from the different species, optionally connected to a cellulose-binding domain. The two divergent cohesins exhibited distinct specificities such that each recognized selectively and bound strongly to its dockerin counterpart. Using this strategy, appropriate dockerin-containing enzymes could be assembled precisely and by design into a desired complex. Compared with the mixture of free cellulases, the resultant cellulosome chimeras exhibited enhanced synergistic action on crystalline cellulose.     

The cellulosome system of Acetivibrio cellulolyticus includes a novel type of adaptor protein and a cell surface anchoring protein

A scaffoldin gene cluster was identified in the mesophilic cellulolytic anaerobe Acetivibrio cellulolyticus. The previously described scaffoldin gene, cipV, encodes an N-terminal family 9 glycoside hydrolase, a family 3b cellulose-binding domain, seven cohesin domains, and a C-terminal dockerin. The gene immediately downstream of cipV was sequenced and designated scaB. The protein encoded by this gene has 942 amino acid residues and a calculated molecular weight of 100,358 and includes an N-terminal signal peptide, four type II cohesions, and a C-terminal dockerin. ScaB cohesins 1 and 2 are very closely linked. Similar, but not identical, 39-residue Thr-rich linker segments separate cohesin 2 from cohesin 3 and cohesin 3 from cohesin 4, and an 84-residue Thr-rich linker connects the fourth cohesin to a C-terminal dockerin. The scaC gene downstream of scaB codes for a 1,237-residue polypeptide that includes a signal peptide, three cohesins, and a C-terminal S-layer homology (SLH) module. A long, ca. 550-residue linker separates the third cohesin and the SLH module of ScaC and is characterized by an 18-residue Pro-Thr-Ala-Ser-rich segment that is repeated 27 times. The calculated molecular weight of the mature ScaC polypeptide (excluding the signal peptide) is 124,162. The presence of the cohesins and the conserved SLH module implies that ScaC acts as an anchoring protein. The ScaC cohesins are on a separate branch of the phylogenetic tree that is close to, but distinct from, the type I cohesins. Affinity blotting with representative recombinant probes revealed the following specific intermodular interactions: (i) an expressed CipV cohesin binds selectively to an enzyme-borne dockerin, (ii) a representative ScaB cohesin binds to the CipV band of the cell-free supernatant fraction, and (iii) a ScaC cohesin binds to the ScaB dockerin. The experimental evidence thus indicates that CipV acts as a primary (enzyme-recognizing) scaffoldin, and the protein was also designated ScaA. In addition, ScaB is thought to assume the role of an adaptor protein, which connects the primary scaffoldin (ScaA) to the cohesin-containing anchoring scaffoldin (ScaC). The cellulosome system of A. cellulolyticus thus appears to exhibit a special type of organization that reflects the function of the ScaB adaptor protein. The intercalation of three multiple cohesin-containing scaffoldins results in marked amplification of the number of enzyme subunits per cellulosome unit. At least 96 enzymes can apparently be incorporated into an individual A. cellulolyticus cellulosome. The role of such amplified enzyme incorporation and the resultant proximity of the enzymes within the cellulosome complex presumably contribute to the enhanced synergistic action and overall efficient digestion of recalcitrant forms of cellulose. Comparison of the emerging organization of the A. cellulolyticus cellulosome with the organizations in other cellulolytic bacteria revealed the diversity of the supramolecular architecture.     

Cellulosomes-structure and ultrastructure

The cellulosome is a macromolecular machine, whose components interact in a synergistic manner to catalyze the efficient degradation of cellulose. The cellulosome complex is composed of numerous kinds of cellulases and related enzyme subunits, which are assembled into the complex by virtue of a unique type of scaffolding subunit (scaffoldin). Each of the cellulosomal subunits consists of a multiple set of modules, two classes of which (dockerin domains on the enzymes and cohesin domains on scaffoldin) govern the incorporation of the enzymatic subunits into the cellulosome complex. Another scaffoldin module-the cellulose-binding domain-is responsible for binding to the substrate. Some cellulosomes appear to be tethered to the cell envelope via similarly intricate, multiple-domain anchoring proteins. The assemblage is organized into dynamic polycellulosomal organelles, which adorn the cell surface. The cellulosome dictates both the binding of the cell to the substrate and its extracellular decomposition to soluble sugars, which are then taken up and assimilated by normal cellular processes.     

Unconventional mode of attachment of the Ruminococcus flavefaciens cellulosome to the cell surface

Sequence extension of the scaffoldin gene cluster from Ruminococcus flavefaciens revealed a new gene (scaE) that encodes a protein with an N-terminal cohesin domain and a C terminus with a typical gram-positive anchoring signal for sortase-mediated attachment to the bacterial cell wall. The recombinant cohesin of ScaE was recovered after expression in Escherichia coli and was shown to bind to the C-terminal domain of the cellulosomal structural protein ScaB, as well as to three unknown polypeptides derived from native cellulose-bound Ruminococcus flavefaciens protein extracts. The ScaB C terminus includes a cryptic dockerin domain that is unusual in its sequence, and considerably larger than conventional dockerins. The ScaB dockerin binds to ScaE, suggesting that this interaction occurs through a novel cohesin-dockerin pairing. The novel ScaB dockerin was expressed as a xylanase fusion protein, which was shown to bind tenaciously and selectively to a recombinant form of the ScaE cohesin. Thus, ScaE appears to play a role in anchoring the cellulosomal complex to the bacterial cell envelope via its interaction with ScaB. This sortase-mediated mechanism for covalent cell-wall anchoring of the cellulosome in R. flavefaciens differs from those reported thus far for any other cellulosome system.