Structural Insight of a Trimodular Halophilic Cellulase with a Family 46 Carbohydrate-Binding Module

Cellulases are the key enzymes used in the biofuel industry. A typical cellulase contains a catalytic domain connected to a carbohydrate-binding module (CBM) through a flexible linker. Here we report the structure of an atypical trimodular cellulase which harbors a catalytic domain, a CBM46 domain and a rigid CBM_X domain between them. The catalytic domain shows the features of GH5 family, while the CBM46 domain has a sandwich-like structure. The catalytic domain and the CBM46 domain form an extended substrate binding cleft, within which several tryptophan residues are well exposed. Mutagenesis assays indicate that these residues are essential for the enzymatic activities. Gel affinity electrophoresis shows that these tryptophan residues are involved in the polysaccharide substrate binding. Also, electrostatic potential analysis indicates that almost the entire solvent accessible surface of CelB is negatively charged, which is consistent with the halophilic nature of this enzyme.     

纸坊病病毒病因的研究

1985年4～10月与1986年6～8月,在贵州省沿河县的纸坊村和崔家坨村先后发生了病因不明的传染病。纸坊村约有1/5的村民发病,病死率为12％,崔家坨村有1/10的村民发病,病死率高达30％。发病波及各年龄组,以青壮年为多,有家庭集聚现象。 本病起病急,轻症者只有头晕、乏力、肌痛、多汗、心悸伴以低热,有的初期有短暂的腹泻。重症者有高热(40℃以上)、大汗、心悸、游走性肌肉痉挛伴有明显疼痛和触痛,以腰骶部及四肢肌肉为好发部位。病人烦燥不安,2～5天内死亡。经实验室检查,排除了食物中毒、农药中毒、钩端螺旋体病和弓形体感染。从病人和接触者的粪便中分离到9株病毒,性状一致,为RNA型25nm的球形颗粒,耐酸,耐乙醚,能凝集人“O”型血球。经血清学鉴定为ECHO3型病毒。16份病人双份血清的检测结果表明,恢复期血清对该病毒中和抗体有4倍以上升高者共8例(纸坊村和崔家坨各4例)。病人单份血清也都有较高的抗体。有理由认为两年中先后在两个村庄发生的传染病与ECHO3型病毒有密切关系。查阅文献,尚未见有关ECHO3型病毒引起以肌痛、游走性肌痉挛为特征的疾病的报道。     

Structural and functional analyses of catalytic domain of GH10 xylanase from Thermoanaerobacterium saccharolyticum JW/SL‐YS485

Xylanases are capable of decomposing xylans, the major components in plant cell wall, and releasing the constituent sugars for further applications. Because xylanase is widely used in various manufacturing processes, high specific activity, and thermostability are desirable. Here, the wild‐type and mutant (E146A and E251A) catalytic domain of xylanase from Thermoanaerobacterium saccharolyticum JW/SL‐YS485 (TsXylA) were expressed in Escherichia coli and purified subsequently. The recombinant protein showed optimal temperature and pH of 75°C and 6.5, respectively, and it remained fully active even after heat treatment at 75°C for 1 h. Furthermore, the crystal structures of apo‐form wild‐type TsXylA and the xylobiose‐, xylotriose‐, and xylotetraose‐bound E146A and E251A mutants were solved by X‐ray diffraction to high resolution (1.32–1.66 Å). The protein forms a classic (β/α)₈ folding of typical GH10 xylanases. The ligands in substrate‐binding groove as well as the interactions between sugars and active‐site residues were clearly elucidated by analyzing the complex structures. According to the structural analyses, TsXylA utilizes a double displacement catalytic machinery to carry out the enzymatic reactions. In conclusion, TsXylA is effective under industrially favored conditions, and our findings provide fundamental knowledge which may contribute to further enhancement of the enzyme performance through molecular engineering. Proteins 2013; 81:1256–1265. © 2013 Wiley Periodicals, Inc.     

Development of thermodynamic optimum searching (TOS) to improve the prediction accuracy of flux balance analysis

Flux balance analysis (FBA) has been widely used in calculating steady‐state flux distributions that provide important information for metabolic engineering. Several thermodynamics‐based methods, for example, quantitative assignment of reaction directionality and energy balance analysis have been developed to improve the prediction accuracy of FBA. However, these methods can only generate a thermodynamically feasible range, rather than the most thermodynamically favorable solution. We therefore developed a novel optimization method termed as thermodynamic optimum searching (TOS) to calculate the thermodynamically optimal solution, based on the second law of thermodynamics, the minimum magnitude of the Gibbs free energy change and the maximum entropy production principle (MEPP). Then, TOS was applied to five physiological conditions of Escherichia coli to evaluate its effectiveness. The resulting prediction accuracy was found significantly improved (10.7–48.5%) by comparing with the ¹³C‐fluxome data, indicating that TOS can be considered an advanced calculation and prediction tool in metabolic engineering. Biotechnol. Bioeng. 2013; 110: 914–923. © 2012 Wiley Periodicals, Inc.     

嗜碱单胞菌的新型羧基转移酶α亚基基因Aa-accA及其抗盐碱性能

[目的]探讨解淀粉嗜碱单胞菌(Alkalimonas amylolytica)N10来源的羧基转移酶α亚基(Acetyl-coenzyme A carboxylase subunit alpha,AccA)基因Aa-accA对细菌及植物细胞耐盐碱性的作用.[方法]通过PCR方法从嗜碱菌N10基因组中扩增基因Aa-accA,并在大肠杆菌(Escherichia coli)K12中表达,通过测定工程菌及对照菌在不同盐浓度[0%,2%,4%,6%(W/V) NaCl]及不同碱性pH(8.0,8.5,9.0,9.5)的LB中生长12 h后的OD600值,以及二者在分别含6%(W/V) NaCl及pH 9的LB中的生长曲线,评价Aa-accA对大肠杆菌耐盐碱性的影响.同时以pPZP111为载体,构建了植物细胞重组表达载体,通过农杆菌介导方法将该基因转入烟草BY-2悬浮细胞表达,利用FDA染色方法测定经盐碱溶液处理后残存的活细胞数量评价该基因对植物细胞耐盐碱性的影响.[结果]PCR扩增得到基因Aa-accA,其ORF含957 bp,编码318个氨基酸的多肽,BLAST比对显示该基因为羧基转移酶α亚基(AccA)家族中的成员,其氨基酸序列与E.coli的AccA具有76%同源性;含有Aa-accA的E.coli K12相较于对照组在不同NaCl浓度及不同碱性pH的LB中表现出了明显的生长优势,特别是在6%(W/V) NaCl及pH 9的LB中培养12 h后,终OD600分别是对照菌的2.6倍和3.5倍;缺失体实验结果显示基因缺失的突变体E.coli K12△accA在6%(W/V) NaCl及pH 9的LB中不能正常生长,而含有Aa-accA基因的重组质粒使得E.coli K12△accA在同样条件下OD600值达到0.5和0.2;转入此基因的烟草BY-2细胞,经盐碱溶液处理后,其存活细胞比例高于野生型.[结论]本研究首次发现了Aa-accA基因与盐碱性的相关性,可提高大肠杆菌及烟草BY-2细胞的耐盐碱能力.     

琥珀酸脱氢酶或琥珀酰辅酶A合成酶缺失促进大肠杆菌积累5-氨基乙酰丙酸

5-氨基乙酰丙酸 (ALA) 是生物体内四吡咯类化合物的合成前体,在农业及医药领域应用广泛,是极具开发价值的高附加值生物基化学品。目前利用外源C4途径的重组大肠杆菌发酵生产ALA的研究主要利用LB培养基并添加葡萄糖和琥珀酸、甘氨酸等合成前体,成本较高。琥珀酸在C4途径中以琥珀酰辅酶A的形式直接参与ALA的合成。文中在以葡萄糖为主要碳源的无机盐培养基中研究了琥珀酰辅酶A下游代谢途径琥珀酸脱氢酶编码基因sdhAB和琥珀酰辅酶A合成酶编码基因sucCD缺失对ALA积累的影响。与仅表达异源ALA合成酶的对照菌株相比,sdhAB和sucCD缺失菌株ALA的产量分别提高了25.59%和12.40%,且ALA的积累不依赖于琥珀酸的添加和LB培养基的使用,从而大幅降低了生产成本,显示出良好的工业应用前景。     

Improved extracellular expression and high-cell-density fed-batch fermentation of chitosanase from <Emphasis Type="Italic">Aspergillus Fumigatus</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Chitosanase (CSN) from Aspergillus fumigatus has good thermal stability, wide pH range duration, and effective hydrolysis for chitosan. Inhere, CSN was successfully expressed in Escherichia coli followed by extracellular secretion under the guidance of an N-terminal signal peptide PelB, which effectively prompted its secretion out of E. coli cells. To facilitate its later purification, N-terminal or C-terminal 6xHis epitope tag was added to the PelB-CSN protein complex. Our results indicated that PelB-CSN without 6xHis-tag (PelB-CSN) or with N-terminal 6xHis-tag (PelB-CSN-N) can both be effectively secreted into the medium, while CSN with 6xHis-tag anchored at C-terminus was expressed as inclusion bodies. Process optimization strategies were further developed to improve the secretion efficiency of recombinant PelB-CSN-N in E. coli. Under the induction of 10 g/L lactose in shake-flask culture, the extracellular activity of CSN reached 6015 U/mL at 25 °C in TB medium containing 1 % glycine. Moreover, a fed-batch fermentation strategy for high-cell-density cultivation was applied in a 5-L fermenter, increasing the extracellular CSN activity to 14,000 U/mL in 2-day fermentation with the optimal addition of lactose and glycine.     

An efficient CRISPR/Cas9-based genome editing system for alkaliphilic Bacillus sp. N16-5 and application in engineering xylose utilization for D-lactic acid production

Alkaliphiles are considered more suitable chassis than traditional neutrophiles due to their excellent resistance to microbial contamination. Alkaliphilic Bacillus sp. N16-5, an industrially interesting strain with great potential for the production of lactic acid and alkaline polysaccharide hydrolases, can only be engineered genetically by the laborious and time-consuming homologous recombination. In this study, we reported the successful development of a CRISPR/Cas9-based genome editing system with high efficiency for single-gene deletion, large gene fragment deletion and exogenous DNA chromosomal insertion. Moreover, based on a catalytically dead variant of Cas9 (dCas9), we also developed a CRISPRi system to efficiently regulate gene expression. Finally, this efficient genome editing system was successfully applied to engineer the xylose metabolic pathway for the efficient bioproduction of D -lactic acid. Compared with the wild-type Bacillus sp. N16-5, the final engineered strain with XylR deletion and AraE overexpression achieved 34.3% and 27.7% increases in xylose consumption and D -lactic acid production respectively. To our knowledge, this is the first report on the development and application of CRISPR/Cas9-based genome editing system in alkaliphilic Bacillus, and this study will significantly facilitate functional genomic studies and genome manipulation in alkaliphilic Bacillus, laying a foundation for the development of more robust microbial chassis.