Preparation and Properties of Clostridium thermocellum Lichenase Deletion Variants and Their Use for Construction of Bifunctional Hybrid Proteins

Major properties (pH and temperature optimum, stability) of lichenase (b-1,3-1,4-glucanase) deletion variants from Clostridium thermocellum were comparatively studied. The deletion variant LicBM2 was used to create hybrid bifunctional proteins by fusion with sequences of the green fluorescent protein (GFP) from Aequorea victoria. The data show that in hybrid proteins both GFP and lichenase retain their major properties, namely, GFP remains a fluorescent protein and the lichenase retains activity and high thermostability. Based on the results of this investigation and results that have been obtained earlier, the use of the deletion variants of lichenase and the bifunctional hybrid proteins as reporter proteins is suggested.     

Manipulation of salicylate content in Arabidopsis thaliana by the expression of an engineered bacterial salicylate synthase

Salicylic acid (SA) plays a central role as a signalling molecule involved in plant defense against microbial attack. Genetic manipulation of SA biosynthesis may therefore help to generate plants that are more disease-resistant. By fusing the two bacterial genes pchA and pchB from Pseudomonas aeruginosa, which encode isochorismate synthase and isochorismate pyruvate-lyase, respectively, we have engineered a novel hybrid enzyme with salicylate synthase (SAS) activity. The pchB-A fusion was expressed in Arabidopsis thaliana under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter, with targeting of the gene product either to the cytosol (c-SAS plants) or to the chloroplast (p-SAS plants). In p-SAS plants, the amount of free and conjugated SA was increased more than 20-fold above wild type (WT) level, indicating that SAS is functional in Arabidopsis. P-SAS plants showed a strongly dwarfed phenotype and produced very few seeds. Dwarfism could be caused by the high SA levels per se or, perhaps more likely, by a depletion of the chorismate or isochorismate pools of the chloroplast. Targeting of SAS to the cytosol caused a slight increase in free SA and a significant threefold increase in conjugated SA, probably reflecting limited chorismate availability in this compartment. Although this modest increase in total SA content did not strongly induce the resistance marker PR-1, it resulted nevertheless in enhanced disease resistance towards a virulent isolate of Peronospora parasitica. Increased resistance of c-SAS lines was paralleled with reduced seed production. Taken together, these results illustrate that SAS is a potent tool for the manipulation of SA levels in plants.     

Two terpene synthases in resistant Pinus massoniana contribute to defence against Bursaphelenchus xylophilus

Pine wood nematode (PWN; Bursaphelenchus xylophilus), a destructive pest of Pinus massoniana, is causing a severe epidemic of pine wilt disease in China. When invaded by PWN, resistant P. massoniana secretes an abundance of oleoresin terpenoids as a defensive strategy. However, regulatory mechanisms of this defence in resistant P. massoniana have yet to be elucidated. Here, we characterized two terpene synthase genes, α‐pinene synthase (PmTPS4) and longifolene synthase (PmTPS21), identified in resistant P. massoniana and investigate the contribution of these genes to the oleoresin defence strategy in resistant masson pines. Up‐regulation of these two genes in the stem supported their involvement in terpene biosynthesis as part of the defence against PWN. Recombinant protein expression revealed catalytic activity for the two PmTPSs, with PmTPS4 primarily producing α‐pinene, while PmTPS21 produced α‐pinene and longifolene simultaneously. The major enzymatic products of the two terpene synthases had inhibitory effects on PWN in vitro. We demonstrated that PmTPS4 and PmTPS21 played positive roles in terpene‐defence mechanisms against PWN infestation. The major products of these terpene synthases could directly inhibit the survival rate of PWN in vitro. We revealed that PmTPS21 was a novel bifunctional enzyme capable of simultaneous production of both monoterpene and sesquiterpene.     

Microwave Quasi-Solid State to Construct Strong Metal-Support Interactions with Interfacial Electron-Enriched Ru for Anion Exchange Membrane Electrolysis

Regulating the metal-support interaction of the anchored metal nanoclusters is recognized as valid approach to optimize the electrocatalytic performance through tuning the interfacial electronic structure. However, developing novel support and understanding the interfacial electron accumulation on modulating the reaction kinetics are still elusive. Herein, highly-dispersed Ruthenium (Ru) nanoclusters anchored onto phosphorous doped molybdenum boride (Ru/P-MoB) is developed through ultrafast microwave-plasma (60 s) approach. The synthesized Ru/P-MoB impressively promote the hydrogen evolution with low overpotentials of 34, 45, and 40 mV to drive 10 mA cm⁻² in alkaline freshwater, alkaline seawater and acid media. Specially, it presents superior turnover frequency and mass/specific activity relative to Pt/C, Ru/C, and Ru/MoB. Moreover, the anion exchange membrane (AEM) electrolyzer cell based on Ru/P-MoB can achieve 500 and 1000 mA cm⁻² with small voltages of 1.71 and 1.78 V with good durability. Experimental and density functional theoretical (DFT) analysis reveal that the strong metal-support interactions (Ru─Mo and Ru─P bonds) with generated interfacial electron-enriched Ru, and then favoring the water-molecule adsorption/dissociation and optimal H intermediate adsorption free energy. This work provides novel designing avenue to exploit electrocatalysts with outstanding catalytic performance under high current density at practical high-temperature.     

Direct asymmetric Michael addition of thioether‐based aryl sulfanyl‐propan‐2‐one to nitroalkenes catalyzed by a chiral amine‐thiourea bifunctional organocatalyst

Although the organocatalytic direct asymmetric Michael reactions of carbonyl compounds to nitroalkenes have been investigated intensely, the Michael reaction of the thioether‐based donors remains a rather undeveloped field. This work concerns the asymmetric Michael addition of aryl sulfanyl‐propan‐2‐one to nitroalkenes with benzoic acid as an additive, and chiral amine‐thiourea as a bifunctional organocatalyst. The reactions provided the highly functionalized chiral adducts with excellent enantioselectivities (up to 96% ee) and good yields. Moreover, the further transformed products exhibited excellent diastereoselectivity. Chirality, 2010. © 2010 Wiley‐Liss, Inc.     

Trinary Layered Double Hydroxides as High‐Performance Bifunctional Materials for Oxygen Electrocatalysis

Layered double hydroxides (LDHs) are a family of high‐profile layer materials with tunable metal species and interlayer spacing, and herein the LDHs are first investigated as bifunctional electrocatalysts. It is found that trinary LDH containing nickel, cobalt, and iron (NiCoFe‐LDH) shows a reasonable bifunctional performance, while exploiting a preoxidation treatment can significantly enhance both oxygen reduction reaction and oxygen evolution reaction activity. This phenomenon is attributed to the partial conversion of Co²⁺ to Co³⁺ state in the preoxidation step, which stimulates the charge transfer to the catalyst surface. The practical application of the optimized material is demonstrated with a small potential hysteresis (800 mV for a reversible current density of 20 mA cm^?2) as well as a high stability, exceeding the performances of noble metal catalysts (commercial Pt/C and Ir/C). The combination of the electrochemical metrics and the facile and cost‐effective synthesis endows the trinary LDH as a promising bifunctional catalyst for a variety of applications, such as next‐generation regenerative fuel cells or metal–air batteries.     

Transcriptional and biochemical regulation of a novel Arabidopsis thaliana bifunctional aspartate kinase-homoserine dehydrogenase gene isolated by functional complementation of a yeast hom6 mutant

An aspartate kinase-homoserine dehydrogenase (AK-HSDH) cDNA of Arabidopsis thaliana has been cloned by functional complementation of a Saccharomyces cerevisiae strain mutated in its homoserine dehydrogenase (HSDH) gene (hom6). Two of the three isolated clones were also able to complement a mutant yeast aspartate kinase (AK) gene (hom3). Sequence analysis showed that the identified gene (akthr2), located on chromosome 4, is different from the previously cloned A. thaliana AK-HSDH gene (akthr1), and corresponds to a novel bifunctional AK-HSDH gene. Expression of the isolated akthr2 cDNA in a HSDH-less hom6 yeast mutant conferred threonine and methionine prototrophy to the cells. Cell-free extracts contained a threonine-sensitive HSDH activity with feedback properties of higher plant type. Correspondingly, cDNA expression in an AK-deficient hom3 yeast mutant resulted in threonine and methionine prototrophy and a threonine-sensitive AK activity was observed in cell-free extracts. These results confirm that akthr2 encodes a threonine-sensitive bifunctional enzyme. Transgenic Arabidopsis thaliana plants (containing a construct with the promoter region of akthr2 in front of the gus reporter gene) were generated to compare the expression pattern of the akthr2 gene with the pattern of akthr1 earlier described in tobacco. The two genes are simultaneously expressed in meristematic cells, leaves and stamens. The main differences between the two genes concern the time-restricted or absent expression of the akthr2 gene in the stem, the gynoecium and during seed formation, while akthr1 is less expressed in roots.