表面展示表达果胶酯酶的重组酿酒酵母构建及乙醇发酵

【目的】以淀粉为原料的乙醇发酵工艺仍然是当前燃料乙醇的主要生产方式。然而,一些原料中含有的果胶物质不仅降低了乙醇产率,而且会导致醪液粘度增大,从而会进一步影响传质和传热、增加设备负担等。构建能够自主降解果胶质的重组酿酒酵母并应用于燃料乙醇生产是值得探索的领域。【方法】论文将来源于黑曲霉的果胶酯酶基因克隆于α因子信号肽下游并通过酵母α-凝集素C-端蛋白的介导构建了在细胞表面锚定表达果胶酯酶的重组酿酒酵母PE。【结果】重组酵母的果胶酯酶表达水平达到2.6 U/g(菌体湿重),并进一步鉴定了重组果胶酯酶性质。以甘薯粉为原料的同步糖化发酵实验中,重组酵母PE的乙醇浓度和乙醇转化率分别达到95 g/L和88.1%,与出发菌株相比提高了2.2%。更重要的是,表面展示果胶酯酶能够显著降低发酵过程中的发酵液粘度。【结论】通过在工业酿酒酵母表面展示表达果胶酯酶不仅能够提高糖化酶等的作用效果和酿酒酵母的代谢能力,而且能够显著降低乙醇生产过程中发酵液的粘度,将对工业规模乙醇生产在降低设备负担、节约能耗方面具有一定的潜在价值。     

Characterization of Ternary Protein Systems In?Vivo with Tricolor Heterospecies Partition Analysis

Tools and assays that characterize protein-protein interactions are of fundamental importance to biology, because protein assemblies play a critical role in the control and regulation of nearly every cellular process. The availability of fluorescent proteins has facilitated the direct and real-time observation of protein-protein interactions inside living cells, but existing methods are mostly limited to binary interactions between two proteins. Because of the scarcity of techniques capable of identifying ternary interactions, we developed tricolor heterospecies partition analysis. The technique is based on brightness analysis of fluorescence fluctuations from three fluorescent proteins that serve as protein labels. We identified three fluorescent proteins suitable for tricolor brightness experiments. In addition, we developed the theory of identifying interactions in a ternary protein system using tricolor heterospecies partition analysis. The theory was verified by experiments on well-characterized protein systems. A graphical representation of the heterospecies partition data was introduced to visualize interactions in ternary protein systems. Lastly, we performed fluorescence fluctuation experiments on cells expressing a coactivator and two nuclear receptors and applied heterospecies partition analysis to explore the interactions of this ternary protein system.     

Comparative genomic and protein sequence analyses of the chemotaxis system of Azorhizobium caulinodans北大核心CSCD

［Objective］ Azorhizobium caulinodans ORS571 can fix nitrogen not only as a free-living organism and an associative-symbiotic bacterium by colonizing the root surface of non-leguminous plants, but also as a symbiotic bacterium by interacting with leguminous plant Sesbania rostrata.Due to its ability to grow and fix nitrogen under three conditions, A.caulinodans uses sophisticated chemotaxis signal transduction systems to transform environmental cues into corresponding behavioral responses.Chemotaxis appears crucial for the growth of A.caulinodansin complicated environment and the construction of associative relationship with the plant.However, little is known about the chemotactic pathway of A.caulinodans.Thus, our study aimed to compare the chemotaxis-like genes of A.caulinodans with those of well-studied species.［Methods］ NCBI protein BLAST was used for searching sequence similarity with default parameter values against the genomes of A.caulinodans.HMMER3, based on Pfam database, was used for comparative analyses of methyl-accepting chemotaxis protein （MCP）.［Results］ There was a major chemotaxis cluster in A.caulinodans and the CheR methylated MCPs independently of pentapeptide motif.There were 43 MCP homologs containing diverse signal-sensing architectures in A.caulinodans.In addition,cytoplasmic domains of these MCPs were all composed of 38 heptad repeats.［Conclusion］ Despite the extremely high homology presented between the chemotactic system of A.caulinodans and those of well-studied species, A.caulinodans shows its own unique characteristics.The classification of these chemotactic pathways by comparative genomics enables us to better understand how A.caulinodansresponds to changes in environment via exquisite signal transductions in chemotaxis system.     

Dual emission fluorescent silver nanoclusters for sensitive detection of the biological coenzyme NAD+/NADH

Fluorescent silver nanoclusters (Ag NCs) displaying dual-excitation and dual-emission properties have been developed for the specific detection of NAD⁺ (nicotinamide adenine dinucleotide, oxidized form). With the increase of NAD⁺ concentrations, the longer wavelength emission (with the peak at 550 nm) was gradually quenched due to the strong interactions between the NAD⁺ and Ag NCs, whereas the shorter wavelength emission (peaking at 395 nm) was linearly enhanced. More important, the dual-emission intensity ratio (I₃₉₅/I₅₅₀), fitting by a single-exponential decay function, can efficiently detect various NAD⁺ levels from 100 to 4000 μM, as well as label NAD⁺/NADH (reduced form of NAD) ratios in the range of 1–50.     

Genomic constitution and taxonomy of the Chinese hexaploids Elymus cylindricus and E. breviaristatus (Poaceae: Triticeae)

Elymus cylindricus (2n = 6x = 42) and E. breviaristatus (2n = 6x = 42) are distributed in grasslands and deserts of northern and north‐western China. Their genomic constitution and taxonomic status are unclear. Elymus cylindricus was crossed with E. wawawaiensis J.R.Carlson & Barkworth ( StH ), Roegneria grandis Keng ( StY ) and Campeiostachys dahurica (Turcz. ex Griseb.) B.R.Baum, J.L. Y ang & C. Y en var. dahurica ( StYH ). Meiotic pairing in the hybrids E. cylindricus × E. wawawaiensis ( StH ), E. cylindricus × R. grandis ( StY ) and E. cylindricus × C. dahurica var. dahurica ( StYH ) showed on average 10.00, 11.30 and 20.92 bivalents per cell, respectively. Elymus breviaristatus was crossed with C. dahurica var. dahurica ( StYH ) and E. cylindricus. Chromosome pairing in the hybrids of E. breviaristatus × C. dahurica var. dahurica and E. breviaristatus × E. cylindricus showed on average 19.60 and 19.27 bivalents, respectively. Genomic in situ hybridization (GI SH ) revealed the presence of St , Y and H genomes in E. cylindricus and E. breviaristatus. An intergenomic rearrangement was observed in E. cylindricus using GI SH . Meiotic pairing data and GI SH indicated that both E. cylindricus and E. breviaristatus are allohexaploids containing the StYH genomes. Elymus cylindricus and E. breviaristatus should be treated as Campeiostachys dahurica var. cylindrica and Campeiostachys breviaristata, respectively.     

Glucose‐ and mannose‐induced stomatal closure is mediated by ROS production,Ca2+ and water channel in Vicia faba

Sugars act as vital signaling molecules that regulate plant growth, development and stress responses. However, the effects of sugars on stomatal movement have been unclear. In our study, we explored the effects of monosaccharides such as glucose and mannose on stomatal aperture. Here, we demonstrate that glucose and mannose trigger stomatal closure in a dose‐ and time‐dependent manner in epidermal peels of broad bean (Vicia faba). Pharmacological studies revealed that glucose‐ and mannose‐induced stomatal closure was almost completely inhibited by two reactive oxygen species (ROS) scavengers, catalase (CAT) and reduced glutathione (GSH), was significantly abolished by an NADPH oxidase inhibitor, diphenylene iodonium chloride (DPI), whereas they were hardly affected by a peroxidase inhibitor, salicylhydroxamic acid (SHAM). Furthermore, glucose‐ and mannose‐induced stomatal closure was strongly inhibited by a Ca²⁺ channel blocker, LaCl₃, a Ca²⁺ chelator, ethyleneglycol‐bis(beta‐aminoethylether)‐N,N'‐tetraacetic acid (EGTA) and two water channel blockers, HgCl₂ and dimethyl sulfoxide (DMSO); whereas the inhibitory effects of the water channel blockers were essentially abolished by the reversing agent β‐mercaptoethanol (β‐ME). These results suggest that ROS production mainly via NADPH oxidases, Ca²⁺ and water channels are involved in glucose‐ and mannose‐induced stomatal closure.