Functional characterization of a cold related flavanone 3-hydroxylase from Tetrastigma hemsleyanum: an in vitro, in silico and in vivo study.

Tetrastigma hemsleyanum Diels et Gilg, a traditional Chinese medicine, frequently suffers from cold damage in the winter, leading to lower yields. There is a pressing need to improve cold resistance; however, the mechanisms underlying T. hemsleyanum responses to cold stress are still not clearly understood. Here, we explored the function of the flavanone 3-hydroxylase gene (ThF3H) in T. hemsleyanum under cold treatment. The open reading frame of ThF3H is 1092 bp and encodes 363 amino acid residues. In vitro, the ThF3H enzyme was expressed in E. coli and successfully catalyzed naringenin and eriodictyol into dihydrokaempferol and dihydroquercetin, respectively. ThF3H exhibited a higher affinity for naringenin than for eriodictyol, which was in accordance with an in silico molecular docking analysis. The optimal pH and temperature for ThF3H activity were 7.0 and 30 °C, respectively. In vivo, overexpression of the ThF3H gene enhanced the cold tolerance of transgenic Arabidopsis lines, which was likely due to the increase in flavonoids. Collectively, the function of a cold-related ThF3H in the flavonoid biosynthesis pathway may be helpful for improving the cold tolerance of T. hemsleyanum through molecular breeding techniques.

Realization of Efficient Phosphorescent Organic Light-Emitting Devices Using Exciplex-Type Co-Host.

High-performance phosphorescent organic light-emitting devices with an exciplex-type co-host were fabricated. The co-host is constituted by 1,3,5-tris(N-phenylbenzimidazol-2-yl) benzene, and 4,4,4-tris (N-carbazolyl) triphenylamine, and has obvious virtues in constructing efficient devices because of the thermally activated delayed fluorescence (TADF) resulting from a reverse intersystem crossing (RISC) process. The highest external quantum efficiency and luminance are 14.60% and 100,900 cd/m2 for the optimal co-host device. For comparison, 9.22% and 25,450 cd/m2 are obtained for a device employing 4,4,4-tris (N-carbazolyl) triphenylamine as a single-host. Moreover, the efficiency roll-off is notably alleviated for the co-host device, indicated by much higher critical current density of 327.8 mA/cm2, compared to 120.8 mA/cm2 for the single-host device. The alleviation of excitons quenching resulting from the captured holes and electrons, together with highly sufficient energy transfer between the co-host and phosphorescent dopant account for the obvious boost in device performances.

Hybrid warm-white organic light-emitting device based on tandem structure.

We realized an efficient hybrid tandem warm white organic light-emitting device with extremely stable color purity in forward direction by employing an easy-fabrication charge generation unit (CGU) combining 1, 4, 5, 8, 9, 11-Hexaazatriphenylene-hexacarbonitrile with ultrathin bilayers of LiF and Al. The tandem white device exhibits maximum efficiencies of 37.3 cd/A, 23.3 lm/W and still remains at high efficiencies of 34.4 cd/A, 17 lm/W at the luminance of 1000 cd/m2. When the operating current density increases from 5 mA/cm2 to 30 mA/cm2, the variations in Commission Internationale de l'Eclairage (CIE) coordinates are only (0.007, 0.003). Moreover, we also studied the cause of color variation in tandem white device by performing systematic optical simulation and discovered the origin of alleviation in efficiency roll-off for tandem white device by applying triplet-polaron quenching (TPQ) model.

NprR-NprX Quorum-Sensing System Regulates the Algicidal Activity of Bacillus sp. Strain S51107 against Bloom-Forming Cyanobacterium Microcystis aeruginosa.

Harmful cyanobacterial blooms have severely impaired freshwater quality and threatened human health worldwide. Here, a Gram-positive bacterium, Bacillus sp. strain S51107, which exhibits strong algicidal activity against Microcystis aeruginosa, was isolated from Lake Taihu. We found that the algicidal activity of strain S51107 was regulated primarily by NprR-NprX quorum sensing (QS), in which the mature form of the signaling peptide NprX was identified as the SKPDIVG heptapeptide. Disruption of the nprR-nprX cassette markedly decreased the algicidal activity, and complemented strains showed significantly recovered algicidal activity. Strain S51107 produced low-molecular-weight algicidal compounds [indole-3-carboxaldehyde and cyclo(Pro-Phe)] and high-molecular-weight algicidal substance(s) (>3 kDa). Moreover, the production of high-molecular-weight algicidal substance(s) was regulated by NprR-NprX QS, but the production of low-molecular-weight algicidal compounds was not. High-molecular-weight algicidal substance(s) played a more important role than low-molecular-weight algicidal compounds in the algicidal activity of strain S51107. The results of this study could increase our knowledge about algicidal characteristics of a potential algicidal bacterium, Bacillus sp. strain S51107, and provide the first evidence that the algicidal activity of Gram-positive algicidal bacteria is regulated by QS, which will greatly enhance our understanding of the interactions between algae and indigenous algicidal bacteria, thereby providing aid in the design and optimization of strategies to control harmful algae blooms.

The algicidal activity of Aeromonas sp. strain GLY-2107 against bloom-forming Microcystis aeruginosa is regulated by N-acyl homoserine lactone-mediated quorum sensing.

Cyanobacterial blooms have disrupted the efficient utilization of freshwater worldwide. A new freshwater bacterial strain with strong algicidal activity, GLY-2107, was isolated from Lake Taihu and identified as Aeromonas sp. It produced two algicidal compounds: 2107-A (3-benzyl-piperazine-2,5-dione) and 2107-B (3-methylindole). Both compounds exhibited potent algicidal activities against Microcystis aeruginosa, the dominant bloom-forming cyanobacterium in Lake Taihu. The EC50 values (concentration for 50% maximal effect) of 3-benzyl-piperazine-2,5-dione and 3-methylindole were 4.72 and 1.10 µg ml-1 respectively. Based on a thin-layer chromatography biosensor assay and ultra-performance liquid chromatography-coupled high resolution-tandem mass spectrometry (UPLC-HRMS/MS), the N-acyl homoserine lactone (AHL) profile of strain GLY-2107 was identified as two short side-chain AHLs: N-butyryl-homoserine lactone (C4-HSL) and N-hexanoyl-homoserine lactone (C6-HSL). The production of the two algicidal compounds was controlled by AHL-mediated quorum sensing (QS), and C4-HSL was the key QS signal for the algicidal activity of the strain GLY-2107. Moreover, 3-methylindole was found to be positively regulated by C4-HSL-mediated QS, whereas 3-benzyl-piperazine-2,5-dione might be negatively controlled by C4-HSL-mediated QS. This study suggests that a QS-regulated algicidal system may have potential use for the development of a novel control strategy for harmful cyanobacterial blooms.

Screening of multi-copy mannanase recombinants of Pichia pastoris based on colony size.

Pichia pastoris pGAP (glyceraldehyde dehydrogenase promoter) expression system was widely used for the expression and production of heterologous proteins. Screening multi-copy recombinants was an effective strategy to improve the heterologous protein production in P. pastoris. Because multiple gene insertion events occurred with a low frequency, hundreds to thousands of antibiotic-resistance recombinants need to be screened. The common way of improving screening efficiency was to increase antibiotic concentration in screening plates. Here we developed a screening method by selecting small colonies from low-concentration antibiotic screening plates. This strategy greatly improved the probability of obtaining multi-copy mannanase gene (man) recombinants and it could replace the common strategy by increasing antibiotic concentration in screening plates. The further study in liquid shake flask cultures revealed that cell concentrations, growth rates and substrate consumption rates of recombinants gradually decreased with the increase in man copy number. This indicated that such a screening strategy was effective to screen multi-copy recombinants based on colony size.

Characterization and constitutive expression of an acidic mesophilic endo-1,4-ß-D-xylanohydrolase with high thermotolerance and catalytic efficiency in Pichia pastoris.

A putative endo-1,4-ß-D-xylanohydrolase gene xyl11 from Aspergillus niger, encoding a 188-residue xylanase of glycosyl hydrolase family 11, was constitutively expressed in Pichia pastoris. The recombinant Xyl11 exhibited optimal activity at pH 5.0 and 50 °C, and displayed more than 68 % of the maximum activity over the temperature range 35-65 °C and 33 % over the pH range 2.2-7.0. It maintained more than 40 % of the original activity after incubation at 90 °C (pH 5.0) for 10 min and more than 75 % of the original activity after incubation at pH 2.2-11.0 (room temperature) for 2 h. The specific activity, K m and V max of purified Xyl11 were 22,253 U mg(-1), 6.57 mg ml(-1) and 51,546.4 µmol min(-1) mg(-1). It could degrade xylan to a series of xylooligosaccharides and no xylose was detected. The recombinant enzyme with high stability and catalytic efficiency could work over wide ranges of pH and temperature and thus has the potential for various industrial applications.

Characterization and constitutive expression of a novel endo-1,4-ß-D-xylanohydrolase from Aspergillus niger in Pichia pastoris.

A putative endo-1,4-ß-D-xylanohydrolase gene xyl10 from Aspergillus niger, encoding a 308-residue mature xylanase belonging to glycosyl hydrolase family 10, was constitutively expressed in Pichia pastoris. The recombinant Xyl10 exhibited optimal activity at pH 5.0 and 60 °C with more than 50 % of the maximum activity from 40 to 70 °C. It retained more than 90 % of the original activity after incubation at 60 °C (pH 5.0) for 30 min and more than 74 % after incubation at pH 3.0-13.0 for 2 h (25 °C). The specific activity, K m and V max values for purified Xyl10 were, respectively, 3.2 × 10(3) U mg(-1), 3.6 mg ml(-1) and 5.4 × 10(3) µmol min(-1 )mg(-1) towards beechwood xylan. The enzyme degraded xylan to a series of xylooligosaccharides and xylose. The recombinant enzyme with these properties has the potential for various industrial applications.

Development of an industrial medium and a novel fed-batch strategy for high-level expression of recombinant ß-mananase by Pichia pastoris.

An industrial medium, Corn Steep Liquor Powder Dextrose (CSD medium) was developed for constitutive expression of recombinant ß-mananase by Pichia pastoris. The ß-mananase activity (513 U/mL) with CSD medium was 1.64- and 2.5-fold higher than with YPD and BSM in shaken flasks. The ß-mananase productivity with CSD medium was 61.0 U/mL h, which was 1.7- and 2.5-fold higher than with YPD and BSM in a 5-L fermenter based on a novel fed-batch strategy combining the real-time exponential feed mode with the DO-stat feed mode. The ß-mananase activity, dry cell weight and the recombinant enzyme reached up to 5132 U/mL, 110.0 g/L and 4.50 g/L after 50 h cultivation in a 50-L fermenter. The high efficient expression of recombinant ß-mananase by P. pastoris indicated that CSD medium and the novel fed-batch strategy have great potential for the production of recombinant ß-mananase in industrial fermentation.