Intracellular imaging of Qdots‐labeled DNA in cyanobacteria

In this contribution, they have attempted to develop a labeling technique for in vivo imaging of functionally active plasmid DNA in cyanobacterial cells through its decoration with semiconductor quantum dots (Qdots) as fluorescent nanoprobes. For that purpose biotinylated plasmid slr2060 DNA was conjugated with Qdots‐streptavidine. The intact DNA was visualized in a single green color by light microscopy. These Qdots‐DNA conjugates were capable of expressing the acyltransferase enzyme. Qdots‐DNA conjugates and confocal microscope imaging technique were adopted to visualize the gene transport across the membrane of the live cyanobacteria cell in real time. Long‐term kinetic study enabled to reveal the steps of extracellular and intracellular microenvironment for plasmid transportation into the live cell. To confirm these processes a confocal microscope and indicator plate assay test were applied in tandem. In this contribution, Qdots‐labeled plasmid DNA was utilized for the first time for long‐term intracellular imaging studies in cyanobacteria species PCC6803. The results showed that the Qdots‐labeled plasmid DNA detection could be used as a powerful labeling technique for visualization of exogenous DNA entry and tracking into living cells by confocal microscopy. Microsc. Res. Tech. 79:447–452, 2016. © 2016 Wiley Periodicals, Inc.     

5,8‐Dihydroxy‐9,12,15(Z,Z,Z)‐Octadecatrienoic Acid Production by Recombinant Cells Expressing Aspergillus nidulans Diol Synthase

Whole cells of recombinant Escherichia coli expressing diol synthase from Aspergillus nidulans produced 5,8‐dihydroxy‐9,12,15(Z,Z,Z)‐octadecatrienoic acid from α‐linolenic acid via 8‐hydroperoxy‐9,12,15(Z,Z,Z)‐octadecatrienoic acid as an intermediate. The optimal conditions for 5,8‐dihydroxy‐9,12,15(Z,Z,Z)‐octadecatrienoic acid production using whole recombinant cells were exhibited at pH 7.0, 40 °C, and 250 rpm with 40 g/L cells, 12 g/L, α‐linolenic acid, and 5 % (v/v) dimethyl sulfoxide in a 250‐mL baffled flask containing 50 mL reaction solution. Under these conditions, whole recombinant cells produced 9.1 g/L 5,8‐dihydroxy‐9,12,15(Z,Z,Z)‐octadecatrienoic acid for 100 min, with a conversion yield of 75 % (w/w), a volumetric productivity of 5.5 g/L/h, and specific productivity of 137 mg/g‐cells/h. As an intermediate, 8‐hydroperoxy‐9,12,15(Z,Z,Z)‐octadecatrienoic acid was observed at approximately 1.4 g/L after 100 min. With regard to dihydroxy fatty acid production, this is the highest reported volumetric and specific productivities thus far. This is the first report on the biotechnological production of 5,8‐dihydroxy‐9,12,15(Z,Z,Z)‐octadecatrienoic acid.     

重组RANK蛋白抑制破骨细胞活性的实验研究

目的研究重组RANK蛋白在体外实验中对破骨细胞的抑制作用。方法成骨细胞与RAW264.7细胞以4∶1比例混合培养6d,以抗酒石酸酸性磷酸酶(TRAP)染色鉴定后,加入3种浓度(10-4g/L、10-5g/L、10-6g/L)重组RANK蛋白,并设空白对照。3d后观察破骨细胞数目和形态,计数骨磨片吸收陷窝。结果混合培养6d后,体系中可见成熟破骨细胞出现。加重组RANK蛋白3d后,与对照组相比,各药物组TRAP阳性细胞个数、骨磨片吸收陷窝计数均明显减少。结论体外实验中重组RANK蛋白可以显著抑制破骨细胞活性及吸收功能。     

Evolutionary adaptation of recombinant shochu yeast for improved xylose utilization

We examined the evolutionary adaptation of recombinant shochu yeast by serial anaerobic cultivation in xylose-based minimal medium. Compared with the parental strain, the adapted strain MA-S4-M1 (M1) markedly improved the growth on xylose and the anaerobic xylose consumption rate. M1 gained improved xylose utilization properties by optimizing the metabolic pathway enzymes and enhancing the uptake of xylose.     

Studies on fermentation with recombinant yeast cells: plasmid stability and kinetics of biomass and protein production

Plasmid stability of the recombinant Saccharomyces cerevisiae YPB‐G strain harbouring a YEp plasmid with α‐amylase and glucoamylase genes as a fusion has been investigated in shake flasks and in a bioreactor using various compositions of media containing glucose or starch as the main carbon source. The medium composition affected both the growth characteristics and the stability of the plasmid. Superior plasmid stability was obtained in yeast minimal medium and in complex medium with glucose. Plasmid stability was substantially increased at high growth rates. Additional data were collected in the same system to investigate the kinetic characteristics of biomass and protein production, and unstructured kinetic models were used to interpret the results. At high initial glucose concentrations, where the biomass and protein production rates were similar, the kinetic models displayed good fits associated with high degrees of correlation. © 2000 Society of Chemical Industry     

高渗漏型AspAT基因工程菌诱导表达的研究

利用异丙基-β-d-硫代半乳糖苷(IPTG)诱导有高渗漏表达现象的天冬氨酸转氨酶(AspAT)工程菌,研究是否可以在这一类基因工程菌中提高酶的产量及活性。在观察IPTG诱导后菌体生长的同时,观察不同的时间及条件下酶活性的变化,发现插入含有天冬氨酸转氨酶基因(AspC,1.2kb)的pUC18质粒的BL21(DE3)在较高渗漏表达的情况下,用IPTG 37℃诱导培养8 h后酶表达开始升高,12 h后趋于稳定表达;同时发现用0.1 mmol/L IPTG诱导后酶的活性提高最多,比未诱导前提高一倍以上。研究认为如果培养条件恰当这类菌应该可以用IPTG诱导高效表达目的酶蛋白。     

Comparison and optimization of poly(3-hydroxybutyrate) recovery fromAlcaligenes eutrophus and recombinantEscherichia coli

The recovery of poly(3-hydroxybutyrate) [PHB] fromAlcaligenes eutrophus and a recombinantEscherichia coli strain harboring theA. eutrophus poly(3-hydroxyalkanoates) biosynthesis genes was studied. When PHB was recovered using sodium hypochlorite or sodium dodecyl sulfate (SDS), non-PHB cell materials (NPCM) of the recombinantE. coli seemed to be more easily digested than those ofA. eutrophus. Furthermore, viscosity increase caused by cell lysis during SDS treatment was negligible for the recombinantE. coli, whereas a very viscous suspension was formed forA. eutrophus. These results, together with our previous finding that PHB in the recombinantE. coli is far less susceptible to molecular degradation by sodium hypochlorite, suggested that the recombinantE. coli was more beneficial than A.eutrophus in terms of PHB recovery. In order to develop an easy and efficient recovery process, we adopted and optimized the SDS treatment since, with the hypochlorite treatment, we could not handle high biomass concentrations effectively. We could obtain a PHB of 95 % purity with 96 % recovery under the optimal condition of a biomass concentration of 5 %, a ratio of SDS to biomass of 0.6, a treatment time of 60 minutes, and a treatment temperature of 30°C.     

Factors affecting the mitotic stability of high-copy-number integration into the ribosomal DNA of Saccharomyces cerevisiae

Yeast vectors suitable for high-level expression of heterologous proteins should combine a high copy number with high mitotic stability. The pMIRY integrative vector system, based upon targeted integration into the yeast rDNA locus, developed in our laboratory satisfies these criteria. However, insertion of a (foreign) gene drastically reduced its mitotic stability of the resulting vector in comparison to its parent. In this paper we have investigated a number of possible reasons for this reduction in stability. The results demonstrate that plasmid size is an important, but not the only, determinant of mitotic stability. Stable maintenance is only observed when the complete plasmid has a size no larger than that of the rDNA unit (9·1 kb). In addition stability depends upon the nature of the rDNA fragment present in the plasmid, required for targeting its integration. On the other hand, it turned out to be irrelevant for mitotic stability whether the heterologous gene was expressed or not. These findings will be important in the design of a pMIRY vector suitable for industrial production of heterologous proteins.