Expression of a high sweetness and heat-resistant mutant of sweet-tasting protein,monellin, in <Emphasis Type="Italic">Pichia pastoris</Emphasis> with a constitutive GAPDH promoter and modified <Emphasis Type="Italic">N</Emphasis>-terminus

Objectives

To improve the stability and sweetness of the sweet-tasting protein, monellin, by using site-directed mutagenesis and a Pichia pastoris expression system with a GAPDH constitutive promoter.

Results

Both wild-type and E2 N mutant of single-chain monellin gene were cloned into the PGAPZαA vector and expressed in Pichia pastoris. The majority of the secreted recombinant protein, at 0.15 g/l supernatant, was monellin. This was purified by Sephadex G50 chromatography. The sweetness threshold of wild-type and E2 N were 30 μg/ml and 20 μg/ml, respectively. Compared with the proteins expressed in Escherichia coli, the thermostability of both proteins was improved. The N-terminal sequence is determinative for the sweetness of the proteins expressed in yeast strains.

Conclusions

Site-directed mutagenesis, modification of the N-terminus of monellin, and without the need of methanol induction in P. pastoris expression system, indicate the possibility for large-scale production of this sweet-tasting protein.

     

Rapid and efficient degradation of bisphenol A by chloroperoxidase from <Emphasis Type="Italic">Caldariomyces fumago</Emphasis>: product analysis and ecotoxicity evaluation of the degraded solution

Objectives

To degrade enzymatically bisphenol A (BPA) that causes serious environmental concerns and is difficult to be degraded by chemical or physical methods.

Results

BPA (150 mg l^?1) was completely degraded by chloroperoxidase (CPO)/H₂O₂ within 7 min at room temperature, atmospheric pressure with the enzyme at 6 μg CPO ml^?1. The degradation products were identified by HPLC–MS, which suggested involvement of multiple steps. Enzymatic treatment followed by existing bioremediation technologies (activated sludge) enhanced removal of COD from 9 to 54 %. Using an ecotoxicity evaluation with Chlorella pyrenoidosa, the degradation products had a lower toxicity than BPA.

Conclusion

BPA can be degraded rapidly and efficiently under mild conditions with chloroperoxidase at 6 μg ml^?1. The degradation products had a lower toxicity than BPA.

     

PCI-24781 can improve in vitro and in vivo developmental capacity of pig somatic cell nuclear transfer embryos

Objective

To examine the effect of PCI-24781 (abexinostat) on the blastocyst formation rate in pig somatic cell nuclear transferred (SCNT) embryos and acetylation levels of the histone H3 lysine 9 and histone H4 lysine 12.

Results

Treatment with 0.5 nM PCI-24781 for 6 h significantly improved the development of cloned embryos, in comparison to the control group (25.3 vs. 10.5 %, P < 0.05). Furthermore, PCI-24781 treatment led to elevated acetylation of H3K9 and H4K12. TUNEL assay and Hoechst 33342 staining revealed that the percentage of apoptotic cells in blastocysts was significantly lower in PCI-24781-treated SCNT embryos than in untreated embryos. Also, PCI-24781-treated embryos were transferred into three surrogate sows, one of whom became pregnant and two fetuses developed.

Conclusion

PCI-24781 improves nuclear reprogramming and the developmental potential of pig SCNT embryos.

     

Deletion of <Emphasis Type="Italic">ldh</Emphasis>A and <Emphasis Type="Italic">ald</Emphasis>H genes in <Emphasis Type="Italic">Klebsiella</Emphasis><Emphasis Type="Italic">pneumoniae</Emphasis> to enhance 1,3-propanediol production

Objectives

To improve 1,3-propanediol (1,3-PD) production and reduce byproduct concentration during the fermentation of Klebsiella pneumonia.

Results

Klebsiella. pneumonia 2-1ΔldhA, K. pneumonia 2-1ΔaldH and K. pneumonia 2-1ΔldhAΔaldH mutant strains were obtained through deletion of the ldhA gene encoding lactate dehydrogenase required for lactate synthesis and the aldH gene encoding acetaldehyde dehydrogenase involved in the synthesis of ethanol. After fed-batch fermentation, the production of 1,3-PD from glycerol was enhanced and the concentrations of byproducts were reduced compared with the original strain K. pneumonia 2-1. The maximum yields of 1,3-PD were 85.7, 82.5 and 87.5 g/l in the respective mutant strains.

Conclusion

Deletion of either aldH or ldhA promoted 1,3-PD production in K. pneumonia.

     

Nanoformulation of curcumin protects HUVEC endothelial cells against ionizing radiation and suppresses their adhesion to monocytes: potential in prevention of radiation-induced atherosclerosis

Objectives

To investigated the potential of a novel dendrosomal nanoformulation of curcumin (DNC) in blocking radiation-induced changes in irradiated human umbilical vein endothelial cells (HUVECs), and their adhesion to human THP-1 monocytoid cells.

Results

Co⁶⁰ gamma rays reduced viability, raised the expression of adhesion molecules, ICAM-1, VCAM-1 and E-selectin (mRNA and protein), augmented the adhesion of THP-1 cells to HUVECs, activated NF-κB binding, increased the release of pro-inflammatory cytokines (IL-6, IL-8 and MCP-1) and induced oxidative damage (reduced glutathione declined, while 8-OHdG and TBARS increased). 5 µM DNC significantly inhibited these radiation-induced changes, activated the Nrf-2 pathway, and effectively suppressed THP-1 adhesion to HUVECs, implicating p38 MAPK signaling.

Conclusion

DNC treatment is a potential preventive method against inflammation and vascular damage from ionizing radiation.

     

Generation of ΔF508-CFTR T84 cell lines by CRISPR/Cas9-mediated genome editing

Objectives: To provide a simple method to make a stable ΔF508-CFTR-expressing T84 cell line that can be used as an efficient screening model system for ΔF508-CFTR rescue. Results: CFTR knockout cell lines were generated by Cas9 with a single-guide RNA (sgRNA) targeting exon 1 of the CFTR genome, which produced indels that abolished CFTR protein expressions. Next, stable ΔF508-CFTR expression was achieved by genome integration of ΔF508-CFTR via the lentivirus infection system. Finally, we showed functional rescue of ΔF508-CFTR not only by growing the cells at a low temperature, but also incubating with VX-809, a ΔF508-CFTR corrector, in the established T84 cells expressing ΔF508-CFTR. Conclusions: This cell system provides an appropriate screening platform for rescue of ΔF508-CFTR, especially related to protein folding, escaped from endoplasmic-reticulum-associated protein degradation, and membrane transport.     

High-level expression and biochemical characterization of a novel cold-active lipase from <Emphasis Type="Italic">Rhizomucor endophyticus</Emphasis>

Objectives

To identify novel cold-active lipases from fungal sources and improve their production by heterologous expression in Pichia pastoris.

Results

A novel cold-active lipase gene (ReLipB) from Rhizomucor endophyticus was cloned. ReLipB was expressed at a high level in Pichia pastoris using high cell-density fermentation in a 5-l fermentor with the highest lipase activity of 1395 U/ml. The recombinant lipase (RelipB) was purified and biochemically characterized. ReLipB was most active at pH 7.5 and 25 °C. It was stable from pH 4.5–9.0. It exhibited broad substrate specificity towards p-nitrophenyl (pNP) esters (C₂–C₁₆) and triacylglycerols (C₂–C₁₂), showing the highest specific activities towards pNP laurate (231 U/mg) and tricaprylin (1840 U/mg), respectively. In addition, the enzyme displayed excellent stability with high concentrations of organic solvents including cyclohexane, n-hexane, n-heptane, isooctane and petroleum ester and surfactants.

Conclusions

A novel cold-active lipase from Rhizomucor endophyticus was identified, expressed at a high level and biochemically characterized. The high yield and unique enzymatic properties make this lipase of some potential for industrial applications.

     

Production of δ-decalactone from linoleic acid via 13-hydroxy-9(Z)-octadecenoic acid intermediate by one-pot reaction using linoleate 13-hydratase and whole <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> cells

Objective

To produce δ-decalactone from linoleic acid by one-pot reaction using linoleate 13-hydratase with supplementation with whole Yarrowia lipolytica cells.

Results

Whole Y. lipolytica cells at 25 g l^?1 produced1.9 g l^?1 δ-decalactone from 7.5 g 13-hydroxy-9(Z)-octadecenoic acid l^?1 at pH 7.5 and 30 °C for 21 h. Linoleate 13-hydratase from Lactobacillus acidophilus at 3.5 g l^?1 with supplementation with 25 g Y. lipolytica cells l^?1 in one pot at 3 h produced 1.9 g l^?1 δ-decalactone from 10 g linoleic acid l^?1 via 13-hydroxy-9(Z)-octadecenoic acid intermediate at pH 7.5 and 30°C after 18 h, with a molar conversion yield of 31 % and productivity of 106 mg l^?1 h^?1.

Conclusion

To the best of our knowledge, this is the first production of δ-decalactone using unsaturated fatty acid.

     

Development of a sensitive molecular detection assay for mango malformation disease caused by <Emphasis Type="Italic">Fusarium mangiferae</Emphasis>

Objectives

To develop a sensitive and specific molecular assay for detection of mango malformation disease (MMD), which is caused primarily by Fusarium mangiferae.

Results

We screened 100 ISSR primers and identified one (UBC888) that directed the stable amplification of a specific gene fragment of 479 bp (GenBank accession number KJ526382). Based on the DNA sequence of this fragment, a pair of SCAR primers (W342 and W1772) were designed to amplify another gene fragment of 1376 bp (GenBank accession number KJ526383), demonstrating the successful conversion of an ISSR marker to a SCAR marker. An effective and simple detection assay for MMD was established based on this pair of PCR primers, with a high level of specificity and sensitivity to the DNA of F. mangiferae and other species of Fusarium both in vitro and in vivo. It can detect as little as 10 pg fungal DNA from the DNA of mango’s tissues.

Conclusions

Our assay provides a practical method for the early diagnosis so that proper prevention of the mango malformation disease can be developed.