Metabolic evolution of Corynebacterium glutamicum for increased production of L-ornithine.

BACKGROUND: L-ornithine is effective in the treatment of liver diseases and helps strengthen the heart. The commercial applications mean that efficient biotechnological production of L-ornithine has become increasingly necessary. Adaptive evolution strategies have been proven a feasible and efficient technique to achieve improved cellular properties without requiring metabolic or regulatory details of the strain. The evolved strains can be further optimised by metabolic engineering. Thus, metabolic evolution strategy was used for engineering Corynebacterium glutamicum to enhance L-ornithine production. RESULTS: A C. glutamicum strain was engineered by using a combination of gene deletions and adaptive evolution with 70 passages of growth-based selection. The metabolically evolved C. glutamicum strain, named ΔAPE6937R42, produced 24.1 g/L of L-ornithine in a 5-L bioreactor. The mechanism used by C. glutamicum ΔAPE6937R42 to produce L-ornithine was investigated by analysing transcriptional levels of select genes and NADPH contents. The upregulation of the transcription levels of genes involved in the upstream pathway of glutamate biosynthesis and the elevated NADPH concentration caused by the upregulation of the transcriptional level of the ppnK gene promoted L-ornithine production in C. glutamicum ΔAPE6937R42. CONCLUSIONS: The availability of NADPH plays an important role in L-ornithine production in C. glutamicum. Our results demonstrated that the combination of growth-coupled evolution with analysis of transcript abundances provides a strategy to engineer microbial strains for improving production of target compounds.

Metabolic engineering of Corynebacterium glutamicum for increasing the production of L-ornithine by increasing NADPH availability.

The experiments presented here were based on the conclusions of our previous proteomic analysis. Increasing the availability of glutamate by overexpression of the genes encoding enzymes in the L-ornithine biosynthesis pathway upstream of glutamate and disruption of speE, which encodes spermidine synthase, improved L-ornithine production by Corynebacterium glutamicum. Production of L-ornithine requires 2 moles of NADPH per mole of L-ornithine. Thus, the effect of NADPH availability on L-ornithine production was also investigated. Expression of Clostridium acetobutylicum gapC, which encodes NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, and Bacillus subtilis rocG, which encodes NAD-dependent glutamate dehydrogenase, led to an increase of L-ornithine concentration caused by greater availability of NADPH. Quantitative real-time PCR analysis demonstrates that the increased levels of NADPH resulted from the expression of the gapC or rocG gene rather than that of genes (gnd, icd, and ppnK) involved in NADPH biosynthesis. The resulting strain, C. glutamicum ΔAPRE::rocG, produced 14.84 g l⁻¹ of L-ornithine. This strategy of overexpression of gapC and rocG will be useful for improving production of target compounds using NADPH as reducing equivalent within their synthetic pathways.

Facile production of cellulose nanofibers from raw elephant grass by an aluminum chloride-enhanced acidic deep eutectic solvent.

To develop a greener and more efficient method for producing cellulose nanofibers (CNFs) from raw plants, an AlCl3-enhanced ternary deep eutectic solvent, DES2 (consisting of choline chloride, citric acid, and AlCl3·6H2O in a molar ratio of 1:0.4:0.08), was synthesized. Raw elephant grass (EG) was pretreated with DES2, followed by sodium chlorite (NaClO2) bleaching and ultrasonic disruption to extract high-performance CNFs. The DES2 and NaClO2 treatments effectively removed hemicellulose and lignin, achieving removal rates of 99.23 % and 99.62 %, respectively, while maintaining a cellulose content of 78.3 %. DES2 demonstrated easy recyclability and maintained excellent biomass pretreatment performance even after multiple cycles. Following a brief 30-min intermittent ultrasound treatment, the resulting CNFs demonstrated superior crystallinity, increased carboxyl content, and a narrower width distribution compared to CNFs obtained from AlCl3-free DES1. Optimized conditions at 110 °C yielded CNFs with 85.3 % crystallinity, 0.64 mmol/g carboxyl content, 5.15 nm width distribution, and excellent dispersion in water for at least six months. Additionally, CNFs enhanced the tensile strength of chia seed mucilage (CM) composite films, showing a significant improvement to 26.6 MPa, representing a 231.3 % increase over the control film. This study offers a promising approach for efficiently producing CNFs from raw plants.

Inhibitory Effects of 5-Aza-2'-Deoxycytidine and Trichostatin A in Combination with p53-Expressing Adenovirus on Human Laryngocarcinoma Cells.

OBJECTIVE: To investigate the effects of 5-Aza-2'-deoxycytidine (5-Aza-Cdr) and trichostatin A (TSA) combined with p53-expressing adenovirus (Ad-p53) on Hep-2 cell line in vivo and in vitro, in order to explore its possibility in biological treatment of laryngocarcinoma. METHODS: Effects of 5-Aza-Cdr and TSA in combination with Ad-p53 on Hep-2 cell line in vivo were determined by Cell Counting Kit-8 (CCK-8) assay. The effect of drug combination was calculated by Jin's formula. Effects on the cell line in vitro were investigated by establishing the nude mice model. RESULTS: 5-Aza-Cdr and TSA showed inhibitory effects on the proliferation of Hep-2 cells in dose- and time-dependent manner. Ad-p53 can inhibit the growth of Hep-2 cells in vivo and in vitro. However, the combination of epigenetic reagents (5-Aza-Cdr/TSA) and Ad-p53 was less effective than individual use of Ad-p53. 5-Aza-Cdr and Ad-p53 inhibited the growth of transplanted tumors and reduced the volume of tumors, and the tumor volume of Ad-p53 group was significantly smaller than that of the control group (P<0.05). CONCLUSION: Both epigenetic reagents (5-Aza-Cdr/TSA) and Ad-p53 can suppress cell proliferation on Hep-2 in vivo and in vitro and there may be some antagonistic mechanism between Ad-p53 and epigenetic reagents (5-Aza-Cdr/ TSA).

A method combining TA cloning and fluorescence screening for rapid acquisition of transgenic seeds.

The establishment of transgenic plants has greatly promoted the progress of plant research. However, traditional selection methods using antibiotics or herbicides may miss any positive transformants with growth defects. Additionally, screening with antibiotics/herbicides requires a huge amount of seeds, sterile work conditions and a large amount of space to germinate plants, making the selection process time- and labor-consuming. In this study, we constructed a novel stable transformation vector, plasmid of OLE1-GFP T-DNA vector (pOGT), which can shorten the steps of cloning foreign genes into expression vectors by using TA cloning. Additionally, selection of transformed seeds with fluorescence overcomes the difficulties of conventional selection with antibiotics/herbicides and simplifies the screening process for transgenic plants.

Biological mechanisms of cadmium accumulation in edible Amaranth (Amaranthus mangostanus L.) cultivars promoted by salinity: A transcriptome analysis.

Strategies to prevent cadmium (Cd) mobilization by crops under salinity conditions differs among distinct genotypes, but the biological mechanisms of Cd accumulation in different genotype crops promoted by salinity have remained scarce. In this study, we investigated the biological mechanisms of Cd accumulation in two quite different amaranth cultivars of low-Cd accumulator Quanhong (QH) and high-Cd accumulator Liuye (LY) in response to salt stress. Transcriptomes analysis was carried out on leaves and roots tissues of LY and QH grown with exchangeable Cd 0.27 mg kg-1 and salinity 3.0 g kg-1 treatment or control conditions, respectively. A total of 3224 differentially expressed genes (DEGs) in LY (1119 in roots, 2105 in leaves) and 848 in QH (207 in roots, 641 in leaves) were identified. Almost in each fold change category (2-25, 25-210, >210), the numbers of DEGs induced by salinity in LY treatments were much more than those in QH treatments, indicating that LY is more salt sensitive. Gene ontology (GO) analysis revealed that salinity stress promoted soil acidification and Cd mobilization in LY treatments through the enhancive expression of genes related to adenine metabolism (84-fold enrichment) and proton pumping ATPase (50-fold enrichment) in roots, and carbohydrate hydrolysis (2.5-fold enrichment) in leaves compared with that of whole genome, respectively. The genes expression of organic acid transporter (ALMT) was promoted by 2.71- to 3.94-fold in roots, facilitating the secretion of organic acids. Salt stress also inhibited the expression of key enzymes related to cell wall biosynthesis in roots, reducing the physical barriers for Cd uptake. All these processes altered in LY were more substantially compared with that of QH, suggesting that salt sensitive cultivars might accumulate more Cd and pose a higher health risk.

Polyploid origins in Gynostemma pentaphyllum (Cucurbitaceae) inferred from multiple gene sequences.

The genus Gynostemma (Cucurbitaceae) constitutes a polyploid group of perennial creeping herbs, in whose evolution polyploidization is a key component. With the largest variety of cytotypes (2n=22, 44, 66 and 88) in Gynostemma, G. pentaphyllum is also the most widespread species in this genus. In the present study, we inferred the origins of polyploids in G. pentaphyllum using sequences of the plastid intergenic spacers (trnL-trnF, psbB-psbF and rpl20-rps12) and cloned DNA sequences from two nuclear regions (RPB2 and nrDNA ITS). Phylogenetic analyses of the separate and the combined nuclear gene datasets all supported autoploid origins of polyploids in G. pentaphyllum. Three polyploid populations were more closely related, indicating that significant genetic differentiation may have occurred between diploids and polyploids. We concluded that polyploidization might be an important evolutionary mechanism in the diversification of G. pentaphyllum. On the other hand, no chloroplast DNA (cpDNA) variation was detected in ingroups except the octoploid DL 8x, which possessed a different cpDNA haplotype from the other populations of G. pentaphyllum. This can be explained by limited sample sizes, possible extinction of its diploid progenitors and/or the occurrence of chloroplast transfer through hybridization with other Gynostemma species. However, the distribution of cytotypes in G. pentaphyllum was not as typical as many other autopolyploid complexes. Polyploidization failed to contribute significantly to the expansion of its geographic range. The geographic distribution of diploids and polyploids in G. pentaphyllum may be associated with the past ecological environments of different areas, especially during the glacial period.

Overexpression of RASAL1 Indicates Poor Prognosis and Promotes Invasion of Ovarian Cancer.

RAS protein activator like-1 (RASAL1) exists in numerous human tissues and has been commonly demonstrated to act as a tumor suppressor in several cancers. This study aimed to identify the functional characteristics of RASAL1 in ovarian adenocarcinoma and a potential mechanism of action. We analyzed RASAL1 gene expression in ovarian adenocarcinoma samples and normal samples gained from the GEO and Oncomine databases respectively. Then the relationship between RASAL1 expression and overall survival (OS) was assessed using the Kaplan-Meier method. Furthermore, the biological effect of RASAL1 in ovarian adenocarcinoma cell lines was assessed by Quantitative real time-PCR (qRT-PCR), Cell Counting Kit-8 (CCK-8), western blot, wound healing and transwell assay. The statistical analysis showed patients with higher RASAL1 expression correlated with worse OS. The in vitro assays suggested knockdown of RASAL1 could inhibit cell proliferation, cell invasion and migration of ovarian adenocarcinoma. Moreover, the key proteins in the mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) signaling pathway were also decreased in ovarian adenocarcinoma cells with RASAL1 silencing. These findings provide promising evidence that RASAL1 may be not only a powerful biomarker but also an effective therapeutic target of ovarian adenocarcinoma.

[Identification of Gynostemma from their adulterant Cayratia japonica by PCR-RFLP].

OBJECTIVE: To establish a convenient and effective method for the identification of Gynostemma and Cayratia japonica. METHOD: Eight species, including Gynostemm pentaphyllum, G. pentagynum, G. cardiospermum, G. longipe, G. yixingense, G. laxiflorum, G. guangxiense and C. japonica were investigated through PCR - RFLP of six chloroplast DNA fragments. The six gene fragments were digested by six restriction endonuclease respectively, including Taq I, Hpa II, EcoR I, Rsa I, Hha I, Hind III. RESULT: Seven species of Gynostemma and their adulterant could be identified by trnK1f-trnK2r and Rsa. CONCLUSION: PCR - RFLP provides a quick, reliable molecular marker technique for identification of Cynostemma and their adulterant Cayratia japonica.

[Optimization for phosphorous removal in thickening and dewatering sludge water by polyaluminum chloride].

Based on the comparison of phosphorous removal in sludge water and its supernatant by polyaluminum chloride (PAC), separate and combined effects of Al/P mole ratio, pH and mixing speed (MS) on phosphorus removal by PAC for the supernatant of thickening and dewatering sludge water were analyzed by the response surface methodology (RSM), and kinetics of phosphorous removal by PAC was also investigated. The results showed that direct addition of PAC into sludge water deteriorated its settling characteristics, and suspended solids in the sludge water could decrease the phosphorus removal efficiency. The RSM analysis results demonstrated that the effect of individual operation parameter on phosphorus removal was followed as the order of Al/P > pH > MS, and the optimal process parameters with phosphorus removal efficiency of 97.8% were Al/P = 2.49, pH = 8.3 and MS 398 r x min(-1), respectively. The verification experiment showed that the RSM model was valid and effective. Kinetic analysis illustrated that phosphorus removal by PAC was divided into two stages, a chemical precipitation and rapid adsorption stage, followed by a chemical precipitation stage that conformed to the second-order kinetics.

[Modeling and dynamic simulation of the multimode anaerobic/anoxic/aerobic wastewater treatment process].

Mathematical modeling is a useful tool for professional education, process development, design evaluation, operational optimization and automatic control of the wastewater treatment system, and has been extensively applied in numerous full-scale wastewater treatment plants. The ASM2d model was calibrated by the process data, and used to simulate 15 operational test runs of the multimode anaerobic/anoxic/aerobic (AAO) process. After calibration, the model was capable of simulating the sludge concentrations and effluent data in 15 test runs of the multimode AAO system. The dynamic simulation results showed an overall good agreement between the measured and simulated data, for both effluent data and sludge concentrations, with a good reproduction of dynamic processes in AO test runs.

[Inhibition of human laryngeal carcinoma growth by gene therapy and epigenetic therapy].

OBJECTIVE: To observe the effects of gene therapy and epigenetic therapy on the tumor growth of laryngeal carcinoma and the underlying mechanisms. METHODS: The animal model of human laryngeal carcinoma was established by the subcutaneous inoculation of Hep-2 cells at the right armpit of BALB/c nu/nu mice. The tumor-bearing mice were randomized into 4 groups, p53 therapy group(rAd-p53), epigenetic therapy group(5-aza-dC), combination therapy group (rAd-p53+5-aza-dC) and control group. The gene and protein expressions of molecular markers p53 and E-cadherin were detected by FQ-PCR and immunohistochemistry. RESULTS: By the day 20 of the treatments, the mean tumor volumes were(106.09 ± 24.40)mm(3) in p53 therapy group, (166.55 ± 40.11) mm(3) in epigenetic therapy group, (126.11 ± 22.49) mm(3) in combination therapy group,and (252.83 ± 54.09) mm(3) in control group. Both gene therapy (F = 37.30, P < 0.05) and epigenetic therapy (F = 4.79, P < 0.05) inhibited the growth of xenografted tumors, with an interaction effect (F = 22.01, P < 0.05) between the two groups. The integral optical density value of p53 protein expression of p53 therapy group (628.07 ± 95.16) was significantly higher than that of combination therapy group (494.76 ± 100.22), (t = 8.72, P < 0.05). The integral optical density values of E-cadherin protein expression were 558.89 ± 97.58 in p53 therapy group, 380.41 ± 90.60 in epigenetic therapy group, 494.76 ± 102.88 in combination therapy group,and 162.60 ± 40.38 in control group respectively, indicating the enhancements of E-cadherin protein expression by gene therapy (F = 45.24, P < 0.05) or epigenetic therapy(F = 5.73, P < 0.05)and the existence of interaction effect (F = 21.82, P < 0.05) between gene therapy and epigenetic therapy. The expression levels of p53 gene were 4.43 ± 0.12 in p53 therapy group, 1.06 ± 0.11 in epigenetic therapy group, 3.51 ± 0.10 in combination therapy group,and 1.09 ± 0.11 in control group, respectively, showing an interaction effect between gene therapy and epigenetic therapy (F = 298.11, P < 0.05). The expression levels of E-cadherin gene were 4.50 ± 0.34 in p53 therapy group, 2.02 ± 0.16 in epigenetic therapy group, 2.99 ± 0.12 in combination therapy group, and 1.00 ± 0.11 in control group, respectively. The expression of E-cadherin gene was enhanced by gene therapy (F = 329.12, P < 0.05)or epigenetic therapy(F = 88.57, P < 0.05), with an interaction effect between the two therapies (F = 122.17, P < 0.05). CONCLUSIONS: Xenografted tumors of human laryngeal carcinoma cells are inhibited by gene therapy, the epigenetic therapy and the combination therapy. The gene therapy was significantly better than the epigenetic therapy or the combination therapy. There might be antagonistic effect between p53 and 5-aza-dC.

Polyethylene glycol-directed SnO2 nanowires for enhanced gas-sensing properties.

SnO(2) nanowires with lengths in the tens of micrometres range have been synthesized on a large scale via a facile polyethylene glycol-directed method at ambient temperature followed by a suitable thermal treatment of the precursor nanowires. The morphology of the precursor of the SnO(2) nanowires is tunable by changing the concentration of either SnCl(2) or polyethylene glycol. After calcination, the resulting SnO(2) nanowires retain a similar shape to the precursor, but with hierarchical architecture, which can be considered as one-dimensional nanowires assembled by interconnected SnO(2) nanoparticles with a high surface-to-volume ratio. The SnO(2) nanowires are investigated with XRD, SEM, TEM, and gas sensing tests for detecting CO and H(2). It is found that the present SnO(2) nanowires exhibit a remarkable sensitivity and low detection limit (10 ppm for H(2)), as well as good reproducibility and short response/recovery times, which benefit from the unique hierarchical structure with a high surface-to-volume ratio and the 3D network formed by the nanowires.

LiFePO4 Nanoparticles Embedded in a Nanoporous Carbon Matrix: Superior Cathode Material for Electrochemical Energy-Storage Devices.

An optimized nanostructure design for high-power, high-energy lithium-ion batteries and supercapacitors is realized by fabricating a nanocomposite with highly dispersed nanoparticles of active materials in a nanoporous carbon matrix. A nano-LiFePO4 /nanoporous carbon matrix nanocomposite forms a bridge between a supercapacitor and a battery electrode and offers a reasonable compromise between rate and capacity.