Showing papers in "Biotechnology Letters in 2013"
TL;DR: Neither the upstream nor downstream bioprocessing unit operations had a discernible effect on cell quality with the harvested cells retaining their immunophenotypic markers, key morphological features and differentiation capacity.
Abstract: For the first time, fully functional human mesenchymal stem cells (hMSCs) have been cultured at the litre-scale on microcarriers in a stirred-tank 5 l bioreactor, (2.5 l working volume) and were harvested via a potentially scalable detachment protocol that allowed for the successful detachment of hMSCs from the cell-microcarrier suspension. Over 12 days, the dissolved O2 concentration was >45 % of saturation and the pH between 7.2 and 6.7 giving a maximum cell density in the 5 l bioreactor of 1.7 × 105 cells/ml; this represents >sixfold expansion of the hMSCs, equivalent to that achievable from 65 fully-confluent T-175 flasks. During this time, the average specific O2 uptake of the cells in the 5 l bioreactor was 8.1 fmol/cell h and, in all cases, the 5 l bioreactors outperformed the equivalent 100 ml spinner-flasks run in parallel with respect to cell yields and growth rates. In addition, yield coefficients, specific growth rates and doubling times were calculated for all systems. Neither the upstream nor downstream bioprocessing unit operations had a discernible effect on cell quality with the harvested cells retaining their immunophenotypic markers, key morphological features and differentiation capacity.
178 citations
TL;DR: Eugenol, the major constituent of clove extract, exhibited QS inhibitory activity and was identified as a novel QS inhibitor in herbal extracts by effective screening systems and evaluated its biological activity.
Abstract: In bacteria, quorum sensing (QS) is a process of chemical communication involving the production, release, and subsequent detection of signaling molecules. QS regulates the production of key virulence factors in pathogens. During the screening of herbal extracts, clove extract was found to inhibit QS-controlled gene expression in Pseudomonas aeruginosa QSIS-lasI and Chromobacterium violaceum CV026 biosensors. Using a bioautographic TLC assay, preparative TLC, and HPLC analysis, eugenol, the major constituent of clove extract, exhibited QS inhibitory activity. Eugenol at sub-inhibitory concentrations inhibited the production of virulence factors, including violacein, elastase, pyocyanin, and biofilm formation. Using two Escherichia coli biosensors, MG4/pKDT17 and pEAL08-2, we confirmed that eugenol inhibited the las and pqs QS systems. Our data identified eugenol as a novel QS inhibitor. The purpose of this study was to track the quorum sensing inhibitor (QSI) in herbal extracts by effective screening systems and evaluate its biological activity. The QSIs from herbal extracts are potential agents for the treatment of bacterial infections.
174 citations
TL;DR: The synthesis of copper nanostructured nanoparticles by bacteria, fungi, and plant extracts is discussed, showing that biogenic synthesis is an economically feasible, simple and non-polluting process.
Abstract: Copper nanoparticles have been the focus of intensive study due to their potential applications in diverse fields including biomedicine, electronics, and optics. Copper-based nanostructured materials have been used in conductive films, lubrification, nanofluids, catalysis, and also as potent antimicrobial agent. The biogenic synthesis of metallic nanostructured nanoparticles is considered to be a green and eco-friendly technology since neither harmful chemicals nor high temperatures are involved in the process. The present review discusses the synthesis of copper nanostructured nanoparticles by bacteria, fungi, and plant extracts, showing that biogenic synthesis is an economically feasible, simple and non-polluting process. Applications for biogenic copper nanoparticles are also discussed.
160 citations
TL;DR: The mechanisms governing acclimation to high CO2 comprise the subject of this review and are discussed in view of the use of CO2 enrichment to increase the productivity of microalgal cultures, as well as the practice of carbon capture from flue gases.
Abstract: Recent developments in the field of microalgal biotechnology, including CO2 biomitigation and the discovery of new species of microalgae that are tolerant to extremely high CO2 levels (40–100 vol%), have renewed interest in the physiological effects and mechanisms of high-CO2 tolerance in photoautotrophs. Photosynthetic apparatus state transitions that increase ATP generation, upregulation of H+-ATPases pumping protons out of the cell, rapid shutdown of CO2-concentrating mechanisms, and adjustment of membranes’ fatty acid composition are currently believed to be the key mechanisms governing cellular pH homeostasis and hence microalgae’s tolerance to high CO2 levels, which is especially characteristic of extremophile and symbiotic species. The mechanisms governing acclimation to high CO2 comprise the subject of this review and are discussed in view of the use of CO2 enrichment to increase the productivity of microalgal cultures, as well as the practice of carbon capture from flue gases.
128 citations
TL;DR: To improve the frequency of HR, the KU70 and KU80 genes responsible for double strand break repair in the NHEJ pathway in Y. lipolytica were identified and disrupted and the number of Ura+ transformants was reduced.
Abstract: In Yarrowia lipolytica, targeted gene replacement occurs only with long length (1 kb) homologous flanking fragments, as this yeast preferentially uses the non-homologous end-joining mechanism (NHEJ) for DNA repair over homologous recombination (HR). To improve the frequency of HR, we identified and disrupted the KU70 and KU80 genes responsible for double strand break repair in the NHEJ pathway in Y. lipolytica. In ku70∆ HR of URA3 marker at the ADE2 locus occurred with 43 % frequency with as little as 50 bp long flanking regions. The number of Ura+ transformants was reduced to 1 % of the Po1d (ura3-302) wild type-like strain level, regardless of the flanking fragment length. On the contrary, even though HR was not improved in ku80∆, Ura+ transformants was 60 % lower compared to the wild type.
127 citations
TL;DR: Three parameters were evaluated after interaction between four Trichoderma species and plant-pathogenic fungi: Fusarium solani, Rhizoctonia solani and Sclerotinia sclerotiorum.
Abstract: Trichoderma spp. are used for biocontrol of several plant pathogens. However, their efficient interaction with the host needs to be accompanied by production of secondary metabolites and cell wall-degrading enzymes. Three parameters were evaluated after interaction between four Trichoderma species and plant-pathogenic fungi: Fusarium solani, Rhizoctonia solani and Sclerotinia sclerotiorum. Trichoderma harzianum and T. asperellum were the most effective antagonists against the pathogens. Most of the Trichoderma species produced toxic volatile metabolites, having significant effects on growth and development of the plant pathogens. When these species were grown in liquid cultures with cell walls from these plant pathogens, they produced and secreted β-1,3-glucanase, NAGAse, chitinase, acid phosphatase, acid proteases and alginate lyase.
117 citations
TL;DR: A novel halophilic strain that could carry out heterotrophic nitrification and aerobic denitrification was isolated and named as Halomonas campisalis ha3, and grew well in the medium containing up to 20 % (w/v) NaCl.
Abstract: A novel halophilic strain that could carry out heterotrophic nitrification and aerobic denitrification was isolated and named as Halomonas campisalis ha3. It removed inorganic nitrogen compounds (e.g. NO3
−, NO2
− and NH4
+) simultaneously, and grew well in the medium containing up to 20 % (w/v) NaCl. PCR revealed four genes in the genome of ha3 related to aerobic denitrification: napA, nirS, norB and nosZ. The optimal conditions for aerobic denitrification were pH 9.0, at 37 °C, with 4 % (w/v) NaCl and sodium succinate as carbon source. The nitrogen removal rate was 87.5 mg NO3
−–N l−1 h−1. Therefore, this strain is a potential aerobic denitrifier for the treatment of saline wastewater.
112 citations
TL;DR: Crops such as tomato, potato, maize, groundnut, mustard, finger millet, cotton, lychee, banana, grape, wheat and rice have been successfully engineered for fungal resistance either with chitinase alone or in combination with other PR proteins.
Abstract: Crop plants have evolved an array of mechanisms to counter biotic and abiotic stresses. Many pathogenesis-related proteins are expressed by plants during the attack of pathogens. Advances in recombinant DNA technology and understanding of plant-microbe interactions at the molecular level have paved the way for isolation and characterization of genes encoding such proteins, including chitinases. Chitinases are included in families 18 and 19 of glycosyl hydrolases (according to www.cazy.org ) and they are further categorized into seven major classes based on their aminoacid sequence homology, three-dimensional structures, and hydrolytic mechanisms of catalytic reactions. Although chitin is not a component of plant cell walls, plant chitinases are involved in development and non-specific stress responses. Also, chitinase genes sourced from plants have been successfully over-expressed in crop plants to combat fungal pathogens. Crops such as tomato, potato, maize, groundnut, mustard, finger millet, cotton, lychee, banana, grape, wheat and rice have been successfully engineered for fungal resistance either with chitinase alone or in combination with other PR proteins.
90 citations
TL;DR: In this article, the malic enzyme gene (YALI0E18634g) was cloned from Y. lipolytica and expressed in Escherichia coli and purified the recombinant protein (YlME).
Abstract: Malic enzyme (EC 1.1.1.40) converts l-malate to pyruvate and CO2 providing NADPH for metabolism especially for lipid biosynthesis in oleaginous microorganisms. However, its role in the oleaginous yeast, Yarrowia lipolytica, is unclear. We have cloned the malic enzyme gene (YALI0E18634g) from Y. lipolytica into pET28a, expressed it in Escherichia coli and purified the recombinant protein (YlME). YlME used NAD+ as the primary cofactor. Km values for NAD+ and NADP+ were 0.63 and 3.9 mM, respectively. Citrate, isocitrate and α-ketoglutaric acid (>5 mM) were inhibitory while succinate (5–15 mM) increased NADP+- but not NAD+-dependent activity. To determine if fatty acid biosynthesis could be increased in Y. lipolytica by providing additional NADPH from an NADP+-dependent malic enzyme, the malic enzyme gene (mce2) from an oleaginous fungus, Mortierella alpina, was expressed in Y. lipolytica. No significant changes occurred in lipid content or fatty acid profiles suggesting that malic enzyme is not the main source of NADPH for lipid accumulation in Y. lipolytica.
88 citations
TL;DR: The current review summarizes the state-of-the-art of the application of Nitritation–anammox systems and discusses the possibilities of utilizing these recent results for wastewater treatment.
Abstract: Anaerobic ammonium-oxidizing (anammox) bacteria convert ammonium to N2 with nitrite as the terminal electron acceptor in the absence of O2. Nitritation-anammox bioreactors provide a cost-effective and environment-friendly alternative to conventional nitrification/denitrification nitrogen removal systems. Currently, this process is only applied for ammonium removal from wastewater with high ammonium load and temperature. Nevertheless, recent results obtained with laboratory-scale bioreactors suggest new possible routes of application of the Nitritation-anammox technology including (1) municipal wastewater treatment, removal of (2) methane in combination with nitrite-reducing methane-oxidizing bacteria, (3) nitrate coupled to organic acid oxidation and (4) nitrogen oxides. The current review summarizes the state-of-the-art of the application of Nitritation-anammox systems and discusses the possibilities of utilizing these recent results for wastewater treatment.
86 citations
TL;DR: The contractility of the system was recovered quickly after the removal of the chemical stimulation, which indicated the flexibility of this system and the recycling potential, and the integrated Bio-MEMS device responded to thechemical stimulation spontaneously which demonstrated the potential as a functional biosensor.
Abstract: Muscle-powered, biological, microelectro-mechanical system is promising for actuator and biosensor applications. Functional conjugation between the cells, tissues, and biomolecules to the microdevice is crucial for this application. Bioprinting as an enabling technology possesses the advantages of high throughput, digital control, and highly accurate delivery of various biological factors to the desired locations for numerous applications such as 3D tissue fabrication. We have now evaluated the feasibility of the precise placement of mouse myoblasts onto micro-sized cantilevers. The evenly aligned printed cells fused with each other and formed mature myotubes after only 4 days. In contrast, it took more than 14 days for randomly deposited cells to do so. The printed myotubes were functional and responded to the electrical stimulation synchronously. Furthermore, the integrated Bio-MEMS device responded to the chemical stimulation spontaneously which demonstrated the potential as a functional biosensor. The contractility of the system was recovered quickly after the removal of the chemical stimulation, which indicated the flexibility of this system and the recycling potential.
TL;DR: The cost of lipases and the relatively slower reaction rate remain the major obstacles for enzymatic production of biodiesel, but this problem can be solved by immobilizing the enzyme on a suitable matrix or support, which increases the chances of re-usability.
Abstract: Biodiesel can be produced by transesterification of vegetable or waste oil catalysed by lipases. Biodiesel is an alternative energy source to conventional fuel. It combines environmental friendliness with biodegradability, low toxicity and renewability. Biodiesel transesterification reactions can be broadly classified into two categories: chemical and enzymatic. The production of biodiesel using the enzymatic route eliminates the reactions catalysed under acid or alkali conditions by yielding product of very high purity. The modification of lipases can improve their stability, activity and tolerance to alcohol. The cost of lipases and the relatively slower reaction rate remain the major obstacles for enzymatic production of biodiesel. However, this problem can be solved by immobilizing the enzyme on a suitable matrix or support, which increases the chances of re-usability. The main factors affecting biodiesel production are composition of fatty acids, catalyst, solvents, molar ratio of alcohol and oil, temperature, water content, type of alcohol and reactor configuration. Optimization of these parameters is necessary to reduce the cost of biodiesel production.
TL;DR: This review focuses on research efforts to utilize glycerol as a carbon source for the production of a variety of fuels and chemicals by both native and metabolically engineered microorganisms.
Abstract: Glycerol has attracted the attention of scientific and industrial communities due to its generation in bulk quantities as a byproduct of biofuel industries. With the rapid growth of these industries in recent years, glycerol is frequently treated as a very low-value byproduct or even a waste product with a disposal cost associated to it. Glycerol is not only abundant and inexpensive but also can generate more reducing equivalents than glucose or xylose. This unique characteristic of glycerol offers a tremendous opportunity for its biological conversion to valuable products at higher yield. This review focuses on research efforts to utilize glycerol as a carbon source for the production of a variety of fuels and chemicals by both native and metabolically engineered microorganisms.
TL;DR: The potential for converting inulin into a variety of high value-added biorefinery products, including biofuels and biochemicals, are reviewed and the feasibility of regarding JA as a model species of an inulin-rich crop is considered.
Abstract: The polysaccharides in Jerusalem artichoke (JA) carry a substantial amount of energy that can be partly accessed through bioconversion into storable fuels. We review the potential for converting inulin into a variety of high value-added biorefinery products, including biofuels and biochemicals, and consider the feasibility of regarding JA as a model species of an inulin-rich crop. We discuss feedstock pretreatment, microorganisms used during fermentation, biorefinery products derived from JA, and how to enhance the economic competitiveness of JA as an energy crop.
TL;DR: The applications of PCR as well as the definition and characteristics for PCR primer design, along with characteristics of population-based methods used in primer design are discussed in detail.
Abstract: The design of primers has a major impact on the success of PCR in relation to the specificity and yield of the amplified product. Here, we introduce the applications of PCR as well as the definition and characteristics for PCR primer design. Recent primer design tools based on Primer3, along with several computational intelligence-based primer design methods which have been applied in primer design, are also reviewed. In addition, characteristics of population-based methods used in primer design are discussed in detail.
TL;DR: LIPUS treatment at an appropriate intensity can, therefore, be an efficient and cost-effective method to enhance cell viability, proliferation and neural differentiation of iPSCs–NCSCs in vitro for peripheral nerve tissue engineering.
Abstract: Low-intensity pulsed ultrasound (LIPUS) acting on induced pluripotent stem cells-derived neural crest stem cells (iPSCs-NCSCs) is considered a promising therapy to improve the efficacy of injured peripheral nerve regeneration. Effects of LIPUS on cell viability, proliferation and neural differentiation of iPSCs-NCSCs were examined respectively in this study. LIPUS at 500 mW cm(-2) enhanced the viability and proliferation of iPSCs-NCSCs after 2 days and, after 4 days, up-regulated gene and protein expressions of NF-M, Tuj1, S100β and GFAP in iPSCs-NCSCs whereas after 7 days expression of only NF-M, S100β and GFAP were up-regulated. LIPUS treatment at an appropriate intensity can, therefore, be an efficient and cost-effective method to enhance cell viability, proliferation and neural differentiation of iPSCs-NCSCs in vitro for peripheral nerve tissue engineering.
TL;DR: Improved understanding of the accumulation of flavonoids in cereal grains would help to improve human nutrition through these staple foods and benefits for human nutrition are reviewed.
Abstract: Flavonoids are plant secondary metabolites that contribute to the adaptation of plants to environmental stresses, including resistance to abiotic and biotic stress. Flavonoids are also beneficial for human health and depress the progression of some chronic diseases. The biosynthesis of flavonoids, which belong to a large family of phenolic compounds, is a complex metabolic process with many pathways that produce different metabolites, controlled by key enzymes. There is limited knowledge about the composition, biosynthesis and regulation of flavonoids in cereals. Improved understanding of the accumulation of flavonoids in cereal grains would help to improve human nutrition through these staple foods. The biosynthesis of flavonoids, scope for altering the flavonoid composition in cereal crops and benefits for human nutrition are reviewed here.
TL;DR: The catalytic activity of MCRAs from four LAB stains suggests that MCRA proteins catalyse the first step in conjugated linoleic acid production, the first report of MCRA from L. rhamnosus with such catalytic function.
Abstract: The 67 kDa myosin-cross-reactive antigen (MCRA) is a member of the MCRA family of proteins present in a wide range of bacteria and was predicted to have fatty acid isomerase function. We have now characterised the catalytic activity of MCRAs from four LAB stains, including Lactobacillus rhamnosus LGG, L. plantarum ST-III, L. acidophilus NCFM and Bifidobacterium animalis subsp. lactis BB-12. MCRA genes from these strains were cloned and expressed in Escherichia coli, and the recombinant protein function was analysed with lipid profiles by GC–MS. The four MCRAs catalysed the conversion of linoleic acid and oleic acid to their respective 10-hydroxy derivatives, which suggests that MCRA proteins catalyse the first step in conjugated linoleic acid production. This is the first report of MCRA from L. rhamnosus with such catalytic function.
TL;DR: The gene coding for d-psicose 3-epimerase (DPEase) from Clostridium sp.
Abstract: The gene coding for d-psicose 3-epimerase (DPEase) from Clostridium sp. BNL1100 was cloned and expressed in Escherichia coli. The recombinant enzyme was purified by Ni-affinity chromatography. It was a metal-dependent enzyme and required Co2+ as optimum cofactor. It displayed catalytic activity maximally at pH 8.0 and 65 °C (as measured over 5 min). The optimum substrate was d-psicose, and the K
m, turnover number (k
cat), and catalytic efficiency (k
cat/K
m) for d-psicose were 227 mM, 32,185 min−1, and 141 min−1 mM−1, respectively. At pH 8.0 and 55 °C, 120 g d-psicose l−1 was produced from 500 g d-fructose l−1 after 5 h.
TL;DR: This combinatorial approach together with selection of an appropriate growth medium resulted in highest astaxanthin biomass contents reported to date for X. dendrorhous.
Abstract: Xanthophyllomyces dendrorhous (Phaffia rhodozyma) is the only yeast or fungus that synthesizes the commercially attractive carotenoid astaxanthin. For a suitable bioprocess, the wild type has to be modified for increasing biomass content. To achieve this, a two step strategy has been followed. At first, random mutagenesis was applied leading to colonies with substantially higher astaxanthin content. Then, the resulting strain was genetically engineered by targeting limiting reactions for further enhancement of astaxanthin biosynthesis. This combinatorial approach together with selection of an appropriate growth medium resulted in highest astaxanthin biomass contents reported to date for X. dendrorhous. In a fermenter culture, its maximum content was 9.7 mg/g dry weight.
TL;DR: An alginate lyase-producing bacterial strain, Vibrio sp.
Abstract: An alginate lyase-producing bacterial strain, Vibrio sp. QY105, was isolated from sea mud of Qingdao. It secreted 90 % of total enzyme activity within the first 20 h of fermentation. An alginate lyase, AlyV5, with an apparent MW of 37 kDa and a specific activity of 2152 U/mg was purified from the culture supernatant. It was most active at 38 °C and pH 7.0 in 20 mM Tris/HCl. The enzyme was stable over a broad pH range (6.0–9.0) and retained ~40 % activity after holding at 90 °C for 10 min. AlyV5 showed activities towards both polyguluronate and polymannuronate, but degraded the former more efficiently. AlyV5 mainly produced disaccharide, trisaccharide and tetrasaccharide from polyguluronate, trisaccharide, tetrasaccharide and pentasaccharide from polymannuronate. The purpose of this study is to find a polyG-preference alginate lyase for the saccharification of alginate combined with our polyM-preference alginate lyases.
TL;DR: The findings imply that productivity in a bioreactor may be increased by reducing the heterogeneity within the culture, and molecular mechanisms underlying productivity of fungi in bioreactors should not be studied at the culture level but at the level of micro-colony populations or even at zonal or hyphal level.
Abstract: Fungi are widely used as cell factories for the production of pharmaceutical compounds, enzymes and metabolites. Fungi form colonies that consist of a network of hyphae. During the last two decades it has become clear that fungal colonies within a liquid culture are heterogeneous in size and gene expression. Heterogeneity in growth, secretion, and RNA composition can even be found between and within zones of colonies. These findings imply that productivity in a bioreactor may be increased by reducing the heterogeneity within the culture. The results also imply that molecular mechanisms underlying productivity of fungi in bioreactors should not be studied at the culture level but at the level of micro-colony populations or even at zonal or hyphal level.
TL;DR: When both VEGF and bFGF were added in the early proliferating stage and BMP-2 wasadded in the late differentiation stage, osteogenic differentiation of MSCs could be enhanced more effectively.
Abstract: Mesenchymal stem cells (MSCs) were treated with bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) dose-dependently and time-dependently. Together they caused a strong synergistic effect on the osteogenic differentiation of MSCs, with lower concentrations of each factor being enough to show the synergistic promotion (50 ng BMP-2/ml, 1 ng VEGF/ml and 10 ng bFGF/ml). When both VEGF and bFGF were added in the early proliferating stage (the first 7 days) and BMP-2 was added in the late differentiation stage (the last 7 days), osteogenic differentiation of MSCs could be enhanced more effectively.
TL;DR: A novel method was developed for extraction of short-chain-length poly(hydroxyalkanoates) (scl-PHA) from microbial biomass by the well-known “scl- PHA anti-solvent” acetone at elevated temperature and pressure in a closed system combining components for extraction, filtration, and product work-up.
Abstract: A novel method was developed for extraction of short-chain-length poly(hydroxyalkanoates) (scl-PHA) from microbial biomass by the well-known “scl-PHA anti-solvent” acetone at elevated temperature and pressure in a closed system combining components for extraction, filtration, and product work-up. Recovery of scl-PHA using this new approach was compared with established methods using chloroform at ambient pressure. The new method performs similar regarding product purity (98.4 vs. 97.7 %) and extraction yield (96.8 % by both methods), and is by far faster than established chloroform extraction (20 min vs. 12 h). Separation of the polymer from acetone is simply achieved by cooling down the acetone solution of scl-PHA, thus allows for a nearly quantitative recovery of the solvent that conveniently can be reused. Characterization of scl-PHA extracted by both methods does not reveal any significant difference in terms of molar mass and thermo analytical parameters.
TL;DR: The adapted strain showed increased stress resistance to diamide and menadione, SDS, Tween 20, HCl, NaOH, and ampicillin and especially the aromatic compound-degrading gene clusters were more than threefold up-regulated.
Abstract: Corynebacterium glutamicum was adapted in a chemostat for 1,900 h with gradually increasing H2O2 stress to understand the oxidative stress response of an industrial host. After 411 generations of adaptation, C. glutamicum developed the ability to grow under stress of 10 mM H2O2, whereas the wild-type did not. The adapted strain also showed increased stress resistance to diamide and menadione, SDS, Tween 20, HCl, NaOH, and ampicillin. A total of 1,180 genes in the RNA-seq transcriptome analysis of the adapted strain were up-regulated twice or higher (corresponding to 38.6 % of the genome), and 126 genes were down-regulated half or less (4.1 % of genome) under 10 mM H2O2-stress conditions compared with those of the wild-type under a no stress condition. Especially the aromatic compound-degrading gene clusters (vanRABK, pcaJIRFLO, and benABCDRKE) were more than threefold up-regulated. Plausible reasons for the H2O2-stress tolerance of the adapted strain are discussed as well as the potential strategy for development of oxidative stress-tolerant strain.
TL;DR: Recent advances in reconstituted cell-free protein synthesis (Protein synthesis Using Recombinant Elements expression systems) are creating new opportunities to tailor the reactions for specialized applications including in vitro protein evolution, printing protein microarrays, isotopic labeling, and incorporating nonnatural amino acids.
Abstract: Cell-free protein synthesis harnesses the synthetic power of biology, programming the ribosomal translational machinery of the cell to create macromolecular products Like PCR, which uses cellular replication machinery to create a DNA amplifier, cell-free protein synthesis is emerging as a transformative technology with broad applications in protein engineering, biopharmaceutical development, and post-genomic research By breaking free from the constraints of cell-based systems, it takes the next step towards synthetic biology Recent advances in reconstituted cell-free protein synthesis (Protein synthesis Using Recombinant Elements expression systems) are creating new opportunities to tailor the reactions for specialized applications including in vitro protein evolution, printing protein microarrays, isotopic labeling, and incorporating nonnatural amino acids
TL;DR: A novel strong constitutive promoter, PGCW14, is selected and identified and tested its promoter activity using enhanced green fluorescent protein (EGFP) as a reporter and exhibited significantly stronger promoter activity than the classicStrong constitutive promoters PTEF1 and PGAP under various carbon sources.
Abstract: The available promoters in the Pichia pastoris expression platform are still limited. We selected and identified a novel strong constitutive promoter, P GCW14 , and tested its promoter activity using enhanced green fluorescent protein (EGFP) as a reporter. Potential promoter regions of P GCW14 were cloned upstream of the EGFP gene and promoter activity was analyzed by measuring fluorescence intensity. P GCW14 exhibited significantly stronger promoter activity than the classic strong constitutive promoters P TEF1 and P GAP under various carbon sources, suggesting that P GCW14 is a strong and constitutive promoter. Hence, P GCW14 can be used as a promoter for high-level expression of heterologous proteins.
TL;DR: For the first time the production of poly(β-l-malic acid) (PMA) has been achieved using agricultural biomass substrates by the yeast-like fungus Aureobasidium pullulans using alkaline H2O2-pretreated corn fiber and wheat straw as sole carbon sources.
Abstract: For the first time the production of poly(β-l
-malic acid) (PMA) has been achieved using agricultural biomass substrates by the yeast-like fungus Aureobasidium pullulans. Strains NRRL Y-2311-1, NRRL 50382, NRRL 50383, and NRRL 50384, representing diverse isolation sources and phylogenetic clades, produced PMA from alkaline H2O2-pretreated corn fiber and wheat straw as sole carbon sources. Pretreated wheat straw was better than pretreated corn fiber, and strain NRRL 50383 gave the highest overall yields of PMA. The addition of CaCO3 plus supplementary hydrolytic enzymes enhanced PMA production. Four basal media were compared for PMA production, and the best was found to be a N-limited pullulan production medium (PM). In this medium, PMA production took place during growth limitation. Under optimal conditions, strain NRRL 50383 produced more than 20 g PMA/l from 5 % (w/v) pretreated wheat straw in PM with 3 % (w/v) CaCO3 and supplementary enzymes.
TL;DR: Improved performances of CadR mutants based E. coli biosensors indicated that truncating C-terminal extension of Cad R could improve the specificity.
Abstract: The metalloprotein, CadR, was redesigned to optimize cadmium and mercury specificity of CadR-based E. coli biosensors. By truncating 10 and 21 amino acids from the C-terminal extension of CadR, CadR-TC10 and CadR-TC21 were obtained, respectively. The genes cadR, cadR-TC10 and cadR-TC21 were used as sensing elements to construct green fluorescent protein based E.coli biosensors. Induction at 30 °C for 4 h in supplemented M9 medium was the optimized condition for the biosensor. Compared with CadR-based biosensor, there was a clear decline in induction coefficient for CadR-TC21-based biosensor (decreased by 86 % in Zn(II), 44 % in Hg(II), and only 37 % in Cd(II)). While in CadR-TC10-based biosensor, the induction coefficient decreased by 95 % in Zn(II), 70 % in Hg(II), and 67 % in Cd(II). Improved performances of CadR mutants based E. coli biosensors indicated that truncating C-terminal extension of CadR could improve the specificity.
TL;DR: It is suggested that Paeoniflorin possesses neuroprotective activity by reducing the production of proinflammatory factors from activated microglial cells.
Abstract: Chronic activation of microglial cells endangers neuronal survival through the release of various proinflammatory and neurotoxic factors. Paeoniflorin (PF), a water-soluble monoterpene glycoside found in the root of Paeonia lactiflora Pall, has a wide range of pharmacological functions, such as anti-oxidant, anti-inflammatory, and anti-cancer effects. Neuroprotective potential of PF has also been demonstrated in animal models of neuropathologies. Here, we have examined the efficacy of PF in the repression of inflammation-induced neurotoxicity and microglial inflammatory response. In organotypic hippocampal slice cultures, PF significantly blocked lipopolysaccharide (LPS)-induced hippocampal cell death and productions of nitric oxide (NO) and interleukin (IL)-1β. PF also inhibited the LPS-stimulated productions of NO, tumor necrosis factor-α, and IL-1β from primary microglial cells. These results suggest that PF possesses neuroprotective activity by reducing the production of proinflammatory factors from activated microglial cells.