Short Hairpin RNA-Induced Myostatin Gene Silencing in Caprine Myoblast Cells In Vitro

Myostatin (MSTN) belongs to the transforming growth factor (TGF)-β superfamily and is a potent negative regulator of skeletal muscle development and growth. Dysfunction of MSTN gene either by natural mutation or induced through genetic manipulation (knockout or knockdown) has been reported to increase the remarkable muscle mass in mammalian species. RNA interference (RNAi) is the most promising method for inhibition of gene expression that can be utilized for MSTN gene knockdown by developing short hairpin RNA (shRNA) construct against it. We utilized three antisense RNA expressing vectors with six constructs to knockdown MSTN gene in in vitro caprine myoblast cell culture system. We observed that all six shRNA constructs were successful in MSTN silencing with efficiency ranging from 7 to 46 % by quantitative real-time PCR and up to 19 % by western blotting. The significant upregulation of interferon response gene OAS1 (5- to 11-fold) in cells transfected with shRNA constructs were indicative of induction of interferon response. This RNAi-based method of increasing muscle mass could provide an alternative strategy to gene knockout methods for improving the production traits and economic properties of livestock.     

Improvement of Solvent Production from Xylose Mother Liquor by Engineering the Xylose Metabolic Pathway in Clostridium acetobutylicum EA 2018

Xylose mother liquor (XML) is a by-product of xylose production through acid hydrolysis from corncobs, which can be used potentially for alternative fermentation feedstock. Sixteen Clostridia including 13 wild-type, 1 industrial strain, and 2 genetically engineered strains were screened in XML, among which the industrial strain Clostridium acetobutylicum EA 2018 showed the highest titer of solvents (12.7 g/L) among non-genetic populations, whereas only 40 % of the xylose was consumed. An engineered strain (2018glcG-TBA) obtained by combination of glcG disruption and expression of the d-xylose proton-symporter, d-xylose isomerase, and xylulokinase was able to completely utilize glucose and l-arabinose, and 88 % xylose in XML. The 2018glcG-TBA produced total solvents up to 21 g/L with a 50 % enhancement of total solvent yield (0.33 g/g sugar) compared to that of EA 2018 (0.21 g/g sugar) in XML. This XML-based acetone–butanol–ethanol fermentation using recombinant 2018glcG-TBA was estimated to be economically promising for future production of solvents.     

Performances of Lactobacillus brevis for Producing Lactic Acid from Hydrolysate of Lignocellulosics

Utilizing all forms of sugars derived from lignocellulosic biomass via various pretreatment and hydrolysis process is a primary criterion for selecting a microorganism to produce biofuels and biochemicals. A broad carbon spectra and potential inhibitors such as furan, phenol compounds and weak acids are two major obstacles that limited the application of dilute-acid hydrolysate of lignocellulosics in lactic acid fermentation. Two strains of bacteria isolated from sour cabbage, S3F4 (Lactobacillus brevis) and XS1T3-4 (Lactobacillus plantrum), exhibited the ability to utilize various sugars present in dilute-acid hydrolysate of biomass. The S3F4 strain also showed strong resistance to potential fermentation inhibitors such as ferulic acid and furfural. Fermentation in flasks by this strain resulted in 39.1 g/l of lactic acid from dilute acid hydrolysates of corncobs that had initial total sugar concentration of 56.9 g/l (xylose, 46.4 g/l; glucose, 4.0 g/l; arabinose, 6.5 g/l). The hydrolysate of corncobs was readily utilized by S3F4 without detoxification, and the lactic acid concentration obtained in this study was higher compared to other reports.     

Enhancing Production of l-Serine by Increasing the glyA Gene Expression in Methylobacterium sp. MB200

Microbial fermentation using methylotrophic bacteria is one of the most promising methods for l-serine production. Here we describe the metabolic engineering of a Methylobacterium strain to increase the production of l-serine. The glyA gene, encoding serine hydroxymethyltransferase (SHMT), was isolated from the genomic DNA of Methylobacterium sp. MB200, using a DNA fragment encoding Methylobacterium extorquens AM1 SHMT as a probe, and inserted into the vector pLAFR3. The resulting construct was transformed into Methylobacterium sp. MB200 using triparental mating. The genetic-engineered strain, designated as Methylobacterium sp. MB202, was shown to produce 11.4?±?0.6 mg/ml serine in resting cell reactions from 30 mg/ml wet cells, 20 mg/ml glycine, and 70 mg/ml methanol in 2 days, representing a 4.4-fold increase from that of the wild strain. The results demonstrated the potential for improving l-serine production by manipulating the glyA in bacteria and should facilitate the production of l-serine using Methylobacterium sp. strains.     

Overproduction of Polygalacturonase by Penicillium griseoroseum Recombinant Strains and Functional Analysis by Targeted Disruption of the pgg2 Gene

Inactivation of the pgg2 gene, a polygalacturonase-encoding gene from Penicillium griseoroseum, reduced the total activity of polygalacturonase (PG) by 90 % in wild-type P. griseoroseum, which indicates that the pgg2 gene is the major gene responsible for PG production in this species. To increase PG production, the coding region of the pgg2 gene was cloned under the control of the glyceraldehyde 3-phosphate dehydrogenase (gpd) promoter and the terminator region of the tryptophan synthase (trpC) gene from Aspergillus nidulans (pAN52pgg2 vector). This vector was then used to transform P. griseoroseum. The transformed strains were characterized according to PG production using glucose, sucrose, or sugar cane juice as the carbon sources. The recombinant P. griseoroseum T146 strain contained an additional copy of the pgg2 gene, which resulted in a 12-fold increase in PG activity when compared with that detected in the supernatant of the control PG63 strain. The proteins secreted by the recombinant strain T146 showed a strong band at 38 kDa, which corresponds to the molecular weight of PG of the P. griseoroseum. The results demonstrate the significant biotechnological potential of recombinant P. griseoroseum T146 for use in PG production.     

Isolation of Biphenyl and Polychlorinated Biphenyl-Degrading Bacteria and Their Degradation Pathway

Four strains of biphenyl-degrading bacteria were isolated from a sewage and identified from the Rhodococcus genus (SK-1, SK-3, and SK-4) and Aquamicrobium genus (SK-2) by 16S rRNA sequence. Among these strains, strain SK-2 was most suitable for biphenyl degradation. When 0.65, 1.3, 2.6, or 3.9 mM of biphenyl was used, the biphenyl was completely degraded within 24 and 96 h of culture, respectively. However, in the case of 6.5 and 9.75 mM of biphenyl, the biphenyl degradation yields were about 80 % and 46.7 % after 120 h of culture, respectively. The isolated strains could degrade a broad spectrum of aromatic compounds including high-chlorinated polychlorinated biphenyl (PCB) congeners in the presence of biphenyl. In addition, strain SK-2 could utilize PCB congeners containing one to six chlorine substituents such as 2,2′,4,4′,5,5′-hexachlorobiphenyl. The PCB utilization rate by the strain SK-2 was increased compared to that of other PCB congener-utilizing bacteria. The four isolates metabolized 4-chlorobiphenyl to 4-chlorobenzoic acid and 2-hydroxy-6-oxo-6-(4′-chlorophenyl)-hexa-2,4-dienoic acid. These results suggest the isolated strains might be good candidates for the bioremediation of PCB-contaminated soil, especially high-saline soils.     

Molecular Cloning and Heterologous Expression of Laccase from Aeromonas hydrophila NIU01 in Escherichia coli with Parameters Optimization in Production

Prior studies disclosed that Aeromonas hydrophila NIU01 was a biodecolorization and bioelectricity bacterium which was isolated from a cross-strait of Taiwan. However, enzymatic function, laccase, involved in this strain had never been reported. This first attempt is to explore its laccase activity, the molecular cloning and heterologous recombinant expression in Escherichia coli. A full-length novel gene of 1,647 bp, LacA, encoding of 549 amino acids was successfully cloned by polymerase chain reaction. The recombinant pET-15b(+)-NIU-LacA expression was compared in different E. coli strains. By applying Taguchi’s L9 in culture optimization, the soluble laccase increased to 22.7 %, in which the conditions were obtained at 22 °C with initial shaking speed at 200 rpm, addition of lactose of 0.2 mM and CuSO₄ of 0.5 mM to the medium, and shaking off while cell mass reached to OD_600nm of 1.5. NIU-LacA was strongly inhibited by chloride ion. The optimal temperature was 60 °C and the optimum pH for ABTS (2,2′-azino-bis (3-ethylbenzthiazolinesulfonic acid) and 2,6-DMP (2,6-dimethoxyphenol) were pH 2.1 and pH 7.5 which enzymatic activity was 274.6 and 44.8 U/L, respectively. Further study in structural modeling of NIU-LacA showed the C terminal domain was the major variance in the three most closely A. hydrophila strains.     

Thermokinetics simulation for multi-walled carbon nanotubes with sodium alginate by advanced kinetics and technology solutions

To accomplish an effective analysis of adsorption, the strong acid dye from aqueous solution of sodium alginate (SA) and multi-walled carbon nanotubes (MWCNTs) composite gel beads were used as important parameters. Differential scanning calorimetry (DSC) was used to measure the heat of breakdown reaction. The experimental conditions were set at 0.5, 1, 2, 4, and 8 °C min^?1, and the temperature range was 30–300 °C. The heating rates and the temperature range were set as follows: Four kinds of proportion in this experiment contained 2 SA % w/v (SA), 0.03, 0.09, 0.18, 0.36 % w/v (MWCNTs), and 10 % w/v calcium chloride, respectively. Four samples, 5, 6, 7, 8, and 9 mg, were used to detect the experimental data. It contributed to understanding the reaction for the distinctive MWCNTs. With the thermokinetic data by isoconversional approach obtained from advanced kinetics and technology solutions (AKTS), the related thermal safety information can be obtained from the thermal reaction of MWCNTs. Valuable parameters, such as activation energy (E _a) and heat of decomposition, can be applied in operation, including adsorption and desorption processes. After DSC tests, and under the four compositions of SA/MWCNTs, at different heating rates of 0.5, 1, 2, 4, and 8 °C min^?1, primarily we found that when the heating rate was increased, exothermic onset temperature would increase gradually. After analyzing E _a value by isoconversional kinetics, we learned that in four different adsorption compositions, SA/MWCNTs_0.03 (161.20 kJ mol^?1) was the minimum. Among them, the highest value was SA/MWCNTs_0.18 (220.48 kJ mol^?1). However, in this study, for SA/MWCNTs compositions we found that E _a value will drop in the final material SA/MWCNTs_0.36. Accordingly, if the ratio of SA and calcium chloride was fixed, then different compositions of the MWCNTs would affect adsorption efficiency of SA/MWCNTs and E _a variation.     

Probiotic Attributes of Autochthonous Lactobacillus rhamnosus Strains of Human Origin

The study was aimed at evaluating the probiotic potential of indigenous autochthonous Lactobacillus rhamnosus strains isolated from infant feces and vaginal mucosa of healthy female. The survival of the selected strains and the two reference strains (L. rhamnosus GG and L. casei Actimel) was 67–81 % at pH 2 and 70–80 % after passage through the simulated gastrointestinal fluid. These strains are able to grow in the presence of 4 % bile salt, 10 % NaCl, and 0.6 % phenol. The cell surface of L. rhamnosus strains is hydrophilic in nature as revealed by bacterial adhesion to hydrocarbons (BATH) assay. Despite this, L. rhamnosus strains showed mucin adherence, autoaggregation and coaggregation properties that are strain-specific. In addition, they produce bile salt hydrolase (BSH) and β-galactosidase activities. L. rhamnosus strains exhibit antimicrobial activity against food spoilage organisms and gastrointestinal pathogens, as well as Candida and Aspergillus spp. L. rhamnosus strains have similar antibiotic susceptibility pattern, and resistance to certain antibiotics is intrinsic or innate. The strains are neither haemolytic nor producer of biogenic amines such as histamine, putrescine, cadaverine and tyramine. Lyophilized cells of L. rhamnosus Fb exhibited probiotic properties demonstrating potential of the strain for technological suitability and in the preparation of diverse probiotic food formulations.     

Curing the Plasmid pMC1 from the Poly (γ-glutamic Acid) Producing Bacillus amyloliquefaciens LL3 Strain Using Plasmid Incompatibility

Bacillus amyloliquefaciens LL3 is a glutamate-independent poly-γ-glutamic acid (γ-PGA) producing strain which consists of a circular chromosome (3,995,227 bp) and an endogenous plasmid pMC1 (6,758 bp). The study of the function of native plasmid and the genome-size reduction of the B. amyloliquefaciens LL3 strain requires elimination of the endogenous plasmid. Traditional plasmid-curing procedures using sodium dodecyl sulfate (SDS) or acridine orange combined with heat treatment have been shown to be ineffective in this strain. Plasmid incompatibility is an effective method for curing which has been studied before. In our research, the hypothetical Rep protein gene and the origin of replication of the endogenous plasmid were cloned into the temperature-sensitive vector yielding the incompatible plasmid pKSV7-rep-ori. This plasmid was transformed into LL3 by electroporation. The analysis of the strain bearing incompatible plasmids after incubation at 30 °C for 30 generations showed the production of plasmid cured strains. High frequency of elimination was achieved with more than 93 % of detected strains showing to be plasmid-cured. This is the first report describing plasmid cured in a γ-PGA producing strain using this method. The plasmid-cured strains showed an increase of γ-PGA production by 6 % and led to a yield of 4.159 g/l, compared to 3.918 g/l in control and cell growth increased during the early stages of the exponential phase. Gel permeation chromatography (GPC) characterization revealed that the γ-PGA produced by plasmid-cured strains and the wild strains were identical in terms of molecular weight. What is more, the further study of plasmid function showed that curing of the endogenous plasmid did not affect its sporulation efficiency.     

Screening and Production Study of Microbial Xylanase Producers from Brazilian Cerrado

Hemicelluloses are polysaccharides of low molecular weight containing 100 to 200 glycosidic residues. In plants, the xylans or the hemicelluloses are situated between the lignin and the collection of cellulose fibers underneath. The xylan is the most common hemicellulosic polysaccharide in cell walls of land plants, comprising a backbone of xylose residues linked by β-1,4-glycosidic bonds. So, xylanolytic enzymes from microorganism have attracted a great deal of attention in the last decade, particularly because of their biotechnological characteristics in various industrial processes, related to food, feed, ethanol, pulp, and paper industries. A microbial screening of xylanase producer was carried out in Brazilian Cerrado area in Selviria city, Mato Grosso do Sul State, Brazil. About 50 bacterial strains and 15 fungal strains were isolated from soil sample at 35 °C. Between these isolated microorganisms, a bacterium Lysinibacillus sp. and a fungus Neosartorya spinosa as good xylanase producers were identified. Based on identification processes, Lysinibacillus sp. is a new species and the xylanase production by this bacterial genus was not reported yet. Similarly, it has not reported about xylanase production from N. spinosa. The bacterial strain P5B1 identified as Lysinibacillus sp. was cultivated on submerged fermentation using as substrate xylan, wheat bran, corn straw, corncob, and sugar cane bagasse. Corn straw and wheat bran show a good xylanase activity after 72 h of fermentation. A fungus identified as N. spinosa (strain P2D16) was cultivated on solid-state fermentation using as substrate source wheat bran, wheat bran plus sawdust, corn straw, corncob, cassava bran, and sugar cane bagasse. Wheat bran and corncobs show the better xylanase production after 72 h of fermentation. Both crude xylanases were characterized and a bacterial xylanase shows optimum pH for enzyme activity at 6.0, whereas a fungal xylanase has optimum pH at 5.0–5.5. They were stable in the pH range 5.0–10.0 and 5.5–8.5 for bacterial and fungal xylanase, respectively. The optimum temperatures were 55C and 60 °C for bacterial and fungal xylanase, respectively, and they were thermally stable up to 50 °C.     

mga Genosensor for Early Detection of Human Rheumatic Heart Disease

The 5′ amino-labeled DNA probe complementary to mga gene of Streptococcus pyogenes was immobilized on carboxylated multiwall carbon nanotubes electrode and hybridized with 0.1–100 ng/6 μl single-stranded genomic DNA (ssG-DNA) of S. pyogenes from throat swab of suspected rheumatic heart disease (RHD) patients. Electrochemical response was measured by cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance (EI). The sensitivity of the sensor was 106.03?(μA/cm²)/ng and limit of detection (LOD) was found 0.014 ng/6 μl with regression coefficient (R ²) of 0.921 using DPV. The genosensor was characterized by FTIR and SEM, and electrode was found stable for 6 months on storage at 4 °C with 5–6 % loss in initial DPV current. mga genosensor is the first report on RHD sensor which can save life of several suspected patients by early diagnosis in 30 min.     

Enhanced Organic Solvent Tolerance of Escherichia coli by 3-Hydroxyacid Dehydrogenase Family Genes

A 3-hydroxyisobutyrate dehydrogenase-encoding gene mmsB has been identified as one of the key genes responsible for the enhanced organic solvent tolerance (OST) of Pseudomonas putida JUCT1. In this study, the OST-related effect of two 3-hydroxyacid dehydrogenase family genes (mmsB and zwf) was investigated in Escherichia coli JM109. It was noted that the growth of E. coli JM109 was severely hampered in 4 % decalin after zwf knockout. Additionally, its complementation resulted in significantly enhanced solvent tolerance compared with its parent strain. Furthermore, E. coli JM109 carrying mmsB showed better OST capacity than that harboring zwf. To construct E. coli strains with an inheritable OST phenotype, mmsB was integrated into the genome of E. coli JM109 by red-mediated recombination. Using E. coli JM109(DE3) (ΔendA::mmsB) as host strain, whole-cell biocatalysis was successfully carried out in an aqueous/butyl acetate biphasic system with a remarkably improved product yield.     

Novel dual inhibitors against FP-2 and PfDHFR as potential antimalarial agents: Design,synthesis and biological evaluation

A series of novel 2,4-diaminopyrimidine-modified compounds was designed and synthesized. Compound 14 showed micromolar dual inhibitory effect on both FP-2 and PfDHFR, and potential inhibition to the proliferation of P. falciparum 3D7 strain and chloroquine-resistant P. falciparum Dd2 strain.     

Xylitol Production by Genetically Engineered Trichoderma reesei Strains Using Barley Straw as Feedstock

Xylitol, a naturally occurring five-carbon sugar alcohol derived from d-xylose, is currently in high demand by industries. Trichoderma reesei, a prolific industrial cellulase and hemicellulase producing fungus, is able to selectively use d-xylose from hemicelluloses for xylitol production. The xylitol production by T. reesei can be enhanced by genetic engineering of blocking further xylitol metabolism in the d-xylose pathway. We have used two different T. reesei strains which are impaired in the further metabolism of xylitol including a single mutant in which the xylitol dehydrogenase gene was deleted (?xdh1) and a double mutant where additionally l-arabinitol-4-dehydrogenase, an enzyme which can partially compensate for xylitol dehydrogenase function, was deleted (?lad1?xdh1). Barely straw was first pretreated using NaOH and Organosolv pretreatment methods. The highest xylitol production of 6.1 and 13.22 g/L was obtained using medium supplemented with 2 % Organosolv-pretreated barley straw and 2 % d-xylose by the ?xdh1 and ?lad1?xdh1 strains, respectively.     

Genome Shuffling of Aspergillus niger for Improving Transglycosylation Activity

Isomaltooligosaccharides (IMO), the glucosylsaccharides used as food additives, are made from saccharified starch by enzymes or microbial cells with transglycosylation activity. This study aimed to generate shuffled futants of Aspergillus niger with enhanced transglycosylation activity for industrial IMO production. The starting mutant population was generated by ⁶⁰Co-γ radiation; mutants with higher transglycosylation activity were selected and subjected to recursive protoplast fusion. The resulting fusants were screened by a novel high-throughput method based on detecting non-fermentable reducing sugar. After three rounds of genome shuffling, the best performing strain GS3-3 was obtained, its transglycosylation activity (14.91 U/mL) was increased by 194.1 % compared to that of original strain C-6181. In fermentor test, transglycosylation activity of GS3-3 was obtained at 16.61 U/mL. The mycelia of GS3-3 were reused ten times to produce IMO syrup from liquefied cassava starch containing about 280 g/L total sugar within 4 days. The conversion of liquefied cassava starch to IMO was at 71.3–72.1 %, which was higher than the best conversion (68 %) ever reported. GS3-3 shows a great potential for industrial IMO production.     

Determination of Hydrogen Peroxide Using a Novel Sensor Based on Fe3O4 Magnetic Nanoparticles

Fe₃O₄ magnetic nanoparticles were synthesized by chemical co-precipitation with sodium citrate as a surfactant and were used with chitosan to construct a novel hydrogen peroxide sensor. The electrochemical behavior of hydrogen peroxide at the sensor was investigated by cyclic voltammetry. The composite film electrocatalyzed the reduction of hydrogen peroxide, and the peak current increased linearly with concentration from 1.00 × 10^?5 to 1.00 × 10^?3 mol · L^?1 (R = 0.9974) with a detection limit of 1.53 × 10^?6 mol · L^?1. This novel nonenzyme sensor provided good sensitivity, stability, and precision with potential applications.