ALBERT

Description: Clostridium acetobutylicum has been considered as an attractive platform host for biorefinery due to its metabolic diversity. Considering its capability to overproduce butanol through butyrate, it was thought that butyric acid can also be efficiently produced by this bacterium through metabolic engineering. The pta-ctfB -deficient C. acetobutylicum CEKW, in which genes encoding phosphotransacetylase and CoA-transferase were knocked out, was assessed for its potential as a butyric acid producer in fermentations with four controlled pH values at 5.0, 5.5, 6.0, and 6.4. Butyric acid could be best produced by fermentation of the CEKW at pH 6.0, resulting in the highest titer of 26.6 g/l, which is 6.4 times higher than that obtained with the wild type. However, due to the remaining solventogenic ability of the CEKW, 3.6 g/l solvents were also produced. Thus, the CEKW was further engineered by knocking out the adhE1 -encoding aldehyde/alcohol dehydrogenase to prevent solvent production. Batch fermentation of the resulting C. acetobutylicum HCEKW at pH 6.0 showed increased butyric acid production to 30.8 g/l with a ratio of butyric-to-acetic acid (BA/AA) of 6.6 g/g and a productivity of 0.72 g/l/h from 86.9 g/l glucose, while negligible solvent (0.8 g/l ethanol only) was produced. The butyric acid titer, BA/AA ratio, and productivity obtained in this study were the highest values reported for C. acetobutylicum , and the BA/AA ratio and productivity were also comparable to those of native butyric acid producer Clostridium tyrobutyricum . These results suggested that the simultaneous deletion of the pta-ctfB-adhE1 in C. acetobutylicum resulted in metabolic switch from biphasic to acidogenic fermentation, which enhanced butyric acid production.

Description: Disruption of spatiotemporal behavior of intracellular signaling cascades including tumor necrosis factor alpha-related apoptosis-inducing ligand (TRAIL)-mediated signaling in prostate cancer has gained tremendous attention in the past few years. There is an increasing effort in translating the emerging information about TRAIL-mediated signaling obtained through experimental and preclinical data to clinic. Fascinatingly, novel targeting approaches are being developed to enhance the tissue- or subcellular-specific delivery of drugs with considerable focus on prostate cancer. These applications have the potential to revolutionize prostate cancer therapeutic strategies and include the accumulation of drugs in target tissue as well as the selection of internalizing ligands for enhanced receptor-mediated uptake of drugs. In this mini-review, we outline outstanding developments in therapeutic strategies based on the regulation and/or targeting of TRAIL pathway for the treatment of prostate cancer. Moreover, microRNAs (miRNAs), with potential transcriptional and posttranscriptional regulation of gene expression, will be presented for their potential in prostate cancer treatment. Emphasis has been given to the use of delivery approaches, especially based on nanotechnology. Considerably, enhanced information regarding miRNA regulation of TRAIL-mediated signaling in prostate cancer cells may provide potential biomarkers for the characterization of patients as responders and nonresponders of TRAIL-based therapy and could provide rationalized basis for combination therapies with TRAIL death receptor-targeting drugs.

Description: The development of cellulase-based bioprocess is afflicted by the processing efficiency of enzymes. To address this issue, a method based on artificial oil bodies (AOBs) was proposed to integrate production and immobilization of recombinant cellulase. First, the heterologous endoglucanase ( celA ), cellobiohydrolase ( celK ), and β-glucosidase ( gls ) genes were individually fused with oleosin, a structural protein of plant seed oils. After expression in Escherichia coli , each fusion protein of insolubility was mixed together with plant oils. AOBs were assembled by subjecting the mixture to sonication. Consequently, active CelA, CelK, and Gls were resumed and co-immobilized on AOBs surface. Finally, the assembly condition (including the protein ratio) and the reaction condition were further optimized by response surface methodology. The resulting AOBs-bound cellulase remained stable for 4 cycles of cellulose–hydrolyzed reactions. Overall, the result shows a promise of this proposed approach for processing recombinant cellulase, which may provide a facile method to investigate optimum combination of cellulase components towards various cellulosic materials.

Description: Separation strategies based on size-selective precipitation of DNA fragments with polyethylene glycol (PEG) have been used for achieving desired DNA interval in automated sample preparation for next-generation sequencing. By varying PEG concentration, DNA fragments of different sizes can be precipitated onto surfaces of carboxyl-coated paramagnetic particles selectively, and therefore, the desired DNA interval can be obtained. However, one of the crucial points in this approach is to determine the critical PEG concentration for DNA fragment of a certain size. The aim of this work was to develop a convenient and reliable method for accurately determining the critical PEG concentration. In our method, at a fixed concentration of sodium chloride (NaCl), recovered DNA samples obtained with different PEG concentrations were directly quantified, and their concentrations as a function of the PEG concentration were fitted by the logistic function. The critical PEG value was easily and accurately determined from the fitted logistic function. The repeatability and stability of the critical PEG value were assessed, showing an excellent reliability of the method. Based on this method, critical PEG values of different-size DNA fragments were determined at different NaCl concentrations. The effectiveness of the method was also demonstrated by selective precipitation of DNA fragments.

Description: A recombinant oleate hydratase from Lysinibacillus fusiformis converted ricinoleic acid to a product, whose chemical structure was identified as the novel compound 10,12-dihydroxystearic acid by gas chromatograph/mass spectrometry, Fourier transform infrared, and nuclear magnetic resonance analysis. The reaction conditions for the production of 10,12-dihydroxystearic acid were optimized as follows: pH 6.5, 30 °C, 15 g l −1 ricinoleic acid, 9 mg ml −1 of enzyme, and 4 % ( v / v ) methanol. Under the optimized conditions, the enzyme produced 13.5 g l −1 10,12-dihydroxystearic acid without detectable byproducts in 3 h, with a conversion of substrate to product of 90 % ( w / w ) and a productivity of 4.5 g l −1  h −1 . The emulsifying activity of 10,12-dihydroxystearic acid was higher than that of oleic acid, ricinoleic acid, stearic acid, and 10-hydroxystearic acid, indicating that 10,12-dihydroxystearic acid can be used as a biosurfactant.

Description: Bacillus amyloliquefaciens FZB42 has been shown to stimulate plant growth and to suppress the growth of plant pathogenic organisms including nematodes. However, the mechanism underlying its effect against nematodes remains unknown. In this study, we screened a random mutant library of B. amyloliquefaciens FZB42 generated by the mariner transposon Tn YLB-1 and identified a mutant strain F5 with attenuated nematicidal activity. Reversible polymerase chain reaction revealed that three candidate genes RAMB_007470 , yhdY , and prkA that were disrupted by the transposon in strain F5 potentially contributed to its decreased nematicidal activity. Bioassay of mutants impaired in the three candidate genes demonstrated that directed deletion of gene RBAM_007470 resulted in loss of nematicidal activity comparable with that of the F5 triple mutant. RBAM_007470 has been reported as being involved in biosynthesis of plantazolicin, a thiazole/oxazole-modified microcin with hitherto unknown function. Electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) analyses of surface extracts revealed that plantazolicin bearing a molecular weight of 1,354 Da was present in wild-type B. amyloliquefaciens FZB42, but absent in the Δ RABM_007470 mutant. Furthermore, bioassay of the organic extract containing plantazolicin also showed a moderate nematicidal activity. We conclude that a novel gene RBAM_007470 and its related metabolite are involved in the antagonistic effect exerted by B. amyloliquefaciens FZB42 against nematodes.

Description: Syngas fermentation is a promising route for resource recovery. Acetate is an important industrial chemical product and also an attractive precursor for liquid biofuels production. This study demonstrated high fraction acetate production from syngas (H 2 and CO 2 ) in a hollow-fiber membrane biofilm reactor, in which the hydrogen utilizing efficiency reached 100 % during the operational period. The maximum concentration of acetate in batch mode was 12.5 g/L, while the acetate concentration in continuous mode with a hydraulic retention time of 9 days was 3.6 ± 0.1 g/L. Since butyrate concentration was rather low and below 0.1 g/L, the acetate fraction was higher than 99 % in both batch and continuous modes. Microbial community analysis showed that the biofilm was dominated by Clostridium spp., such as Clostridium ljungdahlii and Clostridium drakei , the percentage of which was 70.5 %. This study demonstrates a potential technology for the in situ utilization of syngas and valuable chemical production.

Description: Lignosulfonates(LSs), by-products from chemical pulping processes, are low-value products with limited dispersion properties. The ability of commercially available horseradish peroxidase (HRP) to polymerize LS macromolecules and improve the dispersion properties of LSs was investigated. The polymerization of LSs proceeded efficiently under mild reaction conditions in an aqueous solution with HRP/H 2 O 2 . Gel permeation chromatography showed a significant increase in weight-average molecular weight ( M w ) of sulfonated kraft lignin and sodium lignosulfonate (NaLS) by 8.5-fold and 4.7-fold, respectively. The mechanism of polymerization was investigated by elemental analysis, surface charge measurement, headspace gas chromatography, infrared spectroscopy (IR), and hydrogen nuclear magnetic resonance spectrometry ( 1 H-NMR). The functional group measurements indicated that HRP incubation did not reduce the sulfonic group content. However, it decreased the phenolic and methoxyl group contents. As the phenolic group content decreased, M w increased as a power function. The polymerization was proposed to involve the random coupling of phenoxy radical intermediates. The radicals coupled with each other to form different inter-unit linkages, most of which were the β-O-4’ type, as the 1 H-NMR spectra indicated. Moreover, the HRP/H 2 O 2 incubation induced a significant improvement in the adsorption and dispersion properties of LSs. Therefore, the HRP/H 2 O 2 incubation is a promising approach for industrial applications of LSs.

Description: Marine microalga Nannochloropsis oculata possesses nutrients valuable for human health. In this study, we added freeze-dried N. oculata powder to soybean oil and observed a remarkable inhibition in oil oxidation. The amount of microalgae powder added was positively correlated to the increase in oil stability. The addition of 5.0 % ( w / w ) microalgae powder increased the oil stability index (OSI) values of soybean oil more than twofold at the tested temperatures 120 and 130 °C. N . oculata contains high levels of both phenolic compounds and α-tocopherols that could be the contributors to such an increase of the OSI. Two methods were conducted to assay the active ingredients released from microalgae: one employed three solvent systems to extract the microalgae and the other was the soybean oil added with microalgae. Analyses of free radical scavenging and reducing power suggested that the phenolic compounds dominated the antioxidation activities in soybean oil when it was infused with the microalgae powder. Our results suggest that N. oculata could potentially be used as an additive in cooking oil to increase the shelf life and nutritional value of the oil and to reduce the production of free radicals from lipid oxidation when the oil is used at high-temperature cooking processes.

Description: The actinomycete Streptomyces platensis produces two compounds that display antibacterial activity: platensimycin and platencin. These compounds were discovered by the Merck Research Laboratories, and a complex insoluble production medium was reported. We have used this medium as our starting point in our studies. In a previous study, we developed a semi-defined production medium, i.e., PM5. In the present studies, by varying the concentration of the components of PM5, we were able to develop a superior semi-defined medium, i.e., PM6, which contains a higher concentration of lactose. Versions of PM6, containing lower concentrations of all components, were also found to be superior to PM5. The new semi-defined production media contain dextrin, lactose, MOPS buffer, and ammonium sulfate in different concentrations. We determined antibiotic production capabilities using agar diffusion assays and chemical assays via thin-layer silica chromatography and high-performance liquid chromatography. We reduced crude nutrient carryover from the seed medium by washing the cells with distilled water. Using these semi-defined media, we determined that addition of the semi-defined component soluble starch stimulated antibiotic production and that it and dextrin could both be replaced with glucose, resulting in the chemically defined medium, PM7.

Description: Pichia pastoris is widely used as a host system for heterologous protein expression in both academia and industry. Production is typically accomplished by a fed-batch induction process that is known to have negative impacts on cell physiology that impose limits on both protein yields and quality. We have analysed recombinant protein production in chemostat cultures to understand the physiological responses associated with methanol-induced production of two human lysozyme variants with different degrees of misfolding by P . pastoris . Confounding variables associated with nutrient stress or growth-rate are minimised during steady-state growth in chemostats. Comparison of transcriptome-level data obtained during the non-inducing and inducing steady states identified changes in expression of only about 1 % of the genome during production of either an amyloidogenic human lysozyme variant prone to intracellular aggregation (I56T) or a misfolded but secretable variant (T70N), indicating near-complete acclimation to their production. A marked, but temporary, stress response involving both the unfolded protein response (UPR) and ER-associated degradation pathway was observed during the transient between steady states, particularly following induction of the T70N variant synthesis, and was accompanied by changes in expression of around 50 antisense transcripts. The results suggest that optimal heterologous protein production could best be achieved by a continuous process that minimises the number of methanol-induced transients experienced by the cultures. The processing of HAC1 mRNA required for the UPR was found to be constitutive in the culture conditions used, even in the absence of recombinant protein induction.

Description: The Aminobacter sp. strain MSH1 has potential for pesticide bioremediation because it degrades the herbicide metabolite 2,6-dichlorobenzamide (BAM). Production of the BAM-degrading bacterium using aerobic bioreactor fermentation was investigated. A mineral salt medium limited for carbon and with an element composition similar to the strain was generated. The optimal pH and temperature for strain growth were determined using shaker flasks and verified in bioreactors. Glucose, fructose, and glycerol were suitable carbon sources for MSH1 ( μ  = 0.1 h −1 ); slower growth was observed on succinate and acetic acid ( μ  = 0.01 h −1 ). Standard conditions for growth of the MSH1 strain were defined at pH 7 and 25 °C, with glucose as the carbon source. In bioreactors (1 and 5 L), the specific growth rate of MSH1 increased from μ  = 0.1 h −1 on traditional mineral salt medium to μ  = 0.18 h −1 on the optimized mineral salt medium. The biomass yield under standard conditions was 0.47 g dry weight biomass/g glucose consumed. An investigation of the catabolic capacity of MSH1 cells harvested in exponential and stationary growth phases showed a degradation activity per cell of about 3 × 10 −9  μg BAM h −1 . Thus, fast, efficient, large-scale production of herbicide-degrading Aminobacter was possible, bringing the use of this bacterium in bioaugmentation field remediation closer to reality.

Description: Soy sauce is a traditional condiment manufactured by natural inoculation and mixed culture fermentation. As is well known, it is the microbial community that plays an important role in the formation of its flavors. However, to date, its dynamic changes during the long period of fermentation process are still unclear, intensively constraining the improvement and control of the soy sauce quality. In this work, we revealed the dynamic changes of the microbial community by combining a cultured dependent method and a cultured independent method of polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis. Results indicated that the two methods verified and complemented each other in profiling microbial community, and that significant dynamics of the microbial community existed during the fermentation process, especially the strong inhibition of the growth of most of the microbes when entering into the mash stage from the koji stage. In the analysis of bacterial community, Staphylococcus and Bacillus were found to be the dominant bacteria and detected in the whole fermentation process. Kurthia and Klebsiella began to appear in the koji stage and then fade away in the early stage of the mash fermentation. In the analysis of fungal community, Aspergillus sojae and Zygosaccharomyces rouxii were found to be the dominant fungi in the koji and mash fermentation, respectively. It was clearly shown that when A. sojae decreased and disappeared in the middle stage of the mash fermentation, Z. rouxii appeared and increased at the meantime. Aspergillus parasiticus , Trichosporon ovoides and Trichosporon asahii also appeared in the koji and the early period of the mash fermentation and disappeared thereafter. Similar to Z. rouxii , Millerozyma farinosa and Peronospora farinosa were also found spontaneously which appeared in the mid-late period of the mash fermentation. The principal component analysis suggested that the microbial community underwent significant changes in the early period of the fermentation and, thereafter, tended to the stabilization in the mid-late periods. This study gave us important clues to understand the fermentation process and can serve as a foundation for improving the quality of soy sauce in the future.

Description: Galactosidases are widespread enzymes that are used for manifold applications, including production of prebiotics, biosynthesis of different transgalactosylated products, improving lactose tolerance and in various analytical approaches. The nature of these applications often require galactosidases to be present in a purified form with clearly defined properties, including precisely determined substrate specificities, low sensitivity to inhibitors, and high efficiency and stability under distinct conditions. In this study, we present the recombinant expression and purification of two previously uncharacterized β-galactosidases from Aspergillus nidulans as well as one β-galactosidase from Aspergillus niger . All enzymes were active toward p -nitrophenyl-β- d -galactopyranoside as substrate and displayed similar temperature and pH optima. The purified recombinant galactosidases digested various complex substrates containing terminal galactose β-1,4 linked to either N -acetylglucosamine or fucose, such as N -glycans derived from bovine fibrin and Caenorhabditis elegans . In our comparative study of the recombinant galactosidases with the commercially available galactosidase from Aspergillus oryzae , all enzymes also displayed various degrees of activity toward complex oligosaccharides containing β-1,3-linked terminal galactose residues. All recombinant enzymes were found to be robust in the presence of various organic solvents, temperature variations, and freeze/thaw cycles and were also tested for their ability to synthesize galactooligosaccharides. Furthermore, the use of fermentors considerably increased the yield of recombinant galactosidases. Taken together, we demonstrate that purified recombinant galactosidases from A. niger and from A. nidulans are suitable for various glycobiological and biotechnological applications.

Description: A Lolium perenne ice-binding protein ( Lp IBP) demonstrates superior ice recrystallization inhibition (IRI) activity and has proposed applications in cryopreservation, food texturing, as well as in being a “green” gas hydrate inhibitor. Recombinant production of Lp IBP has been previously conducted in bacterial and yeast systems for studies of protein characterization, but large-scale applications have been hitherto limited due to high production costs. In this work, a codon-optimized Lp IBP was recombinantly expressed and secreted in a novel one-step vector system from the nuclear genome of the green microalga Chlamydomonas reinhardtii . Both mixotrophic and photoautotrophic growth regimes supported Lp IBP expression, indicating the feasibility of low-cost production using minimal medium, carbon dioxide, and light energy as input. In addition, multiple growth and bioproduct extraction cycles were performed by repetitive batch cultivation trials, demonstrating the potential for semi-continuous production and biomass harvesting. Concentrations of recombinant protein reached in this proof of concept approach were sufficient to demonstrate IRI activity in culture media without additional purification or concentration, with activity further verified by thermal hysteresis and morphology assays. The incorporation of the recombinant Lp IBP into a model gas hydrate offers the promise that algal production may eventually find application as a “green” hydrate inhibitor.

Description: The interest in biofertilizers is increasing and so is the potential for their use in sustainable agriculture. However, many of the products that are currently available worldwide are often of very poor quality, resulting in the loss of confidence from farmers. The formulation of an inoculant is a crucial multistep process that should result in one or several strains of microorganisms included in a suitable carrier, providing a safe environment to protect them from the often harsh conditions during storage and ensuring survival and establishment after introduction into soils. One of the key issues in formulation development and production is the quality control of the products, at each stage of the process. This review presents the different components and the major steps involved in the formulation of good quality biofertilizers, including the techniques used to assess the quality of the products following production. The quality of currently available inoculants is also reviewed, emphasizing the need for better quality control systems worldwide.

Description: A large number of molds serve as producer strains for the industrial production of pharmaceuticals, foods, or organic chemicals. To optimize strains for production processes, conventional strain development programs use random mutagenesis and, more recently, recombinant technologies to generate microbial strains with novel and advantageous properties. The recent detection of mating type genes in fungal production strains and the discovery of cryptic sexuality in presumably asexual fungi open up novel strategies for generating progeny with new, as yet unobserved properties. Mating type genes, which can be considered as “sex genes,” not only direct sexual development but also regulate a broad range of fungal secondary metabolites. In addition, they control hyphal morphology, which has a direct impact on production processes that are often conducted in huge fermenter tanks. Here, we survey the occurrence and function of mating type genes that have been discovered in a wide range of industrial fungal producer strains. The possibility to obtain progeny from industrial producers by sexual mating provides an exciting alternative to conventional strain improvement programs aiming to generate optimized recombinant production strains.

Description: Endo-1,4-β-xylanases (EC 3.2.1.8) hydrolyze the 1,4-β-D-xylosidic linkages in xylans, the most abundant hemicellulose in plant cell walls. Xylanase enzymes have numerous industrial applications, including the manufacturing of animal feed, bread, juice and wine, pulp and paper, and biofuels. In this study, two glycosyl hydrolase family 10 members designated Gt Xyn10A and Gt Xyn10B and two glycosyl hydrolase family 11 members, Op Xyn11A and Cc Xyn11C, were functionally expressed and subjected to biochemical characterization. The K M , V max , and k cat values of the four xylanases, determined using birchwood xylan, ranged from 0.27 to 1.1 mg/mL, 130 to 980 μmol/min/mg, and 109 to 344 s −1 , respectively, where Op Xyn11A gave the highest and Gt Xyn10B the lowest values for all three parameters. Substrate specificity studies and analysis of the products released during the degradation of xylo-oligosaccharides and three types of xylan revealed significant differences in catalytic properties, particularly between Op Xyn11A and the other xylanases and between the family 10 and the family 11 xylanases. Molecular modeling suggests that the unique substrate specificity of Op Xyn11A can be attributed to the presence of a serine rather that an asparagine or aspartate residue at the +1 substrate binding site. Additionally, all four xylanases exhibited biochemical characteristics of interest for various commercial applications.

Description: Hand, foot, and mouth disease (HFMD) has caused significant morbidity and mortality in the Asia-Pacific regions, particularly in infants and young children. Coxsackievirus A16 (CA16) represents one of the major causative agents for HFMD, and the development of a safe and effective vaccine preventing CA16 infections has become a public health priority. In this study, we have developed a yeast system for the production of virus-like particles (VLPs) for CA16 by co-expressing P1 and 3CD of CA16 in Saccharomyces cerevisiae . These VLPs exhibit similarity in both protein composition and morphology as empty particles from CA16-infected cells. Immunization with CA16 VLPs in mice potently induced CA16-specific IgG and neutralization antibodies in a dose-dependent manner. IgG subclass isotyping revealed that IgG1 and lgG2b were dominantly induced by VLPs. Meanwhile, cytokine profiling demonstrated that immunization with VLPs significantly induced the secretion of IFN-γ, indicating potent cellular immune response. Furthermore, in vivo challenge experiments showed that passive immunization with anti-VLPs sera conferred full protection against lethal CA16 challenge in neonate mice. Taken together, our data demonstrated that VLPs produced in yeast might have the potential to be further developed as a vaccine candidate against HFMD.

Description: Studies were carried out to assess the utility of the cellular and extracellular constituents of Bacillus megaterium for the flotation of sphalerite and galena minerals. Based on the flotation results on the individual minerals, it was observed that sphalerite was preferentially floated compared to galena. A maximum selectivity index (SI) value of 11.7 was achieved in the presence of the soluble fraction of the thermolysed cells, which was higher than that obtained with the intact cells (SI of 6.5) and the insoluble fraction of the thermolysed cells (SI of 9.6). The results of the various enzymatic treatment tests revealed that extracellular DNA played a vital role in the selective flotation of sphalerite. A noteworthy finding was that the single-stranded DNA (ssDNA) had a higher biocollector capacity vis-à-vis the double-stranded DNA (dsDNA), leading to better flotation efficiency. About 95 % recovery of sphalerite could be achieved from the mineral mixture by the combined addition of the ssDNA with the non-DNA components of the bacterial cells, resulting in a maximum SI of 19.1. Calcium and phosphate components of the nutrient media were found to be essential for better selectivity of separation of sphalerite. The mechanisms of microbe–mineral interaction are discussed.

Description: While the use of anaerobic digestion to generate methane as a source of bioenergy is increasing worldwide, our knowledge of the microbial communities that perform biomethanation is very limited. Using next-generation sequencing, bacterial population profiles were determined in three full-scale mesophilic anaerobic digesters operated on dairy farms in the state of Vermont (USA). To our knowledge, this is the first report of a metagenomic analysis on the bacterial population of anaerobic digesters using dairy manure as their main substrate. A total of 20,366 non-chimeric sequence reads, covering the V1-V2 hypervariable regions of the bacterial 16S rRNA gene, were assigned to 2,176 operational taxonomic units (OTUs) at a genetic distance cutoff value of 5 %. Based on their limited sequence identity to validly characterized species, the majority of OTUs identified in our study likely represented novel bacterial species. Using a naïve Bayesian classifier, 1,624 anaerobic digester OTUs could be assigned to 16 bacterial phyla, while 552 OTUs could not be classified and may belong to novel bacterial taxonomic groups that have yet to be described. Firmicutes , Bacteroidetes , and Chloroflexi were the most highly represented bacteria overall, with Bacteroidetes and Chloroflexi showing the least and the most variation in abundance between digesters, respectively. All digesters shared 132 OTUs, which as a “core” group represented 65.4 to 70.6 % of sequences in individual digesters. Our results show that bacterial populations from microbial communities of anaerobic manure digesters can display high levels of diversity despite sharing a common core substrate.

Description: The discovery of Dehalococcoides mccartyi reducing perchloroethene and trichloroethene (TCE) to ethene was a key landmark for bioremediation applications at contaminated sites. D. mccartyi -containing cultures are typically grown in batch-fed reactors. On the other hand, continuous cultivation of these microorganisms has been described only at long hydraulic retention times (HRTs). We report the cultivation of a representative D. mccartyi -containing culture in continuous stirred-tank reactors (CSTRs) at a short, 3-d HRT, using TCE as the electron acceptor. We successfully operated 3-d HRT CSTRs for up to 120 days and observed sustained dechlorination of TCE at influent concentrations of 1 and 2 mM TCE to ≥97 % ethene, coupled to the production of 10 12 D. mccartyi cells L culture −1 . These outcomes were possible in part by using a medium with low bicarbonate concentrations (5 mM) to minimize the excessive proliferation of microorganisms that use bicarbonate as an electron acceptor and compete with D. mccartyi for H 2 . The maximum conversion rates for the CSTR-produced culture were 0.13 ± 0.016, 0.06 ± 0.018, and 0.02 ± 0.007 mmol Cl − L culture −1 h −1 , respectively, for TCE, cis -dichloroethene, and vinyl chloride. The CSTR operation described here provides the fastest laboratory cultivation rate of high-cell density Dehalococcoides cultures reported in the literature to date. This cultivation method provides a fundamental scientific platform for potential future operations of such a system at larger scales.

Description: κ-Carrageenases exhibit apparent distinctions in gene sequence, molecular weight, enzyme properties, and posttranslational processes. In this study, a new κ-carrageenase gene named cgk Z was cloned from the marine bacterium Zobellia sp. ZM-2. The gene comprised an open reading frame of 1,638 bp and encoded 545 amino acids. The natural signal peptide of κ-carrageenase was used successfully for the secretory production of the recombinant enzyme in Escherichia coli . A posttranslational process that removes an amino acid sequence of about 20 kDa from the C-terminal end of κ-carrageenase was first discovered in E. coli . An increase in enzyme activity by 167.3 % in the presence of 5 mM DTT was discovered, and Na + at a certain concentration range was positively correlated with enzyme activity. The κ-carrageenase production of E. coli was 9.0 times higher than that of ZM-2. These results indicate the potential use of the enzyme in the biotechnological industry.

Description: Fifty-five bacterial isolates were obtained from surface-sterilized nodules of woody and shrub legumes growing in Ethiopia: Crotalaria spp., Indigofera spp., and Erythrina brucei , and the food legumes soybean and common bean. Based on partial 16S rRNA gene sequence analysis, the majority of the isolates were identified as Gram-negative bacteria belonging to the genera Achromobacter , Agrobacterium , Burkholderia , Cronobacter , Enterobacter , Mesorhizobium , Novosphingobium , Pantoea , Pseudomonas , Rahnella , Rhizobium , Serratia , and Variovorax . Seven isolates were Gram-positive bacteria belonging to the genera Bacillus , Paenibacillus , Planomicrobium , and Rhodococcus . Amplified fragment length polymorphism (AFLP) fingerprinting showed that each strain was genetically distinct. According to phylogenetic analysis of recA , glnII , rpoB , and 16S rRNA gene sequences, Rhizobium , Mesorhizobium , and Agrobacterium were further classified into six different genospecies: Agrobacterium spp., Agrobacterium radiobacter , Rhizobium sp., Rhizobium phaseoli , Mesorhizobium sp., and putative new Rhizobium species. The strains from R . phaseoli , Rhizobium sp. IAR30, and Mesorhizobium sp. ERR6 induced nodules on their host plants. The other strains did not form nodules on their original host. Nine endophytic bacterial strains representing seven genera, Agrobacterium , Burkholderia , Paenibacillus , Pantoea , Pseudomonas , Rhizobium , and Serratia , were found to colonize nodules of Crotalaria incana and common bean on co-inoculation with symbiotic rhizobia. Four endophytic Rhizobium and two Agrobacterium strains had identical nifH gene sequences with symbiotic Rhizobium strains, suggesting horizontal gene transfer. Most symbiotic and nonsymbiotic endophytic bacteria showed plant growth-promoting properties in vitro, which indicate their potential role in the promotion of plant growth when colonizing plant roots and the rhizosphere.

Description: Escherichia coli BA002, in which the ldhA and pflB genes are deleted, cannot utilize glucose anaerobically due to the inability to regenerate NAD + . To restore glucose utilization, overexpression of nicotinic acid phosphoribosyltransferase (NAPRTase) encoded by the pncB gene, a rate-limiting enzyme of NAD(H) synthesis pathway, resulted in a significant increase in cell mass and succinate production under anaerobic conditions. However, a high concentration of pyruvate accumulated. Thus, co-expression of NAPRTase and the heterologous pyruvate carboxylase (PYC) of Lactococcus lactis subsp. cremoris NZ9000 in recombinant E . coli BA016 was investigated. The total concentration of NAD(H) was 9.8-fold higher in BA016 than in BA002, and the NADH/NAD + ratio decreased from 0.60 to 0.04. Under anaerobic conditions, BA016 consumed 17.50 g l −1 glucose and produced 14.08 g l −1 succinate with a small quantity of pyruvate. Furthermore, when the reducing agent dithiothreitol or reduced carbon source sorbitol was added, the cell growth and carbon source consumption rate of BA016 was reasonably enhanced and succinate productivity increased.

Description: 1,2-Benzenedicarboxaldehyde-3,4,5-trihydroxy-6-methyl (flavipin) was found to be antagonistic against nematodes and fungi. Here we demonstrated that flavipin is a potent antioxidant in vitro and in vivo, which has great potential in the therapy for free radical-associated diseases. Therefore, flavipin-producing bio-source was screened from 80 endophytes in Ginkgo biloba . Seven endophytic fungi were able to synthesize antioxidant substances and identified by ITS rDNA sequences. Among them, Chaetomium globosum CDW7 was a remarkable producer of flavipin. The fermentation parameters of CDW7 were then optimized for high flavipin production. Cultured under the optimal condition (25 °C, 100/250 mL flask, 12 discs/flask, 150 rpm, pH 6.5) for 14 days, CDW7 was able to synthesize flavipin at a production of 315.5 mg/L. In addition, flavipin output was positively correlated to antioxidant activities of crude extracts with a correlation coefficient of 0.8235, indicating that flavipin was the major antioxidant component of CDW7's metabolites. These data demonstrated that CDW7 was a highly yielded bio-source of antioxidant flavipin.

Description: Two bacterial hosts expressing cloned aromatic oxygenases were used to catalyze the oxidation and polymerization of indole and related substrates, creating mixtures of indigoid compounds comprised of novel dimers and trimers. Crude extracts and purified compounds were tested for their ability to inhibit the growth of Gram-positive organisms, in general, and Mycobacterium tuberculosis (TB), in particular. Of the 74 compounds tested against M. tuberculosis , ~66 % had minimum inhibitory concentrations (MIC) of 5 μg/ml or less. The most effective antibiotic found was designated SAB-P1, a heterodimer of indole and anthranil, which had a MIC of 0.16 μg/ml, and did not inhibit kidney cells (IC 50 ) at concentrations of 〉8 μg/ml. Combinatorial biocatalysis was used to create a series of halogenated derivatives of SAB-P1 with a wider therapeutic window. None of the derivatives had MIC values that were superior to SAB-P1, but some had a wider therapeutic window because of decreased kidney cell toxicity. Generally, the indigoid dimers that were effective against TB appeared to be specific for TB. Some of the trimers generated, however, had a broader spectrum of activity inhibiting not only TB (MIC = 1.1 μg/ml) but also the growth of Mycobacterium smegmatis MC2 155, Bacillus cereus , Enterococcus faecalis , Staphylococcus epidermidis , Bacillus subtilis 168, and Clostridium acetobutylicum . The structure of two of the novel dimers (SAB-C4 and SAB-P1) and a trimer (SAB-R1) were solved using X-ray crystallography.

Description: The compartmentalized anaerobic reactor (CAR) is a patent novel high-rate reactor, which consists of three compartments. The reactor has a great potential for application due to its many advantages. In this work, the microbial consortium, spatial distribution, and their relationship with performance of CAR were investigated by means of polymerase chain reaction, denaturing gradient gel electrophoresis, and fluorescence in situ hybridization. The results showed that the predominant archaea were Methanobacterium , Methanosaeta , and Methanospirillum , and the predominant bacteria were Firmicutes , Deltaproteobacteria , Spirochaetes , Actinobacteria , and Gammaproteobacteria in the microbial consortium. The methanogenic archaea (MA), the hydrogen-producing acetogenic bacteria (HAB), and the hydrolytic fermentative bacteria (HFB) were found to be predominant in the upper, middle, and bottom compartments, respectively. The results revealed that the granular sludge took on a stratified microbial structure. The HFB, HAB, and MA were located in the outer shell, middle layer, and core, respectively. The microbial populations from the bottom compartment were relatively homogeneous in the granular sludge, and from the middle and upper compartments, they were relatively heterogeneous in the granular sludge. The microbial consortia and their spatial distribution were in accordance with the organic loading rate and chemical components in the three compartments.

Description: We constructed beta-glucosidase (BGL)-displaying Corynebacterium glutamicum , and direct l -lysine fermentation from cellobiose was demonstrated. After screening active BGLs, Sde1394, which is a BGL from Saccharophagus degradans , was successfully displayed on the C. glutamicum cell surface using porin as an anchor protein, and cellobiose was directly assimilated as a carbon source. The optical density at 600 nm of BGL-displaying C. glutamicum grown on cellobiose as a carbon source reached 23.5 after 48 h of cultivation, which was almost the same as that of glucose after 24 h of cultivation. Finally, Sde1394-displaying C. glutamicum produced 1.08 g/l of l -lysine from 20 g/l of cellobiose after 4 days of cultivation, which was about threefold higher than the amount of produced l -lysine using BGL-secretory C. glutamicum strains (0.38 g/l after 5 days of cultivation). This is the first report on amino acid production using cellobiose as a carbon source by BGL-expressing C. glutamicum .

Description: The risk of antibody-dependent enhancement (ADE) of dengue virus (DENV) infection is a major obstacle for the development of dengue vaccine candidates. Here, we described a novel approach for assessment of ADE by measuring DENV nonstructural protein 1 (NS1) production in culture supernatants with Fcγ receptor-expressing K562 cells in ELISA format (ELISA-ADE). Enhancing activities quantified by measurement of kinetics of NS1 production were in a good agreement with the results of the virus titration assay. In conjunction with the previously established enzyme-linked immunospot-based micro-neutralization test (ELISPOT-MNT) in 96-well format, the observable dose–response profiles of enhancing and neutralizing activities against all four DENV serotypes were produced with two flaviviral envelope cross-reactive monoclonal antibodies and four primary DENV-1-infected human sera. The simple high-throughput ELISA-ADE assay offers advantages for quantitative measurement of infection enhancement that can potentially be applied to large-scale seroepidemiological studies of DENV infection and vaccination.

Description: Increasing concerns over limited petroleum resources and associated environmental problems are motivating the development of efficient cell factories to produce chemicals, fuels, and materials from renewable resources in an environmentally sustainable economical manner. Bacillus spp., the best characterized Gram-positive bacteria, possesses unique advantages as a host for producing microbial enzymes and industrially important biochemicals. With appropriate modifications to heterologous protein expression and metabolic engineering, Bacillus species are favorable industrial candidates for efficiently converting renewable resources to microbial enzymes, fine chemicals, bulk chemicals, and fuels. Here, we summarize the recent advances in developing Bacillus spp. as a cell factory. We review the available genetic tools, engineering strategies, genome sequence, genome-scale structure models, proteome, and secretion pathways, and we list successful examples of enzymes and industrially important biochemicals produced by Bacillus spp. Furthermore, we highlight the limitations and challenges in developing Bacillus spp. as a robust and efficient production host, and we discuss in the context of systems and synthetic biology the emerging opportunities and future research prospects in developing Bacillus spp. as a microbial cell factory.

Description: Glucosamine (GlcN), an amino sugar, is a compound derived from substitution of a hydroxyl group of a glucose molecule with an amino group. GlcN and its acetylated derivative, N -acetylglucosamine (GlcNAc), have been widely used in food, cosmetics, and pharmaceutical industries and are currently produced by acid hydrolysis of chitin (a linear polymer of GlcNAc) extracted from crab and shrimp shells. Microbial fermentation by filamentous fungi or recombinant Escherichia coli , as an alternative method for the production of GlcN and GlcNAc, is attracting increasing attention because it is an environmentally friendly process. Although the microbial production of GlcN and GlcNAc is hampered by low yield and high production cost, considerable advances have been made in recent years. Here we review the applications, commercial market, and production of GlcN and GlcNAc, with emphasis on the metabolic and process engineering strategies used to improve GlcN and GlcNAc production by recombinant microbes.

Description: In this work, the successful coupling of enzymatic oxidation and aldol addition reactions for the synthesis of a Cbz-aminopolyol from a Cbz-amino alcohol was achieved for the first time in a multienzymatic one-pot system. The two-step cascade reaction consisted of the oxidation of Cbz-ethanolamine to Cbz-glycinal catalyzed by chloroperoxidase from the fungus Caldariomyces fumago and aldol addition of dihydroxyacetone phosphate to Cbz-glycinal catalyzed by rhamnulose-1-phosphate aldolase expressed as a recombinant enzyme in Escherichia coli , yielding (3 R ,4 S )-5-{[(benzyloxy)carbonyl]amino}-5-deoxy-1- O -phosphonopent-2-ulose. Tools of enzymatic immobilization, reactor configurations, and modification of the reaction medium were applied to highly increase the production of the target compound. While the use of soluble enzymes yielded only 23.6 % of Cbz-aminopolyol due to rapid enzyme inactivation, the use of immobilized ones permitted an almost complete consumption of Cbz-ethanolamine, reaching Cbz-aminopolyol yields of 69.1 and 71.9 % in the stirred-tank and packed-bed reactor, respectively. Furthermore, the reaction production was 18-fold improved when it was catalyzed by immobilized enzymes in the presence of 5 % ( v / v ) dioxane, reaching a value of 86.6 mM of Cbz-aminopoliol (31 g/L).

Description: The complete genome of Acinetobacter oleivorans DR1 contains AqsR and AqsI genes, which are LuxR and LuxI homolog, respectively. In a previous study, we demonstrated that quorum sensing (QS) signals play an important role in biofilm formation and hexadecane biodegradation. However, the regulation of genes controlled by the QS system in DR1 remains unexplored. We constructed an aqsR mutant and performed RNA sequencing analysis to understand the QS system. A total of 353 genes were differentially expressed during the stationary phase of wild-type cells compared to that of the aqsR mutant. AqsR appears to be an exceptionally important regulator because knockout of aqsR affected global gene expression. Genes involved in posttranslational modification, chaperones, cell wall structure, secondary metabolites biosynthesis, and stress defense were highly upregulated only in the wild type. Among upregulated genes, both the AOLE_03905 (putative surface adhesion protein) and the AOLE_11355 (L-asparaginase) genes have putative LuxR binding sites at their promoter regions. Soluble AqsR proteins were successfully purified in Escherichia coli harboring both aqsR and aqsI . Comparison of QS signals in an AqsI–AqsR co-overexpression strain with N -acyl homoserine lactone standards showed that the cognate N -acyl homoserine lactone binding to AqsR might be 3OH C12HSL. Our electrophoretic mobility shift assays with purified AqsR revealed direct binding of AqsR to those promoter regions. Our data showed that AqsR functions as an important regulator and is associated with several phenotypes, such as hexadecane utilization, biofilm formation, and sensitivity to cumene hydroperoxide.

Description: A membrane-bound, pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenase (ADH) was purified from Frateuria aurantia LMG 1558 T . Although F . aurantia belongs to a group of γ-Proteobacteria, the characteristics of its PQQ-ADH were similar to the enzyme characteristics of the typical high-acetic acid-resistant bacterium Gluconacetobacter europaeus from the group of α-Proteobacteria. The PQQ-dependent ADH was solubilized from the membranes and purified after anionic, cationic, and affinity chromatography with specific activity of 117 U/mg. The purified enzyme was estimated to be composed of two subunits of ca. 72 and 45 kDa, as judged by SDS-polyacrylamide gel electrophoresis. The purified enzyme had maximum activity at pH 4.5 and showed the highest substrate specificity to ethanol, isoamyl alcohol, 1-butanol, and 1-propanol. The deduced sequences of cloned genes adhA and adhB encoding subunits I and II of PQQ-ADH showed 80 % amino acid (AA) identity to AdhA and 68 % AA identity to AdhB of Ga . europaeus V3 (LMG 18494). Because of the high similarity between genes encoding subunits I and II of PQQ-ADH and its homologous genes found in a distantly related taxonomic group of acetic acid bacteria, the results suggest the possibility of horizontal gene transfer between these two groups of genera.

Description: Enantioselective oxidation of racemic phenyl-1,2-ethanediol into ( R )-(−)-mandelic acid by a newly isolated Brevibacterium lutescens CCZU12-1 was demonstrated. It was found that optically active ( R )-(−)-mandelic acid ( e.e.p  〉 99.9 %) is produced leaving the other enantiomer ( S )-(+)-phenyl-1,2-ethanediol intact. Using fed-batch method, a total of 172.9 mM ( R )-(−)-mandelic acid accumulated in the reaction mixture after the seventh feed. Moreover, oxidation of phenyl-1,2-ethanediol using calcium alginate-entrapped resting cells was carried out in the aqueous system, and efficient biocatalyst recycling was achieved as a result of cell immobilization in calcium alginate, with a product-to-biocatalyst ratio of 27.94 g ( R )-(−)-mandelic acid g −1 dry cell weight cell after 16 cycles of repeated use.

Description: The bacterium Bacillus amyloliquefaciens anti-CA isolated from mangrove system was found to be able to actively kill Candida albicans isolated from clinic. The bacterial strain anti-CA could produce high level of bioactive substance, amylase and protease in the cheap medium containing 2.0 % soybean meal, 2.0 % wheat flour, pH 6.5 within 26 h. After purification, the main bioactive substance was confirmed to be a cyclic lipopeptide containing a heptapeptide, L-Asp→L-Leu→L-Leu→L-Val→L-Val→L-Glu→L-Leu and a 3-OH fatty acid (15 carbons). In addition to C. albicans , the purified lipopeptide can also kill many yeast strains including Metschnikowia bicuspidata , Candida tropicalis , Yarrowia lipolytica and Saccharomyces cerevisiae. After treated by the purified lipopeptide, both the whole cells and protoplasts of C. albicans were destroyed.

Description: The epothilones, compounds with anticancer mechanisms similar to that of paclitaxel (Taxol), are produced by strains of the myxobacterium Sorangium cellulosum , and the gene cluster responsible for epothilone biosynthesis is organised as a large operon. In this work, we showed that the 440-bp promoter regions of the operons from eight S. cellulosum strains have 94.27 % DNA sequence identity and 50 % variability in promoter activity in Escherichia coli . A primer extension analysis revealed two transcriptional start sites (TSSs) at 246 (TSS1) and 193 bp (TSS2) upstream of the translation start site (TLS), respectively. Promoter truncation determined that the basal promoter from the So0157-2 strain is located within a 264-bp region containing weak promoter activity; whereas in the 38-bp region upstream, the 264-bp promoter was required for the strong promoter activity, which was dramatically increased by 11-fold in average. There was a conserved stem–loop structure between TSS2 and the TLS, which was identified in E. coli as a negative regulatory element. In addition, the upstream non-conserved 357-bp non-coding region contributes to the promoter activity, increasing it by 1.5-fold. In conclusion, the expression of the epothilone operon non-coding region in E. coli is regulated by a double promoter (with −35 and −10 regions and two distinct TSSs), a stem–loop structure, and a distal non-coding region.

Description: Bacterial community compositions were characterized using denaturing gradient gel electrophoresis analysis of bacterial 16S rRNA gene in the sediments of the Pearl River estuary. Sequencing analyses of the excised bands indicated that Gram-negative bacteria, especially Gammaproteobacteria , were dominant in the Pearl River estuary. The diversity of polycyclic aromatic hydrocarbon ring-hydroxylating dioxygenase (PAH-RHD) gene in this estuary was then assessed by clone library analysis. The phylogenetic analyses showed that all PAH-RHD gene sequences of Gram-negative bacteria (PAH-RHD [GN] ) were closely related to the nagAc gene described for Ralstonia sp. U2 or nahAc gene for Pseudomonas sp. 9816–4, while the PAH-RHD gene sequences of Gram-positive bacteria (PAH-RHD [GP] ) at sampling site A1 showed high sequence similarity to the nidA gene from Mycobacterium species. Meanwhile, molecular diversity of the two functional genes was higher at the upstream of this region, while lower at the downstream. Redundancy analysis indicated that environmental factors, such as NH 4 -N, ∑PAHs, pH, SiO 3 -Si, and water depth, affected the distribution of the PAH-RHD [GN] gene in the Pearl River estuary.

Description: In order to provide a more suitable response to public health concerns, we improved the detection of infectious human adenoviruses in water by optimising the commonly used integrated cell culture–PCR method. Risk evaluation studies seek for rapid detection of infectious adenoviruses, including the enteric types 40 and 41 that are considered as the second most common agents of gastroenteritis in children next to rotaviruses. The here-employed 293A cell line used for infectious status assessment showed its ability to multiply adenoviruses including type 41. Two modifications were moreover applied to the workflow for viral detection. The first occurred at the nucleic acid extraction step performed directly on all infected cells, while the second was the application of real-time quantitative PCR as detection tool. All adaptations led to a 3-day reduction of the response delay and an improved sensitivity especially for the enteric adenoviral types. The infectious status of laboratory strain types 2 and 41 was demonstrated by a more than 2-log 10 increase in genome quantity. These conclusions were confirmed and reinforced by the analysis of water samples applying the improved assay. Naturally occurring infectious adenoviruses were detected in wastewater and river water, within 2 days. Types belonging to the species human adenoviruses C and type 31 were observed, but the most frequently identified type was 41 (71 % of identified sequences, n  = 34). This highlights the usefulness of our method for a wide range of types, and especially for the most prevalent and public health-relevant enteric adenoviruses.

Description: Proteins with a Zn(II) 2 Cys 6 domain, Cys-X 2 -Cys-X 6 -Cys-X 5-12 -Cys-X 2 -Cys-X 6-9 -Cys (hereafter, referred to as the C6 domain), form a subclass of zinc finger proteins found exclusively in fungi and yeast. Genome sequence databases of Saccharomyces cerevisiae and Candida albicans have provided an overview of this family of genes. Annotation of this gene family in most fungal genomes is still far from perfect and refined bioinformatic algorithms are urgently needed. Aspergillus flavus is a saprophytic soil fungus that can produce the carcinogenic aflatoxin. It is the second leading causative agent of invasive aspergillosis. The 37-Mb genome of A . flavus is predicted to encode 12,000 proteins. Two and a half percent of the total proteins are estimated to contain the C6 domain, more than twofold greater than those estimated for yeast, which is about 1 %. The variability in the spacing between cysteines, C 3 -C 4 and C 5 -C 6 , in the zinc cluster enables classification of the domains into distinct subgroups, which are also well conserved in Aspergillus nidulans . Sixty-six percent (202/306) of the A . flavus C6 proteins contain a specific transcription factor domain, and 7 % contain a domain of unknown function, DUF3468. Two A . nidulans C6 proteins containing the DUF3468 are involved in asexual conidiation and another two in sexual differentiation. In the anamorphic A . flavus , a homolog of the latter lacks the C6 domain. A . flavus being heterothallic and reproducing mainly through conidiation appears to have lost some components involved in homothallic sexual development. Of the 55 predicted gene clusters thought to be involved in production of secondary metabolites, only about half have a C6-encoding gene in or near the gene clusters. The features revealed by the A . flavus C6 proteins likely are common for other ascomycete fungi.

Description: A range of isolates of Pseudomonas aeruginosa from widely different environmental sources were examined for their ability to synthesise rhamnolipid biosurfactants. No significant differences in the quantity or composition of the rhamnolipid congeners could be produced by manipulating the growth conditions. Sequences for the rhamnolipid genes indicated low levels of strain variation, and the majority of polymorphisms did lead to amino acid sequence changes that had no evident phenotypic effect. Expression of the rhlB and rhlC rhamnosyltransferase genes showed a fixed sequential expression pattern during growth, and no significant up-regulation could be induced by varying producer strains or growth media. The results indicated that rhamnolipids are highly conserved molecules and that their gene expression has a rather stringent control. This leaves little opportunity to manipulate and greatly increase the yield of rhamnolipids from strains of P. aeruginosa for biotechnological applications.

Description: Streptococcus alactolyticus strain FGM, isolated from chicken cecum, was used to increase the extract yield of polysaccharides during Astragalus membranaceus fermentation. It was previously demonstrated that polysaccharides from fermented A . membranaceus by S . alactolyticus had some similar properties to those from A . membranaceus in terms of its ability to help heal hepatic fibrosis in rats and modulate immunopotentiation of broiler chicken. However, methods to increase the yield of the polysaccharides during fermentation of A . membranaceus are not well understood. In this paper, we investigated the involvement of uridine diphosphate (UDP)-glucose 4-epimerase ( galE ) and glucan-1,6-α-glucosidase ( dexB ) during A . membranaceus fermentation through real-time reverse transcription quantitative PCR. The galE and dexB genes of S . alactolyticus were cloned by homology-based cloning and the genome walking method for the first time, and the 3D structure of dexB was analyzed by Swiss-PdbViewer 4.0.1 software. The expression of both the galE and dexB genes in A . membranaceus fermentation was studied using the determined ideal reference gene ldh for transcript normalization. The results showed that these two genes were both highly induced and peaked after 12 h of fermentation. The expression level of galE was stepwise increased from 48 to 72 h, while dexB transcripts were markedly increased at 60 h and decreased by 72 h. These data suggested that dexB and galE of S . alactolyticus strain FGM were involved in the regulation of A . membranaceus fermentation and they might play some roles in the increase of polysaccharides.

Description: Rhodococcus erythropolis WZ010 was capable of producing optically pure (2 S ,3 S )-2,3-butanediol in alcoholic fermentation. The gene encoding an acetoin(diacetyl) reductase from R. erythropolis WZ010 (ReADR) was cloned, overexpressed in Escherichia coli , and subsequently purified by Ni-affinity chromatography. ReADR in the native form appeared to be a homodimer with a calculated subunit size of 26,864, belonging to the family of the short-chain dehydrogenase/reductases. The enzyme accepted a broad range of substrates including aliphatic and aryl alcohols, aldehydes, and ketones. It exhibited remarkable tolerance to dimethyl sulfoxide (DMSO) and retained 53.6 % of the initial activity after 4 h incubation with 30 % ( v / v ) DMSO. The enzyme displayed absolute stereospecificity in the reduction of diacetyl to (2 S ,3 S )-2,3-butanediol via ( S )-acetoin. The optimal pH and temperature for diacetyl reduction were pH 7.0 and 30 °C, whereas those for (2 S ,3 S )-2,3-butanediol oxidation were pH 9.5 and 25 °C. Under the optimized conditions, the activity of diacetyl reduction was 11.9-fold higher than that of (2 S ,3 S )-2,3-butanediol oxidation. Kinetic parameters of the enzyme showed lower K m values and higher catalytic efficiency for diacetyl and NADH in comparison to those for (2 S ,3 S )-2,3-butanediol and NAD + , suggesting its physiological role in favor of (2 S ,3 S )-2,3-butanediol formation. Interestingly, the enzyme showed higher catalytic efficiency for ( S )-1-phenylethanol oxidation than that for acetophenone reduction. ReADR-catalyzed asymmetric reduction of diacetyl was coupled with stereoselective oxidation of 1-phenylethanol, which simultaneously formed both (2 S ,3 S )-2,3-butanediol and ( R )-1-phenylethanol in great conversions and enantiomeric excess values.

Description: Regioselective glycosylation of flavonoids cannot be easily achieved due to the presence of several hydroxyl groups in flavonoids. This hurdle could be overcome by employing uridine diphosphate-dependent glycosyltransferases (UGTs), which use nucleotide sugars as sugar donors and diverse compounds including flavonoids as sugar acceptors. Quercetin rhamnosides contain antiviral activity. Two quercetin diglycosides, quercetin 3- O -glucoside-7- O -rhamnoside and quercetin 3,7- O -bisrhamnoside, were synthesized using Escherichia coli expressing two UGTs. For the synthesis of quercetin 3- O -glucoside-7- O -rhamnoside, AtUGT78D2, which transfers glucose from UDP-glucose to the 3-hydroxyl group of quercetin, and AtUGT89C1, which transfers rhamnose from UDP-rhamnose to the 7-hydroxyl group of quercetin 3- O -glucoside, were transformed into E. coli . Using this approach, 67 mg/L of quercetin 3- O -glucoside-7- O -rhamnoside was synthesized. For the synthesis of quercetin 3,7- O -bisrhamnoside, AtUGT78D1, which transfers rhamnose to the 3-hydroxy group of quercetin, and AtUGT89C1 were used. The RHM2 gene from Arabidopsis thaliana was coexpressed to supply the sugar donor, UDP-rhamnose. E. coli expressing AtUGT78D1 , AtUGT89C1 , and RHM2 was used to obtain 67.4 mg/L of quercetin 3,7- O -bisrhamnoside.

Description: Biological soil disinfestations (BSDs) were developed separately in Japan and in The Netherlands as an alternative to chemical fumigations. In Japan, it was developed based on the knowledge of irrigated paddy rice and upland crop rotation system that was rather tolerant of soil-borne disease development. The methods consist of application of easily decomposable organic matter, irrigation, and covering the soil surface with plastic film, thereby inducing anaerobic (reductive) soil conditions and suppressing many soil-borne pests including fungi, bacteria, nematodes, and weeds. The methods are widely used by organic farmers in the area where residences and agricultural fields are intermingled. To note one advantage of these methods, maintenance of soil suppressiveness to Fusarium wilt of tomato was suggested, while soil treated with chloropicrin became conducive to the disease. Suppression of soil-borne fungal pathogens by BSDs might be attributed to anaerobicity and high temperature, organic acids generated, and metal ions released into soil water. Contributions of respective factors to suppression of respective pathogens might be diverse. Presumably, these factors might vary on the fungal community structure in BSD-treated soil. These factors also work in paddy fields. Therefore, the BSDs developed in Japan are probably a method to raise the efficacy of paddy–upland rotation through intensive organic matter application and through maintenance of a strongly anaerobic (reductive) soil condition.

Description: The human host cell line, F2N78, is a new somatic hybrid cell line designed for therapeutic antibody production. To verify its potential as a human host cell line, recombinant F2N78 cells that produce antibody against rabies virus (rF2N78) were cultivated at different culture pH (6.8, 7.0, 7.2, 7.4, and 7.6) and temperatures (33.0 °C and 37.0 °C). Regardless of the culture temperature, the highest specific growth rate was obtained at a pH of 7.0–7.4. Lowering the culture temperature from 37.0 °C to 33.0 °C suppressed cell growth while allowing maintenance of high cell viability for a longer period. However, it did not enhance antibody production because specific antibody productivity did not increase at 33.0 °C. The highest maximum antibody concentration was obtained at 37.0 °C and pH 6.8. The N-linked glycosylation of the antibody was affected by the culture pH rather than the temperature. Nevertheless, G1F was dominant and G2F occupied a larger portion than G0F in all culture conditions. Compared to the same antibody produced from recombinant CHO cells, the antibody produced from rF2N78 cells has more galactose capping and was more similar to human plasma IgG. Taken together, the results obtained here demonstrate the potential of F2N78 as an alternative human host cell line for therapeutic antibody production.

Description: Microbial enhanced oil recovery (MEOR) refers to the process of using bacterial activities for more oil recovery from oil reservoirs mainly by interfacial tension reduction and wettability alteration mechanisms. Investigating the impact of these two mechanisms on enhanced oil recovery during MEOR process is the main objective of this work. Different analytical methods such as oil spreading and surface activity measurements were utilized to screen the biosurfactant-producing bacteria isolated from the brine of a specific oil reservoir located in the southwest of Iran. The isolates identified by 16S rDNA and biochemical analysis as Enterobacter cloacae (Persian Type Culture Collection (PTCC) 1798) and Enterobacter hormaechei (PTCC 1799) produce 1.53 g/l of biosurfactant. The produced biosurfactant caused substantial surface tension reduction of the growth medium and interfacial tension reduction between oil and brine to 31 and 3.2 mN/m from the original value of 72 and 29 mN/m, respectively. A novel set of core flooding tests, including in situ and ex situ scenarios, was designed to explore the potential of the isolated consortium as an agent for MEOR process. Besides, the individual effects of wettability alteration and IFT reduction on oil recovery efficiency by this process were investigated. The results show that the wettability alteration of the reservoir rock toward neutrally wet condition in the course of the adsorption of bacteria cells and biofilm formation are the dominant mechanisms on the improvement of oil recovery efficiency.

Description: Bioaugmentation with degrading bacteria is an effective method to improve the treatment of refractory industrial wastewater; nevertheless there were controversial opinions about the fate of inoculated bacteria and microbial community dynamics. In this study, two lab-scale sequencing batch reactors filled with modified zeolite were used to treat a coking wastewater with pyridine and quinoline shock load, and a bacterial consortium containing three degrading strains was added in one reactor for bioaugmentation. During 120-day operation, the bioaugmented reactor removed over 99 % pyridine, 99 % quinoline, 85 % TOC, 65 % COD, and 95 % NO 3 − -N with higher resistance to the shock load than the non-bioaugmented reactor. Based on the terminal restriction fragment length polymorphism of 16S rDNA, bacterial community diversity increased in the bioaugmented reactor. Principal component analysis revealed that, to cope with the shock load, the indigenous bacterial community recovered to the initial structure by acclimatizing itself constantly to the inhospitable environment; but bioaugmentation accelerated the shift of whole bacterial community, resulting in a far different structure from the initial one. Canonical correspondence analysis indicated that the environmental parameters of pyridine, quinoline, TOC, and NO 3 − -N had close negative correlations with bioaugmentation; and NH 3 -N and COD were the main parameters to impact on the bacterial community changes and treatment efficiency.

Description: Pseudomonads are metabolically versatile microbes that employ complex regulatory networks to control gene expression, particularly with respect to carbon and nitrogen metabolism. The aim of this study was to characterise the regulatory networks that control pyrimidine metabolism (hydantoin-hydrolysing activity) in Pseudomonas putida strain RU-KM3 S , focussing on transcriptional activation of dihydropyrimidinase (Dhp) and β-ureidopropionase (Bup), encoding dhp and bup , respectively. The two genes are arranged divergently on the chromosome and are separated by ORF1, encoding a putative transporter, which lies upstream of and in the same orientation as bup . The results from this study reveal that pyrimidine metabolism, as a function of Bup and Dhp activity in P . putida RU-KM3 S , is controlled by a complex regulatory network including several global pathways in addition to induction by the substrate. Three major control pathways act at the level of transcriptional and include: (1) induction of transcriptional activation in the presence of hydantoin, (2) carbon catabolite repression mediated via a pathway independent of Crc and (3) quorum sensing that does not require a putative lux box located upstream of the dhp transcriptional start. Finally, the data suggest a minor role for the global regulators Anr, Vfr and Crc, likely through regulation of the activity of transcription factors interacting directly with the bup /ORF1– dhp promoter.

Description: Bacillus subtilis ferments pyruvate to 2,3-butanediol via α-acetolactate synthase, α-acetolactate decarboxylase, and butanediol dehydrogenase (BDH), encoded by the alsSD operon and the unlinked monocistronic bdhA gene, respectively. Upstream and divergent from alsSD is the alsR gene that encodes AlsR, a member of the LysR-type transcriptional regulator family. AlsR directly stimulates alsSD transcription by binding to characteristic sites preceding the alsS promoter, but its effect on bdhA expression was unknown. The effect of AlsR on bdhA expression was assessed in a wild-type strain and a congenic strain carrying an alsR::spc knockout mutation by measuring: (a) expression of a transcriptional bdhA-lacZ fusion; (b) bdhA mRNA steady-state levels by quantitative reverse transcriptase PCR; and (c) expression of BDH enzymatic activity. Activation of bdhA expression occurred in early stationary phase, and expression was lowered, but not abolished, in the alsR::spc mutant. Mapping the transcriptional start site of bdhA by primer extension revealed a 268-nucleotide 5′-untranslated region preceding the bdhA initiation methionine codon. Transcription initiation was not reduced in the alsR::spc mutant, and by electrophoretic mobility shift assay, purified AlsR protein did not bind to the bdhA promoter region, suggesting that bdhA expression is indirectly under AlsR transcriptional control.

Description: Bacillus atrophaeus CAB-1 displays a high inhibitory activity against various fungal pathogens and suppresses cucumber powdery mildew and tomato gray mold. We extracted and identified lipopeptides and secreted proteins and volatile compounds produced by strain CAB-1 to investigate the mechanisms involved in its biocontrol performance. In vitro assays indicated all three types of products contributed to the antagonistic activity against the fungal pathogen Botrytis cinerea . Each of these components also effectively prevented the occurrence of the cucumber powdery mildew caused by Sphaerotheca fuliginea under greenhouse conditions. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry revealed that the major bioactive lipopeptide was fengycin A (C15–C17). We isolated the crude-secreted proteins of CAB-1 and purified a fraction with antifungal activity. This protein sequence shared a high identity with a putative phage-related pre-neck appendage protein, which has not been reported as an antifungal factor. The volatile compounds produced by CAB-1 were complex, including a range of alcohols, phenols, amines, and alkane amides. O -anisaldehyde represented one of the most abundant volatiles with the highest inhibition on the mycelial growth of B. cinerea . To our knowledge, this is the first report on profiling three types of antifungal substances in Bacilli and demonstrating their contributions to plant disease control.

Description: The re-emergence of tuberculosis in recent years led the World Health Organization (WHO) to launch the Stop TB Strategy program. Beside repurposing the existing drugs and exploring novel molecular combinations, an essential step to face the burden of tuberculosis will be to develop new drugs by identifying vulnerable bacterial targets. Recent studies have focused on decaprenylphosphoryl- d -ribose oxidase (DprE1) of Mycobacterium tuberculosis , an essential enzyme involved in cell wall metabolism, for which new promising molecules have proved efficacy as antitubercular agents. This review summarizes the state of the art concerning DprE1 in terms of structure, enzymatic activity and inhibitors. This enzyme is emerging as one of the most vulnerable target in M. tuberculosis .

Description: Arthrobacter simplex 156 is a microorganism that is used for steroid drug biotransformation of cortisone acetate (CA) to prednisone acetate (PA). The enzyme 3-ketosteroid-△ 1 -dehydrogenase encoded by the ksdD gene plays an important role in the bioconversion process. To further improve the biotransformation efficiencies of the industrial strain, a genetic manipulation system for A. simplex 156 was developed. Additional copies of the ksdD gene under the control of the cat promoter (from pXMJ19) were transferred into the strain A. simplex 156 and integrated into the 16S rDNA sites, yielding a series of recombinant strains. One of these recombinant strains, designated A. simplex M158, exhibited superior properties for CA biotransformation. At the substrate concentration of 83.6 g/l, the highest PA production of the recombinant strain reached 66.7 g/l, which is approximately 32.9 % higher than that of wild-type strains, and the incubation time for CA to PA bioconversion was reduced by 20 h. Southern blotting analysis of the recombinant strain indicated two copies of deregulated ksdD genes were integrated into the 16S rDNA sites, which means two of five 16S rRNA operons were insertionally disrupted in the recombinant strain. However, the disruption of the two 16S rRNA operons did not affect the growth rate of the recombinant strain, which survived and thrived under desired conditions. In addition, the new strain was genetically stable for more than 100 generations without the use of antibiotics for selection. These superior characteristics make the new strain more suitable than the wild-type strain for PA production.

Description: Nicotine is a significant toxic waste generated in tobacco manufacturing. Biological methods for the degradation of nicotine waste are in high demand. In this study, we report the identification and characterization of the novel nicotine-degrading strain Shinella sp. HZN7. This strain can degrade 500 mg/L nicotine completely within 3 h at 30 °C and pH values of 6.5 ∼ 8.0. The biodegradation of nicotine by Shinella sp. HZN7 involves five intermediate metabolites: 6-hydroxy-nicotine (6HN), 6-hydroxy- N -methylmyosmine, 6-hydroxypseudooxynicotine (6HPON), 6-hydroxy-3-succinoyl-pyridine (HSP), and 2,5-dihydroxypyridine, as detected by ultraviolet spectrophotometry, HPLC, and LC-MS. We generated three mutants, N7-W18, N7-X5, and N7-M17, by transposon mutagenesis, in which the nicotine-degrading pathway terminated at 6HN, 6HPON, and HSP, respectively. The production of the five intermediate metabolites and their order in the degradation pathway were confirmed in the three mutants, indicating that strain HZN7 degrades nicotine via a variant of the pyridine and pyrrolidine pathways. The mutant gene from strain N7-X5, orf2 , was cloned by self-formed adaptor PCR, but the nucleotide and amino acid sequence showed no similarity to any gene or gene product with defined function. However, orf2 disruption and complementation suggested that the orf2 gene is essential for the conversion of 6HPON to HSP in strain HZN7. This is the first study to provide genetic evidence for this variant nicotine degradation pathway.

Description: AcT (perhydrolase) containing paint composites were prepared leading to broad-spectrum decontamination. AcT was immobilized onto multi-walled carbon nanotubes (MWNTs) and then incorporated into latex-based paints to form catalytic coatings. These AcT-based paint composites showed a 6-log reduction in the viability of spores of Bacillus cereus and Bacillus anthracis (Sterne) within 60 min. The paint composites also showed 〉4-log reduction in the titer of influenza virus (X-31) within 10 min (initially challenged with 10 7 PFU/mL). AcT-based paint composites were also tested using various perhydrolase acyl donor substrates, including propylene glycol diacetate (PGD), glyceryl triacetate, and ethyl acetate, with PGD observed to be the best among the substrates tested for generation of peracetic acid and killing of bacillus spores. The operational stability of paint composites was also studied at different relative humidities and temperatures to simulate real-life operation.

Description: To investigate how the microbial community in activated sludge responded to high antibiotic levels, a bench-scale aerobic wastewater treatment system was used to treat oxytetracycline (OTC) mother liquor (OTC-ML). Removal efficiency of chemical oxygen demand decreased from 64.9 to 51.0 % when the OTC level increased from 191.6 to 620.5 mg/L, respectively. According to the cloning results, Psychrobacter and Cryptophyta were the dominant bacterium and eukaryote in the inoculated sludge, respectively, both of which related to low temperature. After OTC exposure, Alphaproteobacteria and Betaproteobacteria became the dominant bacteria, with a small proportion of Firmicutes , Actinobacteria appeared, and fungi (mainly Saccharomycotina ) became the dominant eukaryotes, indicating the possible functions of these microorganisms in the wastewater treatment of OTC-ML. The relative abundance of nine tetracycline resistance genes and four mobile elements (class 1 integron, class 2 integron, transposon Tn916/1545, and pattern 1 insertion sequence common region) significantly increased from undetectable to 2.1 × 10 −3 in the inoculated sludge to 1.7 × 10 −4 –9.8 × 10 −1 in sludge exposed to 620.5 mg/L OTC by using real-time PCR. The variety of gene cassette arrays of class 1 integron in the sludge samples increased with increasing OTC exposure concentration.

Description: Biologically active β-1,3-oligosaccharides with rapidly growing biomedical applications are produced from hydrolysis of curdlan polysaccharide. The water-insoluble curdlan impedes its hydrolysis efficiency which is enhanced by our newly developed alkali-neutralization treatment process to increase the stability of curdlan suspension to more than 20 days, while the untreated control settled within 5 min. A putative double-layer structure model comprising of a compact core and a hydrated outer layer was proposed to describe the treated curdlan particles based on sedimentation and scanning electron microscopy observation. This model was verified by single- and two-step acid hydrolysis, indicative of the reduced susceptibility to hydrolysis when close to the compact core. Electrospray ionization-mass spectrometry, thin-layer chromatography analyses, and effective HPLC procedure led to the development of improved process to produce purified individual β-1,3-oligosaccharides with degrees of polymerization from 2 to 10 and potential for biomedical applications from curdlan hydrolyzate. Our new curdlan oligosaccharide production process offers an even better alternative to the previously published processes.

Description: Enantiomerically pure l -homophenylalanine ( l -HPA) is a key building block for the synthesis of angiotensin-converting enzyme inhibitors and other chiral pharmaceuticals. Among the processes developed for the l -HPA production, biocatalytic synthesis employing phenylalanine dehydrogenase has been proven as the most promising route. However, similar to other dehydrogenase-catalyzed reactions, the viability of this process is markedly affected by insufficient substrate loading and high costs of the indispensable cofactors. In the present work, a highly efficient and economic biocatalytic process for l -HPA was established by coupling genetically modified phenylalanine dehydrogenase and formate dehydrogenase. Combination of fed-batch substrate addition and a continuous product removal greatly increased substrate loading and cofactor utilization. After systemic optimization, 40 g (0.22 mol) of keto acid substrate was transformed to l -HPA within 24 h and a total of 0.2 mM NAD + was reused effectively in eight cycles of fed-batch operation, consequently giving an average substrate concentration of 510 mM and a productivity of 84.1 g l −1  day −1 for l -HPA. The present study provides an efficient and feasible enzymatic process for the production of l -HPA and a general solution for the increase of substrate loading.

Description: Cyclodipeptides and their derivatives, the diketopiperazines (DKPs), constitute a large class of natural products that exhibit various biological properties. Until recently, there are a few characterized DKP biosynthetic pathways. In all these cases, the formation of the cyclodipeptides that harbor the DKP scaffold is catalyzed either by nonribosomal peptide synthetases or by cyclodipeptide synthases. This review focuses on the DKP biosynthetic pathways and their associated molecular mechanisms.

Description: Halophilic β-lactamase (BLA) has been successfully used as a novel fusion partner for soluble expression of aggregation-prone foreign proteins in Escherichia coli cytoplasm (Appl Microbiol Biotechnol 86:649–658, 2010b ). This halophilic BLA fusion technology was applied here for secretory expression in Brevibacillus . The “ Brevibacillus in vivo cloning” method, recently developed by Higeta Shoyu group, for the construction and transformation of Brevibacillus expression vectors facilitates efficient screening of the production conditions of Brevibacillus expression system. Two single-chain antibodies (scFv), HyHEL-10 single chain scFv (scFvHEL) and anti-fluorescein single chain scFv (scFvFLU), were successfully secreted to culture supernatant as a fusion protein with halophilic BLA. The scFvHEL-His, purified after cleavage of BLA portion with thrombin, was fully active: it formed a stoichiometric complex with the antigen, lysozyme, and inhibited the enzymatic activity. The scFvFLU-His, similarly expressed and purified, stoichiometrically inhibited fluorescence intensity of fluorescein. The molecular mass of scFvHEL-His was determined to be 27,800 Da by light scattering measurements, indicating its monomeric structure in solution.

Description: The effect of dietary nitrate supplementation on rumen bacterial community composition was examined in beef steers fed either a nitrate-N diet or urea-N diet. An automated method of ribosomal intergenic spacer analysis was applied to solid and liquid fractions of ruminal contents to allow comparison of bacterial communities. Supplemental N source affected relative population size of four amplicon lengths (ALs) in the liquid fraction and three ALs in the solid fraction. Five ALs were more prevalent after adaptation to nitrate. Correspondence analysis indicated that feeding the steers the nitrate-N diet versus urea-N diet changed the bacterial community composition in the liquid but not in the solid fraction. This led to an investigation of the relative sizes of potential nitrate-reducing populations. Mannheimia succiniciproducens , Veillonella parvula , and Campylobacter fetus were obtained from nitrate enrichment culture and quantified by real-time PCR based on 16S rRNA sequence. Nitrate supplementation increased the percentage of C . fetus in the liquid and solid phases, and in solid phase, the percentage of M . succiniciproducens increased. No change in species prevalence was observed for V . parvula . However, even after adaptation to dietary nitrate, the relative population sizes for all three putative nitrate-reducing species were very low (〈0.06 % of 16S rRNA gene copy number). The liquid-associated bacterial community composition changed due to nitrate supplementation, and at least part of this change reflects an increase in the species prevalence of C . fetus , a species which is not typically regarded as a ruminal inhabitant.

Description: Streptococcus zooepidemicus is a bacterial pathogen used for production of hyaluronan in industry. Intensive research has significantly contributed to our understanding of S . zooepidemicus biology and pathogenesis. However, the lack of an effective targeted gene inactivation system in S . zooepidemicus has notably prevented the functional genomics analysis of this gram-positive bacterium. Here, we report the development of a markerless gene deletion system in S . zooepidemicus . We constructed a sacB expression cassette on the thermosensitive suicide vector pSET4s and demonstrated its use as a counterselection marker in S . zooepidemicus . We validated the efficiency of this system by deletion of hasA , which synthesizes the important virulence factor hyaluronic acid (HA) capsule. The genotype of the resultant hasA mutant was confirmed by polymerase chain reaction and sequencing. Deletion of hasA resulted in non-mucoid morphology, loss of HA capsule formation, and HA production. These defects can be rescued by introduction of a plasmid containing wild-type hasA expression cassette. Moreover, compared with wild type, hasA mutant showed no significant difference in expressions of other members of the hasABCDE operon, further suggesting that the loss of hasA contributed to the defects observed with Δ hasA mutant. Our results describe the first establishment of a sacB -based counterselection system in S . zooepidemicus , along with the first demonstration of hasA that is the only gene encoding a functional hyaluronan synthase in this bacterium.

Description: Escherichia coli cells that express the full six carotenoid biosynthesis genes ( crtE , crtB , crtI , crtY , crtZ , and crtX ) of the bacterium Pantoea ananatis have been shown to biosynthesize zeaxanthin 3,3′-β- d -diglucoside. We found that this recombinant E. coli also produced a novel carotenoid glycoside that contained a rare carbohydrate moiety, quinovose (chinovose; 6-deoxy- d -glucose), which was identified as 3-β-glucosyl-3′-β-quinovosyl zeaxanthin by chromatographic and spectroscopic analyses. The chirality of the aglycone of these zeaxanthin glycosides had been shown to be 3 R ,3′ R , in which the hydroxyl groups were formed with the CrtZ enzyme. It was here demonstrated that zeaxanthin synthesized from β-carotene with CrtR or CYP175A1, the other hydroxylase with similar catalytic function to CrtZ, possessed the same stereochemistry. It was also suggested that the singlet oxygen-quenching activity of zeaxanthin 3,3′-β- d -diglucoside, which has a chemical structure close to the new carotenoid glycoside, was superior to that of zeaxanthin.

Description: The baculovirus expression vector system is widely used for the production of recombinant proteins. However, the yield of membrane-bound or secreted proteins is relatively low when compared with intracellular or nuclear proteins. In a previous study, we had demonstrated that the co-expression of the human chaperones calreticulin (CALR) or β-synuclein (β-syn) increased the production of a secreted protein considerably. A similar effect was also seen when co-expressing insect translation initiation factor eIF4E. In this study, different combinations of the three genes were tested (CALR alone, β-syn + CALR, or β-syn + CALR + eIF4E) to further improve secretory protein production by assessing the expression level of a recombinant secreted alkaline phosphatase (SEFP). An additional 1.8-fold increment of SEFP production was obtained when cells co-expressed all the three “helper” genes, compared to cells, in which only CALR was co-produced with SEFP. Moreover, the duration of the SEFP production lasted much longer in cells that co-expressed these three “helper” genes, up to 10 dpi was observed. Utilization of this “triple-supporters” containing vector offers significant advantages when producing secreted proteins and is likely to have benefits for the production of viral vaccines and other pharmaceutical products.

Description: Root colonization by antagonistic bacteria is a prerequisite for successful biological control, and the instability of colonization under varying environmental conditions has accentuated the need to improve the colonization activity. Root colonization by Bacillus spp. is mainly determined by chemotaxis and biofilm formation, and both functions are negatively controlled by the global transcription regulator AbrB. Here, we disrupted the gene abrB in B acillus amyloliquefaciens SQR9, which has been proven to be a promising biocontrol agent of cucumber and watermelon wilt disease. Chemotaxis, biofilm formation, and colonization activities as well as biocontrol efficiency were measured and compared between the wild-type strain of SQR9 and the abrB mutant. The data presented in this article demonstrate that the colonization and biocontrol activity of B . amyloliquefaciens SQR9 could be significantly improved by abrB gene disruption. The results offer a new strategy to enhance the biocontrol efficacy of B. amyloliquefaciens SQR9.

Description: The protein 3D8 single-chain variable fragment (3D8 scFv) has potential anti-viral activity due to its ability to penetrate into cells and hydrolyze nucleic acids. Probiotic Lactobacillus paracasei engineered to secrete 3D8 scFv for oral administration was used to test the anti-viral effects of 3D8 scFv against gastrointestinal virus infections. We found that injection of 3D8 scFv into the intestinal lumen resulted in the penetration of 3D8 scFv into the intestinal villi and lamina propria. 3D8 scFv secreted from engineered L. paracasei retained its cell-penetrating and nucleic acid-hydrolyzing activities, which were previously shown with 3D8 scFv expressed in Escherichia coli . Pretreatment of RAW264.7 cells with 3D8 scFv purified from L. paracasei prevented apoptosis induction by murine norovirus infection and decreased messenger RNA (mRNA) expression of the viral capsid protein VP1. In a mouse model, oral administration of the engineered L. paracasei prior to murine norovirus infection reduced the expression level of mRNA encoding viral polymerase. Taken together, these results suggest that L. paracasei secreting 3D8 scFv provides a basis for the development of ingestible anti-viral probiotics active against gastrointestinal viral infection.

Description: Bacterial contamination and biomass harvesting are still challenges associated with coupling of microalgae and wastewater treatment technology. This study investigated aggregation, bacterial growth, lipid production, and pollutant removal during bacteria contaminated Chlorella regularis cultivation under nutrient starvation stress, by supposing the C/N/P ratios of the medium to 14/1.4/1 (MB 2.5 ) and 44/1.4/1 (MB 4.0 ), respectively. Granules of 500–650 μm were formed in the bacteria contaminated inoculum; however, purified C. regularis were generally suspended freely in the medium, indicating that bacterial presence was a prerequisite for granulation. Extracellular polymeric substance (EPS) analysis showed that polysaccharides were dominant in granules, while protein mainly distributed in the outer layer. Denaturing gradient gel electrophoresis (DGGE) results revealed Sphingobacteriales bacterium and Sphingobacterium sp . are vital organisms involved in the flocculation of microalgae, and nitrifiers ( Stenotrophomonas maltophilia ) could co-exist in the granular. Both EPS and DGGE results further supported that bacteria played key roles in granulation. C. regularis was always dominant and determined the total biomass concentration during co-cultivation, but bacterial growth was limited owing to nutrient deficiency. Starvation strategy also contributed to enhancement of lipid accumulation, as lipid content in MB 4.0 with a greater C/N/P led to the greatest increase in the starvation period, and the maximum lipid productivity reached 0.057 g/(L·day). Chemical oxygen demand and nitrogen removal in MB 4.0 reached 92 and 96 %, respectively, after 3 days of cultivation. Thus, cultivation of microalgae in high C/N/P wastewater enabled simultaneous realization of biomass granulation, bacterial overgrowth limitation, enhanced lipid accumulation, and wastewater purification.

Description: Understanding the impact of polycyclic aromatic hydrocarbons (PAHs) on soil environments is of increasingly important concern. Therefore, the microbial degradation of PAHs in soils has drawn considerable attention, but little is known about the PAH degradation genes in urban soils. In this study, we examined the diversity and abundance of the PAH degradation bacteria and evaluated whether the specific bacteria can reflect PAH contents in the soils from urban roadsides directly receiving traffic emission. The results of phylogenetic analysis indicated that low PAH degradation bacterial diversity occurred in the urban roadside soils, only including Mycobacterium sp., Terrabacter sp., and one novel cluster. The community composition diversity of PAH degradation bacteria did not show a significant difference across the sampling sites. The abundance of PAH degradation genes ranged from 5.70 × 10 6 to 6.44 × 10 7  gene copies g −1 dry soil, with an average abundance of 1.43 × 10 7  gene copies g −1 dry soil, and their spatial variations were related significantly to PAH contents in the soils. The Mycobacterium sp. was the most widely detected and estimated to occupy 65.9–100 % of the total PAH degradation bacteria at most of the soil samples, implying that the Mycobacterium sp. might play a primary role in degrading PAHs in the contaminated urban soil environments.

Description: The ability of acetic acid bacteria (AAB) to produce cellulose has gained much industrial interest due to the physical and chemical characteristics of bacterial cellulose. The production of cellulose occurs in the presence of oxygen and in a glucose-containing medium, but it can also occur during vinegar elaboration by the traditional method. The vinegar biofilm produced by AAB on the air-liquid interface is primarily composed of cellulose and maintains the cells in close contact with oxygen. In this study, we screened for the ability of AAB to produce cellulose using different carbon sources in the presence or absence of ethanol. The presence of cellulose in biofilms was confirmed using the fluorochrome Calcofluor by microscopy. Moreover, the process of biofilm formation was monitored under epifluorescence microscopy using the Live/Dead BacLight Kit. A total of 77 AAB strains belonging to 35 species of Acetobacter , Komagataeibacter , Gluconacetobacter , and Gluconobacter were analysed, and 30 strains were able to produce a cellulose biofilm in at least one condition. This cellulose production was correlated with the PCR amplification of the bcsA gene that encodes cellulose synthase. A total of eight degenerated primers were designed, resulting in one primer pair that was able to detect the presence of this gene in 27 AAB strains, 26 of which formed cellulose.

Description: Pseudomonas aeruginosa is an opportunistic pathogen that localizes to and colonizes mucosal tissue. Thus, vaccines that elicit a strong mucosal response against P. aeruginosa should be superior to other vaccination strategies. In this study, to stimulate rapid and enhanced mucosal immune responses, mannose-modified chitosan microspheres loaded with the recombinant outer membrane protein OprF 190–342 -OprI 21–83 (FI) (FI-MCS-MPs) of P. aeruginosa were developed as a potent subunit vaccine for mucosal delivery. FI-MCS-MPs were successfully obtained via the tripolyphosphate ionic crosslinking method. Confocal and immunohistochemical analyses indicated that FI-MCS-MPs exhibited the ability to bind the macrophage mannose receptor (MMR, CD206) in vitro and in vivo. After intranasal immunization of mice with FI-MCS-MPs, FI-specific humoral immune responses were detected, measured as local IgM antibody titers in lung tissue slurry; IgA antibody titers in nasal washes, bronchoalveolar lavage (BAL), and intestinal lavage; and systemic IgA and IgG antibody titers in serum. FI-MCS-MPs induced early and high mucosal and systemic humoral antibody responses comparable to those in the group vaccinated with unmodified mannose. High levels of IFN-γ and IL-4 in addition to T lymphocyte subsets induced a mixed Th1/Th2 response in mice immunized with FI-MCS-MPs, resulting in the establishment of cellular immunity. Additionally, when immunized mice were challenged with P. aeruginosa via the nasal cavity, FI-MCS-MPs demonstrated 75 % protective efficacy. Together, these data indicate that mannose-modified chitosan microspheres are a promising subunit delivery system for vaccines against P. aeruginosa infection.

Description: The methylotrophic yeast Pichia pastoris is an attractive expression system due to its ability to secrete large amounts of recombinant protein, with the potential for glycosylation. Advances in glycoengineering of P. pastoris have successfully demonstrated the humanization of both the N- and O-linked glycosylation pathways in this organism. However, in certain cases, the presence of O-linked glycans on a therapeutic protein may not be desirable. Recently, we have reported the in vitro utility of jack bean α-1,2/3/6-mannosidase to remove O-linked mannose from intact undenatured glycoproteins produced in glycoengineered P. pastoris . However, one caveat of this strategy is that jack bean mannosidase has yet to be cloned and as such is only available as crude cellular extracts. This raises several concerns for using this reagent to treat large preparations of therapeutic proteins generated in P. pastoris . Therefore, we postulated that lysosomal mannosidases which have been cloned and demonstrated to have similar activities to jack bean mannosidase on N-linked glycans would also process O-linked glycans in a similar fashion. To this end, we screened a panel of recombinant lysosomal mannosidases from different organisms and identified several which cannot only reduce extended O-linked mannose chains but which can also hydrolyze the Man-α- O -Ser/Thr glycosidic bond on intact glycoproteins. As such, not only do we show for the first time the utility of lysosomal mannosidase for O-linked mannose processing, but since this is a recombinant enzyme, it has several benefits over the use of crude jack bean mannosidase extracts.

Description: The unfolded protein response (UPR) represents a mechanism to preserve endoplasmic reticulum (ER) homeostasis that is conserved in eukaryotes. ER stress caused by the accumulation of potentially toxic un- or misfolded proteins in the ER triggers UPR activation and the induction of genes important for protein folding in the ER, ER expansion, and transport from and to the ER. Along with this adaptation, the overall capacity for protein secretion is markedly increased by the UPR. In filamentous fungi, various approaches to employ the UPR for improved production of homologous and heterologous proteins have been investigated. As the effects on protein production were strongly dependent on the expressed protein, generally applicable strategies have to be developed. A combination of transcriptomic approaches monitoring secretion stress and basic research on the UPR mechanism provided novel and important insight into the complex regulatory cross-connections between UPR signalling, cellular physiology, and developmental processes. It will be discussed how this increasing knowledge on the UPR might stimulate the development of novel strategies for using the UPR as a tool in biotechnology.

Description: So far, the contribution of ammonia-oxidizing archaea (AOA) to ammonia oxidation in wastewater treatment processes has not been well understood. In this study, two soil aquifer treatment (SATs) systems were built up to treat synthetic domestic wastewater (column 1) and secondary effluent (column 4), accomplishing an average of 95 % ammonia removal during over 550 days of operation. Except at day 322, archaeal amoA genes always outnumbered bacterial amoA genes in both SATs as determined by using quantitative polymerase chain reaction (q-PCR). The ratios of archaeal amoA to 16S rRNA gene averaged at 0.70 ± 0.56 and 0.82 ± 0.62 in column 1 and column 4, respectively, indicating that all the archaea could be AOA carrying amoA gene in the SATs. The results of MiSeq-pyrosequencing targeting on archaeal and bacterial 16S rRNA genes with the primer pair of modified 515R/806R indicated that Nitrososphaera cluster affiliated with thaumarchaeal group I.1b was the dominant AOA species, while Nitrosospira cluster was the dominant ammonia-oxidizing bacteria (AOB). The statistical analysis showed significant relationship between AOA abundance (compared to AOB abundance) and inorganic and total nitrogen concentrations. Based on the mathematical model calculation for microbial growth, AOA had much greater capacity of ammonia oxidation as compared to the specific influent ammonia loading for AOA in the SATs, implying that a small fraction of the total AOA would actively work to oxidize ammonia chemoautotrophically whereas most of AOA would exhibit some level of functional redundancy. These results all pointed that AOA involved in microbial ammonia oxidation in the SATs.

Description: Selective capture of transcribed sequences (SCOTS) is an effective method to identify bacterial genes differentially expressed during different biological processes, including pathogenic interactions with a host species. The method can be used to elucidate molecular mechanisms driving and maintaining such interactions. The method is a powerful genetic tool that overcomes limitations found in other methods, by working with small amounts of mRNA and allowing for the separation of bacterial cDNA from host cDNA. It has been increasingly used in the discovery of genes involved in the bacterium-host interaction. In this review, we briefly introduce the SCOTS method, outline the technical advances offered in the method, and focus on the method’s applications in several microbial pathogens.

Description: A maltotriose-producing α-amylase, AmyA, from a newly isolated bacterial strain Microbulbifer thermotolerans DAU221 was purified and characterized in the heterologous host, Escherichia coli , using the pCold I vector. The amyA gene encoded a 761-residue protein composed of a 33 amino acid secretion signal peptide. The purified α-amylase with a molecular mass of 80 kDa, approximately, shared a sequence motif characteristic of the glycoside hydrolase family 13. The enzyme was optimally active, at 50 °C in sodium phosphate buffer (pH 6.0), by the traditional one factor-at-a-time method. But the optimal conditions of time, temperature, and pH for production of maltotriose from soluble starch were 1.76 h, 44.95 °C, and pH 6.35 by response surface methodology, respectively. Maltotriose, as the major enzyme reaction product, was analyzed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The enzyme was found to be inhibited by the addition of 10 mM Cu 2+ , Fe 3+ , Hg 2+ , Zn 2+ , and EDTA, but exhibited extreme stability toward hexane. The K m and V max values for the hydrolysis of soluble starch were 1.08 mg/mL and 1.736 mmol maltotriose/mg protein/min, respectively.

Description: The single-copy genes encoding putative polyphosphate–glucose phosphotransferases (PPGK, EC 2.7.1.63) from two nitrogen-fixing Cyanobacteria , Nostoc sp. PCC7120 and Nostoc punctiforme PCC73102, were cloned and functionally characterized. In contrast to their actinobacterial counterparts, the cyanobacterial PPGKs have shown the ability to phosphorylate glucose using strictly inorganic polyphosphates (polyP) as phosphoryl donors. This has proven to be an economically attractive reagent in contrast to the more costly ATP. Cyanobacterial PPGKs had a higher affinity for medium–long-sized polyP (greater than ten phosphoryl residues). Thus, longer polyP resulted in higher catalytic efficiency. Also in contrast to most their homologs in Actinobacteria , both cyanobacterial PPGKs exhibited a modest but significant polyP-mannokinase activity as well. Specific activities were in the range of 180–230 and 2–3 μmol min −1  mg −1 with glucose and mannose as substrates, respectively. No polyP-fructokinase activity was detected. Cyanobacterial PPGKs required a divalent metal cofactor and exhibited alkaline pH optima (approx. 9.0) and a remarkable thermostability (optimum temperature, 45 °C). The preference for Mg 2+ was noted with an affinity constant of 1.3 mM. Both recombinant PPGKs are homodimers with a subunit molecular mass of ca. 27 kDa. Based on database searches and experimental data from Southern blots and activity assays, closely related PPGK homologs appear to be widespread among unicellular and filamentous mostly nitrogen-fixing Cyanobacteria . Overall, these findings indicate that polyP may be metabolized in these photosynthetic prokaryotes to yield glucose (or mannose) 6-phosphate. They also provide evidence for a novel group-specific subfamily of strictly polyP-dependent gluco(manno)kinases with ancestral features and high biotechnological potential, capable of efficiently using polyP as an alternative and cheap source of energy-rich phosphate instead of costly ATP. Finally, these results could shed new light on the evolutionary origin of sugar kinases.

Description: Estuarine and tidal wetlands with high primary productivity and biological activity play a crucial role in coastal nutrient dynamics. Here, to better reveal the effects of extracellular enzymes and microbial community on carbon (C) and nitrogen (N) mineralization, the incubation experiments with different C and N addition patterns to the tidal sediments of the Yangtze Estuary (China) were conducted. The results suggested a significant increase in cumulative CO 2 effluxes in the C and CN treatment experiments, while no significant difference in cumulative CO 2 effluxes between the N treatment and control (CK) experiments was observed. In addition, the nutrient addition patterns had a great influence on dissolve organic C and N levels, but a small effect on microbial biomass C and N. Microbial community composition and microbial activity were found to be positively correlated with organic C (OC) and the molar ratio of C to N (C/N). Partial correlation analysis, controlling for C/N, supported direct effects of OC on the activity of carbon-cycling extracellular enzymes (cellulase and polyphenol oxidase), while C/N exhibited negatively correlations with urease and Gram-positive bacteria to Gram-negative bacteria (G+/G−). Strong relationships were found between CO 2 efflux and mineral nitrogen with the activity of specific enzymes (sucrase, cellulase, and polyphenol oxidase) and abundances of Gram-negative bacteria, arbuscular mycorrhizal fungi, and fungi, suggesting the significant influences of microbial community and enzyme activity on C and N mineralization in the estuarine and tidal wetlands. Furthermore, this study could highlight the need to explore effects of nutrient supply on microbial communities and enzyme activity changes associated with the C and N mineralization in these wetlands induced by the climate change.

Description: In this study, the process of pyrite colonization and leaching by three iron-oxidizing Acidithiobacillus species was investigated by fluorescence microscopy, bacterial attachment, and leaching assays. Within the first 4–5 days, only the biofilm subpopulation was responsible for pyrite dissolution. Pyrite-grown cells, in contrast to iron-grown cells, were able to oxidize iron(II) ions or pyrite after 24 h iron starvation and incubation with 1 mM H 2 O 2 , indicating that these cells were adapted to the presence of enhanced levels of reactive oxygen species (ROS), which are generated on metal sulfide surfaces. Acidithiobacillus ferrivorans SS3 and Acidithiobacillus ferrooxidans R1 showed enhanced pyrite colonization and biofilm formation compared to A. ferrooxidans T . A broad range of factors influencing the biofilm formation on pyrite were also identified, some of them were strain-specific. Cultivation at non-optimum growth temperatures or increased ionic strength led to a decreased colonization of pyrite. The presence of iron(III) ions increased pyrite colonization, especially when pyrite-grown cells were used, while the addition of 20 mM copper(II) ions resulted in reduced biofilm formation on pyrite. This observation correlated with a different extracellular polymeric substance (EPS) composition of copper-exposed cells. Interestingly, the addition of 1 mM sodium glucuronate in combination with iron(III) ions led to a 5-fold and 7-fold increased cell attachment after 1 and 8 days of incubation, respectively, in A. ferrooxidans T . In addition, sodium glucuronate addition enhanced pyrite dissolution by 25 %.

Description: Background Monoacetylated xylosyl residues of the main hardwood hemicellulose acetylglucuronoxylan undergo acetyl group migration between positions 2 and 3, and predominantly to position 4 of the non-reducing end xylopyranosyl (NRE-Xyl p ) residues which are amplified by saccharifying enzymes. On monoacetylated non-reducing end xylopyranosyl (NRE-Xyl p ) residues of xylooligosaccharides the acetyl group migrates predominantly to position 4 and hinders their hydrolysis by β-xylosidase. Methods Acetyl migration on the NRE-Xyl p residues and their enzymatic deacetylation by various xylan deacetylases was followed by 1 H-NMR spectroscopy and TLC. Results A 5-min heat treatment of 4-nitrophenyl 3- O -acetyl-β-D-xylopyranoside was sufficient to establish equilibrium between monoacetate derivatives acetylated at positions 2, 3 and 4. Rapid acetyl migration along the NRE-Xyl p ring at elevated temperature was confirmed in derivatives of methyl β-1,4-xylotrioside (Xyl 3 Me) monoacetylated solely on the NRE-Xyl p residue, the analogues of naturally occurring acetylated xylooligosaccharides. The Xyl 3 Me monoacetates were used as substrates to study regioselectivity of the NRE-Xyl p residue deacetylation by various acetylxylan esterases (AcXEs) of distinct carbohydrate esterase (CE) families. CE1, CE4 and CE6 AcXEs hydrolyzed considerably faster the 2″- O -acetyl derivative than the 3″- O -acetyl derivative. In contrast, the CE16 acetyl esterase preferred to attack the ester bond at position 3 followed by position 4. Conclusions Redistribution of acetyl group on the NRE-Xyl p residues is extremely rapid at elevated temperature and includes the formation of 4-acetate. Regioselectivity of AcXEs and CE16 acetyl esterase on these monoacetates is complementary. General significance The formation of all isomers of acetylated xylosyl residues must be taken into account after a long-term incubation of acetylxylan and acetylated xylooligosaccharides solutions or upon their treatment at elevated temperatures. This phenomenon emphasizes requirement of both types of xylan deacetylases to enable a rapid saccharification of xylooligosaccharides by glycoside hydrolases.

Description: Foot-and-mouth disease (FMD) remains a major threat to livestock worldwide, especially in developing countries. To improve the efficacy of vaccination against FMD, various types of vaccines have been developed, including synthetic peptide vaccines. We designed three synthetic peptide vaccines, 59 to 87 aa in size, based on immunogenic epitopes in the VP1, 3A, and 3D proteins of the A/HuBWH/CHA/2009 strain of the foot-and-mouth disease virus (FMDV), corresponding to amino acid positions 129 to 169 of VP1, 21 to 35 of 3A, and 346 to 370 of 3D. The efficacies of the vaccines were evaluated in cattle and guinea pigs challenged with serotype-A FMDV. All of the vaccines elicited the production of virus-neutralizing antibodies. The PB peptide, which contained sequences corresponding to positions 129 to 169 of V P1 and 346 to 370 of 3D, demonstrated the highest levels of immunogenicity and immunoprotection against FMDV. Two doses of 50 μg of the synthetic PB peptide vaccine provided 100 % protection against FMDV infection in guinea pigs, and a single dose of 100 μg provided 60 % protection in cattle. These findings provide empirical data for facilitating the development of synthetic peptide vaccines against FMD.

Description: Corynebacterium glutamicum can consume glucose to excrete glycerol under oxygen deprivation. Although glycerol synthesis from 1,3-dihydroxyacetone (DHA) has been speculated, no direct evidence has yet been provided in C. glutamicum . Enzymatic and genetic investigations here indicate that the glycerol is largely produced from DHA and, unexpectedly, the reaction is catalyzed by ( S , S )-butanediol dehydrogenase (ButA) that inherently catalyzes the interconversion between S -acetoin and ( S , S )-2,3-butanediol. Consequently, the following pathway for glycerol biosynthesis in the bacterium emerges: dihydroxyacetone phosphate is dephosphorylated by HdpA to DHA, which is subsequently reduced to glycerol by ButA. This study emphasizes the importance of promiscuous activity of the enzyme in vivo.

Description: Cofactor is especially important for biotransformation catalyzed by oxidoreductases. Many attempts in enhancing performance of the reactions by improving cofactor utilization have been reported. In this study, efficiency of cofactor-requiring biocatalysis was enhanced by improving cofactor recycling via spatially programmed assembling glycerol dehydrogenase (GlyDH, Escherichia coli MG1655) and glutamate dehydrogenase (GluDH, Bacillus subtilis str168), with the aid of single-stranded DNA (ssDNA). The two enzymes were first independently expressed as molecules fused with a phage protein A* that could covalently link ssDNA with certain features. After an enzymatic cross-linking reaction taking place under mild conditions, the conjugate of fused enzyme and ssDNA was assembled into desired structures through base pairing enabled by the ssDNA. Results showed that, to some extent, the fusion with protein A* could improve the specific activity of the enzymes (GlyDH-A*/GlyDH = 116.0 %; GluDH-A*/GluDH = 105.2 %). Additionally, in the coupled reaction system with glycerol and α-ketoglutaric acid as substrates, regarding the production of glutamic acid based on HPLC analysis, the efficiency of cofactor utilization was significantly enhanced (by 23.8- to 41.9-folds), indicating the existence of a substrate-channeling mechanism for cofactor utilization in the assembled reaction system due to the proximity effects. The degree of substrate channeling was calculated as from 1.65 to 1.73. Furthermore, the efficiency of cofactor utilization was influenced in an architecture-dependent manner when complexes with different stoichiometry of GlyDH and GluDH were utilized in biotransformation. This study demonstrated a novel strategy of cofactor recycling for enhanced performance of coupled oxidoreductive reactions.

Description: In the present study, the use of Rhodococcus erythropolis mutant strain RG9 expressing the cytochrome P450 BM3 mutant M02 enzyme has been evaluated for whole-cell biotransformation of a 17-ketosteroid, norandrostenedione, as a model substrate. Purified P450 BM3 mutant M02 enzyme hydroxylated the steroid with 〉95 % regioselectivity to form 16-β-OH norandrostenedione, as confirmed by NMR analysis. Whole cells of R. erythropolis RG9 expressing P450 BM3 M02 enzyme also converted norandrostenedione into the 16-β-hydroxylated product, resulting in the formation of about 0.35 g/L. Whole cells of strain RG9 itself did not convert norandrostenedione, indicating that metabolite formation is P450 BM3 M02 enzyme mediated. This study shows that R. erythropolis is a novel and interesting host for the heterologous expression of highly selective and active P450 BM3 M02 enzyme variants able to perform steroid bioconversions.

Description: 2,4-Dichlorophenol (2,4-DCP) is considered as an important pollutant because of its high toxicity and wide distribution in wastewaters. Innocuous remediation technologies have been studied for the removal of this pollutant. This study investigated the feasibility of using garlic roots as a plant system for the removal of 2,4-DCP. The optimal conditions for its removal were established based on orthogonal experiments (OA 25 matrix). Significant factors that affect removal efficiency, arranged from high to low importance, include pH, reaction time, 2,4-DCP concentration, and H 2 O 2 concentration. In addition, garlic roots could be re-used for as much as three consecutive cycles. The decrease in pH and the increase of Cl − ion content in the post-removal solutions indicated that 2,4-DCP dehalogenation occurred during transformation. Changes in the deposition pattern of lignin in roots exposed to 2,4-DCP suggested that several of the products deposited were lignin-type polymers. The acute toxicity test revealed that the post-removal solutions were less toxic than the parent solutions. Therefore, garlic roots have considerable potential to effectively and safely remove 2,4-DCP from wastewater.

Description: Yro2 and its paralogous protein Mrh1 of Saccharomyces cerevisiae have seven predicted transmembrane domains and predominantly localize to the plasma membrane. Their physiological functions and regulation of gene expression have not yet been elucidated in detail. We herein demonstrated that MRH1 was constitutively expressed, whereas the expression of YRO2 was induced by acetic acid stress and entering the stationary phase. Fluorescence microscopic analysis revealed that Mrh1 and Yro2 were distributed as small foci in the plasma membrane under acetic acid stress conditions. The null mutants of these genes ( mrh1 ∆, yro2 ∆, and mrh1 ∆ yro2 ∆) showed delayed growth and a decrease in the productivity of ethanol in the presence of acetic acid, indicating that Yro2 and Mrh1 are involved in tolerance to acetic acid stress.

Description: The use of the food-grade bacterium Lactococcus lactis as a vehicle for the oral delivery of DNA vaccine plasmids constitutes a promising strategy for vaccination. The delivery of DNA plasmids into eukaryotic cells is of critical importance for subsequent DNA expression and effectiveness of the vaccine. In this context, the use of the recombinant invasive L. lactis FnBPA+ (fibronectin-binding protein A) strain for the oral delivery of the eukaryotic expression vector vaccination using lactic acid bacteria (pValac), coding for the 6-kDa early secreted antigenic target (ESAT-6) gene of Mycobacterium tuberculosis , could represent a new DNA vaccine strategy against tuberculosis. To this end, the ESAT-6 sequence was cloned into the pValac vector; the L. lactis fibronectin-binding protein A (FnBPA)+ (pValac: ESAT -6) strain was obtained, and its immunological profile was checked in BALB/c mice. This strain was able to significantly increase interferon gamma (IFN-γ) production in spleen cells, showing a systemic T helper 1 (Th1) cell response. The mice also showed a significant increase in specific secretory immunoglobulin A (sIgA) production in colon tissue and fecal extracts. Thus, this is the first time that L. lactis has been used to deliver a plasmid DNA harboring a gene that encodes an antigen against tuberculosis through mucous membranes.

Description: Diacylglycerol acyltransferase (DGAT) catalyzes the acyl-CoA-dependent acylation of sn -1,2-diacylglycerol to produce triacylglycerol (TAG). This enzyme, which is critical to numerous facets of oilseed development, has been highlighted as a genetic engineering target to increase storage lipid production in microorganisms designed for biofuel applications. Here, four transcriptionally active DGAT1 genes were identified and characterized from the oil crop Brassica napus . Overexpression of each BnaDGAT1 in Saccharomyces cerevisiae increased TAG biosynthesis. Further studies showed that adding an N-terminal tag could mask the deleterious influence of the DGATs’ native N-terminal sequences, resulting in increased in vivo accumulation of the polypeptides and an increase of up to about 150-fold in in vitro enzyme activity. The levels of TAG and total lipid fatty acids in S. cerevisiae producing the N-terminally tagged BnaDGAT1.b at 72 h were 53 and 28 % higher than those in cultures producing untagged BnaA.DGAT1.b, respectively. These modified DGATs catalyzed the synthesis of up to 453 mg fatty acid/L by this time point. The results will be of benefit in the biochemical analysis of recombinant DGAT1 produced through heterologous expression in yeast and offer a new approach to increase storage lipid content in yeast for industrial applications.

Description: Naphtho-γ-pyrones (NGPs) are secondary metabolites mainly produced by filamentous fungi ( Fusarium sp., Aspergillus sp.) that should be considered by industrials. Indeed, these natural biomolecules show various biological activities: anti-oxidant, anti-microbial, anti-cancer, anti-HIV, anti-hyperuricuric, anti-tubercular, or mammalian triacylglycerol synthesis inhibition which could be useful for pharmaceutical, cosmetic, and/or food industries. In this review, we draw an overview on the interest in studying fungal NGPs by presenting their biological activities and their potential values for industrials, their biochemical properties, and what is currently known on their biosynthetic pathway. Finally, we will present what remains to be discovered about NGPs.

Description: In Bacillus subtilis , natural genetic competence is subject to complex genetic regulation and quorum sensing dependent. Upon extracellular accumulation of the peptide-pheromone ComX, the membrane-bound sensor histidine kinase ComP initiates diverse signaling pathways by activating—among others—DegQ and ComS. While DegQ favors the expression of extracellular enzymes rather than competence development, ComS is crucial for competence development as it prevents proteolytic degradation of ComK, the key transcriptional activator of all genes required for the uptake and integration of DNA. In Bacillus licheniformis , ComX/ComP sensed cell density negatively influences competence development, suggesting differences from the quorum-sensing-dependent control mechanism in Bacillus subtilis . Here, we show that each of six investigated strains possesses both of two different, recently identified putative comS genes. When expressed from an inducible promoter, none of the comS candidate genes displayed an impact on competence development neither in B. subtilis nor in B. licheniformis . Moreover, disruption of the genes did not reduce transformation efficiency. While the putative comS homologs do not contribute to competence development, we provide evidence that the degQ gene as for B. subtilis negatively influences genetic competency in B. licheniformis .

Description: To better understand the quantitative relationships between messenger RNA (mRNA) and protein biomarkers relevant to bioremediation, we quantified and compared respiration-associated gene products in an anaerobic syntrophic community. Respiration biomarkers for Dehalococcoides , an organohalide reducer, and Methanospirillum , a hydrogenotrophic methanogen, were quantified via qRT-PCR for mRNA and multiple reaction monitoring (MRM) of proteotypic peptides for protein. mRNA transcripts of the Dehalococcoides reductive dehalogenases PceA, TceA, and DMC1545, and hydrogenase HupL, as well as the Methanospirillum oxidoreductases MvrD and FrcA were shown to be similarly regulated with respect to their temporal responses to substrate addition. However, MvrD was two orders of magnitude lower in mRNA abundance. Per cell, Dehalococcoides protein biomarkers quantified were more abundant than Methanospirillum proteins. Comparing mRNA with protein abundance, poor correlations were observed between mRNA transcript levels and the net protein produced. For example, Dehalococcoides HupL and TceA transcripts were similarly abundant though TceA was far more abundant at the protein level (167 ± 121 vs. 1095 ± 337 proteins per cell, respectively). In Methanospirillum , MvrD maintained comparable per-cell protein abundance to FrcA (42 ± 14 vs. 60 ± 1 proteins per cell, respectively) despite the significantly lower transcript levels. Though no variability in protein decay rates was observed, the mRNA translation rate quantified for TceA was greater than the other Dehalococcoides targets monitored. These data suggest that there is considerable variation in the relationship between mRNA abundance and protein production both across transcripts within an organism and across organisms. This highlights the importance of empirically based studies for interpreting biomarker levels in environmentally relevant organisms.

Description: Pyroligneous acid (PA) is a complex highly oxygenated aqueous liquid fraction obtained by the condensation of pyrolysis vapors, which result from the thermochemical breakdown or pyrolysis of plant biomass components such as cellulose, hemicellulose, and lignin. PA produced by the slow pyrolysis of plant biomass is a yellowish brown or dark brown liquid with acidic pH and usually comprises a complex mixture of guaiacols, catechols, syringols, phenols, vanillins, furans, pyrans, carboxaldehydes, hydroxyketones, sugars, alkyl aryl ethers, nitrogenated derivatives, alcohols, acetic acid, and other carboxylic acids. The phenolic components, namely guaiacol, alkyl guaiacols, syringol, and alkyl syringols, contribute to the smoky odor of PA. PA finds application in diverse areas, as antioxidant, antimicrobial, antiinflammatory, plant growth stimulator, coagulant for natural rubber, and termiticidal and pesticidal agent; is a source for valuable chemicals; and imparts a smoky flavor for food.

Description: Two-stage process based on photofermentation of dark fermentation effluents is widely recognized as the most effective method for biological production of hydrogen from organic substrates. Recently, it was described an alternative mechanism, named capnophilic lactic fermentation, for sugar fermentation by the hyperthermophilic bacterium Thermotoga neapolitana in CO 2 -rich atmosphere. Here, we report the first application of this novel process to two-stage biological production of hydrogen. The microbial system based on T. neapolitana DSM 4359 T and Rhodopseudomonas palustris 42OL gave 9.4 mol of hydrogen per mole of glucose consumed during the anaerobic process, which is the best production yield so far reported for conventional two-stage batch cultivations. The improvement of hydrogen yield correlates with the increase in lactic production during capnophilic lactic fermentation and takes also advantage of the introduction of original conditions for culturing both microorganisms in minimal media based on diluted sea water. The use of CO 2 during the first step of the combined process establishes a novel strategy for biohydrogen technology. Moreover, this study opens the way to cost reduction and use of salt-rich waste as feedstock.

Description: Mycoinsecticides application within Integral Pest Management requires high quantities of conidia, with the proper quality and resistance against environmental conditions. Metarhizium anisopliae var. lepidiotum conidia were produced in normal atmospheric conditions (21 % O 2 ) and different concentrations of oxygen pulses (16, 26, 30, and 40 %); conidia obtained under hypoxic conditions showed significantly lower viability, hydrophobicity, and virulence against Tenebrio molitor larvae or mealworm, compared with those obtained under normal atmospheric conditions. Higher concentrations of oxygen (26 and 30 %) improved conidial production. However, when a 30 % oxygen concentration was applied, maximal conidial yields were obtained at earlier times (132 h) relative to 26 % oxygen pulses (156 h); additionally, with 30 % oxygen pulses, conidia thermotolerance was improved, maintaining viability, hydrophobicity, and virulence. Although conidial production was not affected when 40 % oxygen pulses were applied, viability and virulence were diminished in those conidia. In order to find a critical time for mycelia competence to respond to these oxidant conditions, oxygen pulses were first applied either at 36, 48, 60, and 72 h. A critical time of 60 h was determined to be the best time for the M. anisopliae var. lepidiotum mycelia to respond to oxygen pulses in order to increase conidial production and also to maintain the quality features. Therefore, oxygen-enriched (30 %) pulses starting at 60 h are recommended for a high production without the impairment of quality of M. anisopliae var. lepidiotum conidia.

Description: Butenoic acid is a C 4 short-chain unsaturated fatty acid mainly used in the preparation of resins, pharmaceuticals, and fine chemicals. However, butenoic acid derived from petroleum is costly and unfriendly to the environment. Here, we report a novel biosynthetic strategy to produce butenoic acid by utilizing the intermediate of fatty acid biosynthesis pathway in engineered Escherichia coli . A thioesterase gene ( B. thetaiotaomicron thioesterase ( bTE )) from Bacteroides thetaiotaomicron was heterologously expressed in E. coli to specifically convert butenoyl-acyl carrier protein (ACP), a fatty acid biosynthesis intermediate, to butenoic acid. The titer of butenoic acid ranged from 0.07 to 11.4 mg/L in four different E. coli strains with varied expressing vectors. Deletion of endogenous fadD gene (encoding acyl-CoA synthetase) to block fatty acid oxidation improved the butenoic acid production in all strains to some extent. The highest butenoic acid accumulation of 18.7 mg/L was obtained in strain XP-2 (BL21-∆ fadD /pET28a- bTE ). Moreover, partially inhibiting the enoyl-ACP reductase (FabI) of strain XP-2 by triclosan increased butenoic acid production by threefold, and the butenoic acid titer was further increased to 161.4 mg/L by supplying glucose and tryptone in the M9 medium. Fed-batch fermentation of this strain further enhanced butenoic acid production to 4.0 g/L within 48 h. The butenoic acid tolerance assay revealed that this strain could tolerate 15–20 g/L of butenoic acid.

Description: Bacillus amyloliquefaciens strains FZBREP and FZBSPA were derived from the wild-type FZB42 by replacement of the native bacilysin operon promoter with constitutive promoters P repB and P spac from plasmids pMK3 and pLOSS, respectively. These strains contained two antibiotic resistance genes, and markerless strains were constructed by deleting the chloramphenicol resistance cassette and promoter region bordered by two lox sites ( lox 71 and lox 66) using Cre recombinase expressed from the temperature-sensitive vector pLOSS-cre. The vector-encoded spectinomycin resistance gene was removed by high temperature (50 °C) treatment. RT-PCR and qRT-PCR results indicated that P repB and especially P spac significantly increased expression of the bac operon, and FZBREP and FZBSPA strains produced up to 170.4 and 315.6 % more bacilysin than wild type, respectively. Bacilysin overproduction was accompanied by enhancement of the antagonistic activities against Staphylococcus aureus (an indicator of bacilysin) and Clavibacter michiganense subsp. sepedonicum (the causative agent of potato ring rot). Both the size and degree of ring rot-associated necrotic tubers were decreased compared with the wild-type strain, which confirmed the protective effects and biocontrol potential of these genetically engineered strains.

Description: Highly specific and fast multiplex detection methods are essential to conduct reasonable DNA-based diagnostics and are especially important to characterise infectious diseases. More than 1000 genetic targets such as antibiotic resistance genes, virulence factors and phylogenetic markers have to be identified as fast as possible to facilitate the correct treatment of a patient. In the present work, we developed a novel ligation-based DNA probe concept that was combined with the microarray technology and used it for the detection of bacterial pathogens. The novel linear chain (LNC) probes identified all tested species correctly within 1 h based on their 16S rRNA gene in a 25-multiplex reaction. Genomic DNA was used directly as template in the ligation reaction identifying as little as 10 7 cells without any pre-amplification. The high specificity was further demonstrated characterising a single nucleotide polymorphism leading to no false positive fluorescence signals of the untargeted single nucleotide polymorphism (SNP) variants. In comparison to conventional microarray probes, the sensitivity of the novel LNC3 probes was higher by a factor of 10 or more. In summary, we present a fast, simple, highly specific and sensitive multiplex detection method adaptable for a wide range of applications.