Expression of Trichoderma reesei β-Mannanase in Tobacco Chloroplasts and Its Utilization in Lignocellulosic Woody Biomass Hydrolysis

Lignocellulosic ethanol offers a promising alternative to conventional fossil fuels. One among the major limitations in the lignocellulosic biomass hydrolysis is unavailability of efficient and environmentally biomass degrading technologies. Plant-based production of these enzymes on large scale offers a cost-effective solution. Cellulases, hemicellulases including mannanases and other accessory enzymes are required for conversion of lignocellulosic biomass into fermentable sugars. β-mannanase catalyzes endo-hydrolysis of the mannan backbone, a major constituent of woody biomass. In this study, the man1 gene encoding β-mannanase was isolated from Trichoderma reesei and expressed via the chloroplast genome. PCR and Southern hybridization analysis confirmed site-specific transgene integration into the tobacco chloroplast genomes and homoplasmy. Transplastomic plants were fertile and set viable seeds. Germination of seeds in the selection medium showed inheritance of transgenes into the progeny without any Mendelian segregation. Expression of endo-β-mannanase for the first time in plants facilitated its characterization for use in enhanced lignocellulosic biomass hydrolysis. Gel diffusion assay for endo-β-mannanase showed the zone of clearance confirming functionality of chloroplast-derived mannanase. Endo-β-mannanase expression levels reached up to 25 units per gram of leaf (fresh weight). Chloroplast-derived mannanase had higher temperature stability (40°C to 70°C) and wider pH optima (pH 3.0 to 7.0) than E.coli enzyme extracts. Plant crude extracts showed 6–7 fold higher enzyme activity than E.coli extracts due to the formation of disulfide bonds in chloroplasts, thereby facilitating their direct utilization in enzyme cocktails without any purification. Chloroplast-derived mannanase when added to the enzyme cocktail containing a combination of different plant-derived enzymes yielded 20% more glucose equivalents from pinewood than the cocktail without mannanase. Our results demonstrate that chloroplast-derived mannanase is an important component of enzymatic cocktail for woody biomass hydrolysis and should provide a cost-effective solution for its diverse applications in the biofuel, paper, oil, pharmaceutical, coffee and detergent industries

Low Cost Tuberculosis Vaccine Antigens in Capsules: Expression in Chloroplasts, Bio-Encapsulation, Stability and Functional Evaluation In Vitro

Tuberculosis (TB) caused by Mycobacterium tuberculosis is one of the leading fatal infectious diseases. The development of TB vaccines has been recognized as a major public health priority by the World Health Organization. In this study, three candidate antigens, ESAT-6 (6kDa early secretory antigenic target) and Mtb72F (a fusion polyprotein from two TB antigens, Mtb32 and Mtb39) fused with cholera toxin B-subunit (CTB) and LipY (a cell wall protein) were expressed in tobacco and/or lettuce chloroplasts to facilitate bioencapsulation/oral delivery. Site-specific transgene integration into the chloroplast genome was confirmed by Southern blot analysis. In transplastomic leaves, CTB fusion proteins existed in soluble monomeric or multimeric forms of expected sizes and their expression levels varied depending upon the developmental stage and time of leaf harvest, with the highest-level of accumulation in mature leaves harvested at 6PM. The CTB-ESAT6 and CTB-Mtb72F expression levels reached up to 7.5% and 1.2% of total soluble protein respectively in mature tobacco leaves. Transplastomic CTB-ESAT6 lettuce plants accumulated up to 0.75% of total leaf protein. Western blot analysis of lyophilized lettuce leaves stored at room temperature for up to six months showed that the CTB-ESAT6 fusion protein was stable and preserved proper folding, disulfide bonds and assembly into pentamers for prolonged periods. Also, antigen concentration per gram of leaf tissue was increased 22 fold after lyophilization. Hemolysis assay with purified CTB-ESAT6 protein showed partial hemolysis of red blood cells and confirmed functionality of the ESAT-6 antigen. GM1-binding assay demonstrated that the CTB-ESAT6 fusion protein formed pentamers to bind with the GM1-ganglioside receptor. The expression of functional Mycobacterium tuberculosis antigens in transplastomic plants should facilitate development of a cost-effective and orally deliverable TB booster vaccine with potential for long-term storage at room temperature. To our knowledge, this is the first report of expression of TB vaccine antigens in chloroplasts

Many analysts, one data set: making transparent how variations in analytic choices affect results

Twenty-nine teams involving 61 analysts used the same data set to address the same research question: whether soccer referees are more likely to give red cards to dark-skin-toned players than to light-skin-toned players. Analytic approaches varied widely across the teams, and the estimated effect sizes ranged from 0.89 to 2.93 (Mdn = 1.31) in odds-ratio units. Twenty teams (69%) found a statistically significant positive effect, and 9 teams (31%) did not observe a significant relationship. Overall, the 29 different analyses used 21 unique combinations of covariates. Neither analysts’ prior beliefs about the effect of interest nor their level of expertise readily explained the variation in the outcomes of the analyses. Peer ratings of the quality of the analyses also did not account for the variability. These findings suggest that significant variation in the results of analyses of complex data may be difficult to avoid, even by experts with honest intentions. Crowdsourcing data analysis, a strategy in which numerous research teams are recruited to simultaneously investigate the same research question, makes transparent how defensible, yet subjective, analytic choices influence research results

Origin of the hot gas in low-mass protostars, Herschel-PACS spectroscopy of HH 46

Aims. “Water In Star-forming regions with Herschel” (WISH) is a Herschel key programme aimed at understanding the physical and chemical structure of young stellar objects (YSOs) with a focus on water and related species. Methods. The low-mass protostar HH 46 was observed with the Photodetector Array Camera and Spectrometer (PACS) on the Herschel Space Observatory to measure emission in H2O, CO, OH, [O i], and [C ii] lines located between 63 and 186 μm. The excitation and spatial distribution of emission can disentangle the different heating mechanisms of YSOs, with better spatial resolution and sensitivity than previously possible. Results. Far-IR line emission is detected at the position of the protostar and along the outflow axis. The OH emission is concentrated at the central position, CO emission is bright at the central position and along the outflow, and H2O emission is concentrated in the outflow. In addition, [O i] emission is seen in low-velocity gas, assumed to be related to the envelope, and is also seen shifted up to 170 km s−1 in both the red- and blue-shifted jets. Envelope models are constructed based on previous observational constraints. They indicate that passive heating of a spherical envelope by the protostellar luminosity cannot explain the high-excitation molecular gas detected with PACS, including CO lines with upper levels at >2500 K above the ground state. Instead, warm CO and H2O emission is probably produced in the walls of an outflow-carved cavity in the envelope, which are heated by UV photons and non-dissociative C-type shocks. The bright OH and [Oi] emission is attributed to J-type shocks in dense gas close to the protostar. In the scenario described here, the combined cooling by far-IR lines within the central spatial pixel is estimated to be 2 × 10−2 L, with 60–80% attributed to J- and C-type shocks produced by interactions between the jet and the envelope

Production of a recombinant bacterial lipoprotein in higher plant chloroplasts

Little is known about the potential of plastids to accomplish post-translational modifications of foreign proteins. In the present study we generated transplastomic tobacco plants that accumulate the outer surface lipoprotein A (OspA)-the basic constituent of the first generation monovalent human vaccine against Lyme disease. The recombinant OspA exhibits a lipid modification typical for bacteria and induced protective antibodies in mice, demonstrating that functionally active bacterial lipoproteins can be processed in plants

Production of a recombinant bacterial lipoprotein in higher plant chloroplasts.

Little is known about the potential of plastids to accomplish post-translational modifications of foreign proteins. In the present study we generated transplastomic tobacco plants that accumulate the outer surface lipoprotein A (OspA)-the basic constituent of the first generation monovalent human vaccine against Lyme disease. The recombinant OspA exhibits a lipid modification typical for bacteria and induced protective antibodies in mice, demonstrating that functionally active bacterial lipoproteins can be processed in plants