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Vamvaka E.,University of Lleida | Twyman R.M.,TRM Ltd | Murad A.M.,Laboratory of Synthetic Biology | Melnik S.,University of Natural Resources and Life Sciences, Vienna | And 8 more authors.
Plant Biotechnology Journal | Year: 2016

Protein microbicides against HIV can help to prevent infection but they are required in large, repetitive doses. This makes current fermenter-based production systems prohibitively expensive. Plants are advantageous as production platforms because they offer a safe, economical and scalable alternative, and cereals such as rice are particularly attractive because they could allow pharmaceutical proteins to be produced economically and on a large scale in developing countries. Pharmaceutical proteins can also be stored as unprocessed seed, circumventing the need for a cold chain. Here, we report the development of transgenic rice plants expressing the HIV-neutralizing antibody 2G12 in the endosperm. Surprisingly for an antibody expressed in plants, the heavy chain was predominantly aglycosylated. Nevertheless, the heavy and light chains assembled into functional antibodies with more potent HIV-neutralizing activity than other plant-derived forms of 2G12 bearing typical high-mannose or plant complex-type glycans. Immunolocalization experiments showed that the assembled antibody accumulated predominantly in protein storage vacuoles but also induced the formation of novel, spherical storage compartments surrounded by ribosomes indicating that they originated from the endoplasmic reticulum. The comparison of wild-type and transgenic plants at the transcriptomic and proteomic levels indicated that endogenous genes related to starch biosynthesis were down-regulated in the endosperm of the transgenic plants, whereas genes encoding prolamin and glutaredoxin-C8 were up-regulated. Our data provide insight into factors that affect the functional efficacy of neutralizing antibodies in plants and the impact of recombinant proteins on endogenous gene expression. © 2016 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd. Source

« Uno-X Hydrogen to build 1st hydrogen refueling station w/ hydrogen produced by surplus renewable energy from neighboring building | Main | H2 Logic to develop large-scale production plant for H2 refueling stations » Researchers from the Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences have identified and characterized the first thermophilic bacterium capable (Defluviitalea phaphyphila) of direct conversion of brown algae to ethanol. D. phaphyphila Alg1 can simultaneously utilize mannitol, glucose, and alginate to produce ethanol. In an open access paper on their work published in the journal Biotechnology for Biofuels, they report high ethanol yields of 0.47 g/g-mannitol, 0.44 g/g-glucose, and 0.3 g/g-alginate. Brown algae are a large group of marine seaweeds including almost 1800 species of macroalgae with a characteristic olive-green to dark brown color derived from fucoxanthin. … The technology for the mass production of macroalgae has been developed significantly in China and Asia over the last 50 years. Notably, China contributes 72% of global aquaculture-based macroalgae production, including the genera of Laminaria (reclassified as Saccharina for some species, brown algae), Undaria (green algae), Porphyra, and Gracilaria (red algae). Brown algae have complex sugar composition, mainly including alginate, mannitol, and laminarin. Alginate is the unique structural polysaccharides in brown algae… The content of alginate varied from 20 to 40% of dry weight among different species. Mannitol and laminarin are considered as reserve carbohydrates in many brown algae species, which are mostly accumulated in summer. Mannitol is a sugar alcohol form of mannose, while laminarin is a linear polysaccharide… The content of mannitol and laminarin in some species can reach as high as 25 and 30 %, respectively, at the beginning of autumn. In earlier work, the identified a strain of Defluviitalea phaphyphila from coastal sediments. Analysis of the genome indicated that the strain has an integrated brown algae-degrading system. They evaluated the growth and ethanol fermentation properties of D. phaphyphila by employing alginate, mannitol, or glucose (laminarin monomer) as substrates. When glucose (1 %, w/v) was used as sole carbon substrate, all glucose was exhausted in less than 48 h. After 108 h cultivation, 3.8 g/L ethanol and 0.4 g/L acetic acid were produced, with an ethanol-to-acetate ratio of 9.5:1. The ethanol yield was 0.38 g/g-glucose which accounted for 74% of the maximal yield of 0.51 g/g-glucose. In the case of mannitol (1 %, w/v) as substrate, the concentrations of ethanol and acetic acid could be as high as 4.3 and 0.3 g/L, respectively, with an ethanol-to-acetate ratio around 14:1. The ethanol yield from mannitol was about 0.44 g/g-mannitol, which accounted for 86% of the theoretical maximal yield of 0.51 g/g-mannitol. Mannitol was exhausted until 108 h indicating a slower substrate assimilation rate than glucose. Alginate could not be fully dissolved in the medium; total protein concentration of cells was used to monitor the growth of D. phaphyphila in the case of alginate fermentation. A total of 7.6 g/L alginate was consumed after 108 h. Totally, 2.7 g/L ethanol and 3 g/L acetic acid were produced, with an ethanol-to-acetate ratio of 0.9:1. It can be concluded that D. phaphyphila Alg1 could successfully convert alginate into ethanol and acetate, and its products contained more acetic acid than those from glucose and mannitol. They also used an artificial mixed sugar simulating the main components of brown algae with a ratio of alginate:mannitol:glucose = 5:8:1 (3% total sugar). A total of 7.8 g/L ethanol and 1.2 g/L acetic acid were produced with a consumption of 24.2 g/L total sugars. The ethanol-to-acetate ratio and ethanol yield were calculated to be 6.5:1 and 0.32 g/g-total sugar, respectively. It can be concluded that D. phaphyphila Alg1 could simultaneously saccharify and ferment the three main components of brown algae, and although these components have different oxidoreduction potentials, reducing equivalents are well balanced and metabolic flux is directed into ethanol production in this strain. They also obtained an ethanol yield of 0.25 g/g-biomass in fermenting unpretreated kelp.

Murad A.M.,Laboratory of Synthetic Biology | Vianna G.R.,Laboratory of Synthetic Biology | Machado A.M.,Laboratory of Synthetic Biology | Da Cunha N.B.,Laboratory of Synthetic Biology | And 4 more authors.
Analytical and Bioanalytical Chemistry | Year: 2014

Improving the quality and performance of soybean oil as biodiesel depends on the chemical composition of its fatty acids and requires an increase in monounsaturated acids and a reduction in polyunsaturated acids. Despite its current use as a source of biofuel, soybean oil contains an average of 25 % oleic acid and 13 % palmitic acid, which negatively impacts its oxidative stability and freezing point, causing a high rate of nitrogen oxide emission. Gas chromatography and ion mobility mass spectrometry were conducted on soybean fatty acids from metabolically engineered seed extracts to determine the nature of the structural oleic and palmitic acids. The soybean genes FAD2-1 and FatB were placed under the control of the 35SCaMV constitutive promoter, introduced to soybean embryonic axes by particle bombardment and downregulated using RNA interference technology. Results indicate that the metabolically engineered plants exhibited a significant increase in oleic acid (up to 94.58 %) and a reduction in palmitic acid (to <3 %) in their seed oil content. No structural differences were observed between the fatty acids of the transgenic and non-transgenic oil extracts. Source

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