The O-mannosylation and production of recombinant APA (45/47 KDa) protein from Mycobacterium tuberculosis in Streptomyces lividans is affected by culture conditions in shake flasks

<p>Abstract</p> <p>Background</p> <p>The Ala-Pro-rich <it>O</it>-glycoprotein known as the 45/47 kDa or APA antigen from <it>Mycobacterium tuberculosis </it>is an immunodominant adhesin restricted to mycobacterium genus and has been proposed as an alternative candidate to generate a new vaccine against tuberculosis or for diagnosis kits. In this work, the recombinant <it>O</it>-glycoprotein APA was produced by the non-pathogenic filamentous bacteria <it>Streptomyces lividans</it>, evaluating three different culture conditions. This strain is known for its ability to produce heterologous proteins in a shorter time compared to <it>M. tuberculosis</it>.</p> <p>Results</p> <p>Three different shake flask geometries were used to provide different shear and oxygenation conditions; and the impact of those conditions on the morphology of <it>S. lividans </it>and the production of rAPA was characterized and evaluated. Small unbranched free filaments and mycelial clumps were found in baffled and coiled shake flasks, but one order of magnitude larger pellets were found in conventional shake flasks. The production of rAPA is around 3 times higher in small mycelia than in larger pellets, most probably due to difficulties in mass transfer inside pellets. Moreover, there are four putative sites of <it>O</it>-mannosylation in native APA, one of which is located at the carboxy-terminal region. The carbohydrate composition of this site was determined for rAPA by mass spectrometry analysis, and was found to contain different glycoforms depending on culture conditions. Up to two mannoses residues were found in cultures carried out in conventional shake flasks, and up to five mannoses residues were determined in coiled and baffled shake flasks.</p> <p>Conclusions</p> <p>The shear and/or oxygenation parameters determine the bacterial morphology, the productivity, and the <it>O</it>-mannosylation of rAPA in <it>S. lividans</it>. As demonstrated here, culture conditions have to be carefully controlled in order to obtain recombinant <it>O</it>-glycosylated proteins with similar "quality" in bacteria, particularly, if the protein activity depends on the glycosylation pattern. Furthermore, it will be an interesting exercise to determine the effect of shear and oxygen in shake flasks, to obtain evidences that may be useful in scaling-up these processes to bioreactors. Another approach will be using lab-scale bioreactors under well-controlled conditions, and study the impact of those on rAPA productivity and quality.</p

Phenotyping of Urochloa humidicola grass hybrids for agronomic and environmental performance in the Piedmont region of the Orinoquian savannas of Colombia

In the low fertility acid soils of the Orinoquian savannas of Colombia, Urochloa humidicola cv. Tully or Humidicola is one of the most widely planted tropical forage grasses for improving livestock productivity. Low nutritional quality of this grass limits sustainable livestock production in this region. In this study, we conducted a phenotypic evaluation under field and greenhouse conditions of one of the first hybrid populations of U. humidicola generated from the forage breeding program of CIAT. Our objective was to identify a set of new hybrids of U. humidicola that combine improved productivity and nutritional quality plus the biological nitrification inhibition (BNI) trait/ability to reduce nitrogen (N) losses via leaching and nitrous oxide (N2O) emissions. To this end, we tested 118 hybrids (planted in pots) in the greenhouse for over 6 months and measured potential nitrification rates (NR) using soil microcosm incubation. NR values observed ranged from 0.27 to 5.75 mg N-NO3- kg soil(-1) day(-1). Later, 12 hybrids with different levels of NR were selected and field-tested in the Orinoquia region over a 4 years period (2013-2017) for dry matter production, nutrition quality (crude protein, in vitro digestibility and fibres content) and NR in each year. In the rainy season of 2018, two hybrids with superior agronomic performance and contrasting field level NR (Uh08/1149 and 0450) were subjected to analysis of soil-borne N2O emissions after fertilization during 13 days. The NR values recorded were not directly correlated with the forage quality parameters evaluated, however, the two grasses with the lowest NR values were among those with the highest biomass production, crude protein content, and N uptake. The grass hybrid Uh08/1149 and the germplasm accession CIAT 16888 were found as materials with superior forage value, with production of 14.1 and 14.6 tons dry matter ha(-1) year(-1) (up to 8% higher than the cv. Tully), crude protein of 11.5 and 9.1% per cut (up to 20% higher than the cv. Tully), and N uptake of 31.6 and 25.7 kg N ha(-1) cut(-1) (up to 30% higher than the cv. Tully). Additionally, these two grasses are likely to exhibit high-BNI ability, with potential to improve N use efficiency in managed pastures.ISSN:0142-5242ISSN:1365-249

Biological Nitrification Inhibition (BNI) in \u3cem\u3eBrachiaria\u3c/em\u3e Pastures: A Novel Strategy to Improve Eco-Efficiency of Crop-Livestock Systems and to Mitigate Climate Change

Up to 70% of the nitrogen (N) fertilizers applied to agricultural systems are lost due to nitrification and denitrification. Nitrification is a microbiological process that generates nitrate (NO3-) and promotes the losses of N fertilizers by leaching and denitrification. Nitrification and denitrification are the only known biological processes that generate nitrous oxide (N2O), a powerful greenhouse gas contributing to global warming. There is an urgent need to suppress nitrification process in soil to improve N-recovery and N use efficiency (NUE) of agricultural systems and to mitigate climate change (Subbarao et al. 2012). Certain Brachiaria grasses (B. humidicola) can suppress soil-nitrification by releasing biological nitrification inhibitors (BNIs) from roots, thereby reducing N2O emissions. This phenomenon, termed biological nitrification inhibition (BNI), has been the subject of recent research to characterize and validate the concept under field conditions (Subbarao et al. 2009). Advances on three aspects of BNI research are reported here: (1) gene quantification of soil nitrifying microorganisms to determine BNI activity in B. humidicola; (2) screening of B. humidicola breeding materials to identify hybrids with contrasting levels of BNI: and (3) quantification of the BNI-residual effect from B. humidicola on N-recovery and agronomic-NUE of the subsequent maize crop

Climate-Smart Crop-Livestock Systems for Smallholders in the Tropics: Integration of New Forage Hybrids to Intensify Agriculture and to Mitigate Climate Change through Regulation of Nitrification in Soil

It is widely recognized that less than 50% of applied nitrogen (N) fertilizer is recovered by crops, and based on current fertilizer prices the economic value of this “wasted N” globally is currently estimated as US$81 billion annually. Worse still, this wasted N has major effects on the environment (Subbarao et al. 2012). CIAT researchers and their collaborators in Japan reported a major breakthrough in managing N to benefit both agriculture and the environment (Subbarao et al. 2009). Termed Biological Nitrification Inhibition (BNI), this natural phenomenon has been the subject of long-term collaborative research that revealed the mechanism by which certain plants (and in particular the tropical pasture grass B. humidicola) naturally inhibit the conversion of N in the soil from a stable form to forms subject to leaching loss (NO3) or to the potent greenhouse gas N2O (Subbarao et al. 2012). Brachiaria humidicola which is well adapted to the low-nitrogen soils of South American savannas has shown high BNI-capacity among the tropical grasses tested (Subbarao et al. 2007). The major nitrification inhibitor in Brachiaria forage grasses is brachialactone, a cyclic diterpene (Subbarao et al. 2009). Reduction of N loss from the soil under a B. humidicola pasture has a direct and beneficial environmental effect. We hypothesize that this conservation of soil N will have an additional positive impact on a subsequent crop (e.g. maize). At present, recovery of fertilizer N and the impact on crop yield is not known. The main purpose of our inter-institutional and multi-disciplinary project, targeting small-scale farmers, is to develop the innovative approach of BNI using B. humidicola forage grass hybrids to realize sustainable economic and environmental benefits from integrated crop-livestock production systems