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Currently, industrial bioproducts are less competitive than chemically produced goods due to the shortcomings of conventional microbial hosts. Thus, is essential developing robust bacteria for improved cell tolerance to process-specific parameters. In this context, metagenomic approaches from extreme environments can provide useful biological parts to improve bacterial robustness. Here, in order to build genetic constructs that increase bacterial resistance to diverse stress conditions, we recovered novel protein-encoding sequences related to stress-resistance from metagenomic databases using an in silico approach based on Hidden-Markov-Model profiles. For this purpose, we used metagenomic shotgun sequencing data from microbial communities of extreme environments to identify genes encoding chaperones and other proteins that confer resistance to stress conditions. We identified and characterized 10 novel protein-encoding sequences related to the DNA-binding protein HU, the ATP-dependent protease ClpP, and the chaperone protein DnaJ. By expressing these genes in Escherichia coli under several stress conditions (including high temperature, acidity, oxidative and osmotic stress, and UV radiation), we identified five genes conferring resistance to at least two stress conditions when expressed in E. coli. Moreover, one of the identified HU coding-genes which was retrieved from an acidic soil metagenome increased E. coli tolerance to four different stress conditions, implying its suitability for the construction of a synthetic circuit directed to expand broad bacterial resistance.
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BACKGROUND: The need to mitigate and substitute the use of fossil fuels as the main energy matrix has led to the study and development of biofuels as an alternative. Second-generation (2G) ethanol arises as one biofuel with great potential, due to not only maintaining food security, but also as a product from economically interesting crops such as energy-cane. One of the main challenges of 2G ethanol is the inefficient uptake of pentose sugars by industrial yeast Saccharomyces cerevisiae, the main organism used for ethanol production. Understanding the main drivers for xylose assimilation and identify novel and efficient transporters is a key step to make the 2G process economically viable. RESULTS: By implementing a strategy of searching for present motifs that may be responsible for xylose transport and past adaptations of sugar transporters in xylose fermenting species, we obtained a classifying model which was successfully used to select four different candidate transporters for evaluation in the S. cerevisiae hxt-null strain, EBY.VW4000, harbouring the xylose consumption pathway. Yeast cells expressing the transporters SpX, SpH and SpG showed a superior uptake performance in xylose compared to traditional literature control Gxf1. CONCLUSIONS: Modelling xylose transport with the small data available for yeast and bacteria proved a challenge that was overcome through different statistical strategies. Through this strategy, we present four novel xylose transporters which expands the repertoire of candidates targeting yeast genetic engineering for industrial fermentation. The repeated use of the model for characterizing new transporters will be useful both into finding the best candidates for industrial utilization and to increase the model's predictive capabilities.
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The ethanol yield on sugar during alcoholic fermentation allows for diverse interpretation in academia and industry. There are several different ways to calculate this parameter, which is the most important one in this industrial bioprocess and the one that should be maximized, as reported by Pereira, Rodrigues, Sonego, Cruz and Badino (A new methodology to calculate the ethanol fermentation efficiency at bench and industrial scales. Ind Eng Chem Res 2018; 57: 16182-91). On the one hand, the various methods currently employed in industry provide dissimilar results, and recent evidence shows that yield has been consistently overestimated in Brazilian sugarcane biorefineries. On the other hand, in academia, researchers often lack information on all the intricate aspects involved in calculating the ethanol yield in industry. Here, we comment on these two aspects, using fuel ethanol production from sugarcane in Brazilian biorefineries as an example, and taking the work of Pereira, Rodrigues, Sonego, Cruz and Badino (A new methodology to calculate the ethanol fermentation efficiency at bench and industrial scales. Ind Eng Chem Res 2018; 57: 16182-91.) as a starting point. Our work is an attempt to demystify some common beliefs and to foster closer interaction between academic and industrial professionals from the fermentation sector. Pereira, Rodrigues, Sonego, Cruz and Badino (A new methodology to calculate the ethanol fermentation efficiency at bench and industrial scales. Ind Eng Chem Res 2018; 57: 16182-91).
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Etanol , Saccharum , Brasil , Fermentação , Microbiologia IndustrialRESUMO
Biofilm formation on exposed surfaces is a serious issue for the food industry and medical health facilities. There are many proposed strategies to delay, reduce, or even eliminate biofilm formation on surfaces. The present study focuses on the applicability of fire ant venom alkaloids (aka 'solenopsins', from Solenopsis invicta) tested on polystyrene and stainless steel surfaces relative to the adhesion and biofilm-formation by the bacterium Pseudomonas fluorescens. Conditioning with solenopsins demonstrates significant reduction of bacterial adhesion. Inhibition rates were 62.7% on polystyrene and 59.0% on stainless steel surfaces. In addition, solenopsins drastically reduced cell populations already growing on conditioned surfaces. Contrary to assumptions by previous authors, solenopsins tested negative for amphipathic properties, thus understanding the mechanisms behind the observed effects still relies on further investigation.
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Alcaloides/farmacologia , Venenos de Formiga/farmacologia , Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Pseudomonas fluorescens/efeitos dos fármacos , Animais , Formigas , Aderência Bacteriana/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Poliestirenos , Pseudomonas fluorescens/fisiologia , Aço InoxidávelRESUMO
Caffeic acid (CA; 3,4-dihydroxycinnamic acid) is an aromatic compound obtained by the phenylpropanoid pathway. This natural product has antioxidant, antitumor, antiviral, and anti-inflammatory activities. It is also a precursor of CA phenethyl ester (CAPE), a compound with potential as an antidiabetic and liver-protective agent. CA can be found at low concentrations in plant tissues, and hence, its purification is difficult and expensive. Knowledge regarding the pathways, enzymes, and genes involved in CA biosynthesis has paved the way for enabling the design and construction of microbial strains with the capacity of synthesizing this metabolite. In this review, metabolic engineering strategies for the generation of Escherichia coli strains for the biotechnological production of CA are presented and discussed.
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Ácidos Cafeicos/metabolismo , Escherichia coli/metabolismo , Engenharia Metabólica/métodos , Produtos Biológicos , Biotecnologia , Ácidos CumáricosRESUMO
Background: Invert sugar is used greatly in food and pharmaceutical industries. This paper describes scaling-up batch conditions for sucrose inversion catalyzed by the recombinant Pichia pastoris BfrA4X whole cells expressing Thermotoga maritima invertase entrapped in calcium alginate beads. For the first time, we describe the application of a kinetic model to predict the fractional conversion expected during sucrose hydrolysis reaction in both, a model and a prototype bioreactor with 0.5- and 5-L working volume, respectively. Results: Different scaled-up criteria used to operate the 0.5-L bioreactor were analyzed to explore the invert sugar large scale production. After model inversion studies, a 5-L scaled-up reaction system was performed in a 7-L stirred reactor. Both scaled-up criteria, immobilized biocatalyst dosage and stirring speed, were analyzed in each type of bioreactors and the collected data were used to ensure an efficient scale-up of this biocatalyst. Conclusions: To date, there is not enough information to describe the large-scale production of invert sugar using different scaled-up criteria such as dose of immobilized biocatalyst and stirring speed effect on mass transfer. The present study results constitute a valuable tool to successfully carry out this type of high-scale operation for industrial purposes.
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Pichia/metabolismo , Sacarose/metabolismo , Biotecnologia/métodos , Pichia/citologia , Sacarose/química , Cinética , Reatores Biológicos , Thermotoga maritima/enzimologia , Alginatos , Enzimas Imobilizadas , Biocatálise , HidróliseRESUMO
Background: The effect of diverse oxygen transfer coefficient on the L-erythrulose production from meso-erythritol by a newly isolated strain, Gluconobacter kondonii CGMCC8391 was investigated. In order to elucidate the effects of volumetric mass transfer coefficient (K La) on the fermentations, baffled and unbaffled flask cultures, and fed-batch cultures were developed in present work. Results: With the increase of the K La value in the fed-batch culture, L-erythrulose concentration, productivity and yield were significantly improved, while cell growth was not the best in the high K La. Thus, a two-stage oxygen supply control strategy was proposed, aimed at achieving high concentration and high productivity of L-erythrulose. During the first 12 h, Klawas controlled at 40.28 h-1 to obtain high value for cell growth, subsequently K La was controlled at 86.31 h-1 to allow for high L-erythrulose accumulation. Conclusions: Under optimal conditions, the L-erythrulose concentration, productivity, yield and DCW reached 207.9 ± 7.78 g/L, 6.50 g/L/h, 0.94 g/g, 2.68 ± 0.17 g/L, respectively. At the end of fermentation, the L-erythrulose concentration and productivity were higher than those in the previous similar reports.
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Gluconobacter/metabolismo , Oxigênio/metabolismo , Tetroses/biossíntese , Reatores Biológicos , Eritritol , Fermentação , CurtumeRESUMO
Fourteen indigenous Saccharomyces cerevisiae strains isolated from the barks of three tree species located in the Atlantic Rain Forest and Cerrado biomes in Brazil were genetically and physiologically compared to laboratory strains and to strains from the Brazilian fuel ethanol industry. Although no clear correlation could be found either between phenotype and isolation spot or between phenotype and genomic lineage, a set of indigenous strains with superior industrially relevant traits over commonly known industrial and laboratory strains was identified: strain UFMG-CM-Y257 has a very high specific growth rate on sucrose (0.57 ± 0.02 h-1), high ethanol yield (1.65 ± 0.02 mol ethanol mol hexose equivalent-1), high ethanol productivity (0.19 ± 0.00 mol L-1 h-1), high tolerance to acetic acid (10 g L-1) and to high temperature (40°C). Strain UFMG-CM-Y260 displayed high ethanol yield (1.67 ± 0.13 mol ethanol mol hexose equivalent-1), high tolerance to ethanol and to low pH, a trait which is important for non-aseptic industrial processes. Strain UFMG-CM-Y267 showed high tolerance to acetic acid and to high temperature (40°C), which is of particular interest to second generation industrial processes.
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Biodiversidade , Microbiologia Industrial/métodos , Saccharomyces cerevisiae/isolamento & purificação , Saccharomyces cerevisiae/fisiologia , Ácido Acético/toxicidade , Brasil , Tolerância a Medicamentos , Etanol/metabolismo , Temperatura Alta , Saccharomyces cerevisiae/classificação , Sacarose/metabolismo , Árvores/microbiologiaRESUMO
El objetivo del presente trabajo es cuestionar las falsas promesas hechas por la industria de la ingeniería genética agrícola, al prometer que los cultivos producidos mediante esta tecnología generarían una agricultura menos dependendiente en insumos químicos, aumentarían la productividad y ayudarían a reducir los problemas ambientales. Este artículo también analiza las críticas ambientalistas a la biotecnología, expresadas en la preocupación por sus efectos sobre las condiciones sociales y económicas, y los valores culturales, religiosos y morales de las diferentes culturas, que han sido ignorados reiteradamente en el desarrollo tecnológico de los OvGM. El autor concluye afirmando que ha llegado el momento de enfrentar socialmente el reto y la realidad de la ingeniería genética. Las compañías de biotecnología deben sentir el impacto de los movimientos ambientalistas, laborales y campesinos, de modo que reorienten su trabajo para el beneficio de toda la sociedad y de la naturaleza.
The goal of this report is to put into question the false promises that agroindustry has made about genetic engineering in agriculture. They have promised that the transgenic crops should generate agriculture less dependent of chemical supplies, it should increase the productivity and it would help to reduce the environmental problems. On the other hand, this paper analyzes the environmentalist criticism to the biotechnology, specially the deep worry about the social, economic moral, religious, and cultural effects or values that have been ignored in the biotechnological development of the agrobiotechnology. The author concludes starting that it has arrived the social moment to challenge the reality of genetic engineering. The biotechnology companies must feel the impact of environmentalist, labor and rural movements, so that they may reorient their work for the benefit of society and nature.
O objetivo do presente trabalho é questionar as falsas promessas feitos pela indústria da engenharia genética agrícola, ao prometer que os cultivos produzidos mediante esta tecnologia gerariam uma agricultura menos dependente de insumos químicos, aumentariam a produtividade e ajudariam a reduzir os problemas ambientais. Este artigo também analisa as críticas ambientalistas à biotecnologia, expressas na preocupação por seus efeitos sobre as condições sociais e econômicas, e os valores culturais, religiosos e morais das diferentes culturas, que foram ignorados sistematicamente no desenvolvimento tecnológico dos OGMs. O autor conclui afirmando que chegou o momento de enfrentar socialmente o desafio e a realidade da engenharia genética. As empresas de biotecnologia devem sentir o impacto dos movimentos ambientalistas, laborais e rurais, de modo que reorietem seu trabalho para o benefício de toda a sociedade e da natureza.