Metabolic footprint analysis of recombinant Escherichia coli strains during fed-batch fermentations

Metabolic footprinting has become a valuable analytical approach for the characterization of phenotypes and the distinction of specific metabolic states resulting from environmental and/or genetic alterations. The metabolic impact of heterologous protein production in Escherichia coli cells is of particular interest, since there are numerous cellular stresses triggered during this process that limit the overall productivity. Because the knowledge on the metabolic responses in recombinant bioprocesses is still scarce, metabolic footprinting can provide relevant information on the intrinsic metabolic adjustments. Thus, the metabolic footprints generated by Escherichia coli W3110 and the ΔrelA mutant strain during recombinant fed-batch fermentations at different experimental conditions, were measured and interpreted. The IPTG-induction of the heterologous protein expression resulted in the rapid accumulation of inhibitors of the glyoxylate shunt in the culture broth, suggesting the clearance of this anaplerotic route to replenish the TCA intermediaries withdrawn for the additional formation of heterologous protein. Nutritional shifts were also critical in the recombinant cellular metabolism, indicating that cells employ diverse strategies to counteract imbalances in the cellular metabolism, including the secretion of certain metabolites that are, most likely, used as a metabolic relief to survival processes.The authors thank to Raphael Aggio for assisting in the automatic refinement and correction of the GC-MS data. This work was supported in part by the research project Bridging Systems and Synthetic Biology for the development of Improved Microbial Cell Factories (MIT-Pt/BS-BB/0082/2008) and HeliSysBio-Molecular Systems Biology Helicobacter pylori (FCT PTDC/EBB-EBI/104235/2008), both financed by the Portuguese Fundacao para a Ciencia e Tecnologia. Sonia Carneiro was also supported by a PhD grant from the same institution (ref. SFRH/BD/22863/2005)

Applying a metabolic footprinting approach to characterize the impact of the recombinant protein production in Escherichia coli

In this study metabolic footprinting was applied to evaluate the metabolic consequences of protein overproduction at slow growth conditions (μ = 0.1 h-1). The extracellular metabolites detected by gas chromatography-mass spectrometry characterized the metabolic footprints before and after the induction of the recombinant protein production (i.e. pre- and post-induction phases). Metabolic footprinting enabled the discrimination between the two growth phases and ex-posed significant metabolic alterations in the extracellular milieu during the re-combinant processes.This work is partly funded by the Portuguese FCT (Fundacao para a Ciencia e Tecnologia) funded MIT-Portugal Program in Bioengineering (MIT-Pt/BSBB/0082/2008). The work of Sonia Carneiro is supported by a PhD grant from FCT (ref. SFRH/BD/22863/2005)

Influence of the RelA activity on E. coli metabolism by metabolite profiling of glucose-limited chemostat cultures

Metabolite profiling of E. coli W3110 and the isogenic ∆relA mutant cells was used to characterize the RelA-dependent stringent control of metabolism under different growth conditions. Metabolic profiles were obtained by gas chromatography–mass spectrometry (GC-MS) analysis and revealed significant differences between E. coli strains grown at different conditions. Major differences between the two strains were assessed in the levels of amino acids and fatty acids and their precursor metabolites, especially when growing at the lower dilution rates, demonstrating differences in their metabolic behavior. Despite the fatty acid biosynthesis being the most affected due to the lack of the RelA activity, other metabolic pathways involving succinate, lactate and threonine were also affected. Overall, metabolite profiles indicate that under nutrient-limiting conditions the RelA-dependent stringent response may be elicited and promotes key changes in the E. coli metabolism.The authors thank to Raphael Aggio for assisting in the automatic refinement and correction of the GC-MS data, Katie Smart for performing acetate analyses and Clark Ehlers for his support with the bioreactor set up. This work was supported by the Portuguese FCT (Fundacao para a Ciencia e Tecnologia) funded MIT-Portugal Program in Bioengineering (MIT-Pt/BS-BB/0082/2008) and by ERDF-European Regional Development Fund through the COMPETE Programme (operational programme for competitiveness) and by National Funds through the FCT (Portuguese Foundation for Science and Technology) within the project FCOMP-01-0124-FEDER-009707 (HeliSysBio. molecular Systems Biology in Helicobacter pylori). The work was also supported by a PhD grant from FCT (ref. SFRH/BD/22863/2005)

Equality of opportunity and educational achievement in Portugal

Portugal has one of the highest levels of income inequality in Europe, and low wages and unemployment are concentrated among low skill individuals. Education is an important determinant of inequality. However, there are large differences in the educational attainment of different individuals in the population, and the sources of these differences emerge early in the life-cycle when families play a central role in individual development. We estimate that most of the variance of school achievement at age 15 is explained by family characteristics. Observed school inputs explain very little of adolescent performance. Children from highly educated parents benefit of rich cultural environments in the home and become highly educated adults. Education policy needs to be innovative: (1) it needs to explicitly recognize the fundamental long run role of families on child development; (2) it needs to acknowledge the failure of traditional input based policies

Podridão parda da haste: avaliação de genótipos de soja, safra 2013/2014.

bitstream/item/114978/1/2014-Documentos-online-151-p47.pd

Gene flow from transgenic common beans expressing the bar gene.

Gene flow is a common phenomenon even in self-pollinated plant species. With the advent of genetically modified plants this subject has become of the utmost importance due to the need for controlling the spread of transgenes. This study was conducted to determine the occurrence and intensity of outcrossing in transgenic common beans. In order to evaluate the outcross rates, four experiments were conducted in Santo Antonio de Goiás (GO, Brazil) and one in Londrina (PR, Brazil), using transgenic cultivars resistant to the herbicide glufosinate ammonium and their conventional counterparts as recipients of the transgene. Experiments with cv. Olathe pinto and the transgenic line Olathe M1/4 were conducted in a completely randomized design with ten replications for three years in one location, whereas the experiments with cv. Pérola and the transgenic line Pérola M1/4 were conducted at two locations for one year, with the transgenic cultivar surrounded on all sides by the conventional counterpart. The outcross occurred at a negligible rate of 0.00741% in cv. Pérola, while none was observed (0.0%) in cv. Olathe pinto. The frequency of gene flow was cultivar dependent and most of the observed outcross was within 2.5 m from the edge of the pollen source

Vortex-line liquid phases: Longitudinal superconductivity in the lattice London model

We study the vortex-line lattice and liquid phases of a clean type-II superconductor by means of Monte Carlo simulations of the lattice London model. Motivated by a recent controversy regarding the presence, within this model, of a vortex-liquid regime with longitudinal superconducting coherence over long length scales, we directly compare two different ways to calculate the longitudinal coherence. For an isotropic superconductor, we interpret our results in terms of a temperature regime within the liquid phase in which longitudinal superconducting coherence extends over length scales larger than the system thickness studied. We note that this regime disappears in the moderately anisotropic case due to a proliferation, close to the flux-line lattice melting temperature, of vortex loops between the layers.Comment: 8 pages, Revtex, with eps figures. To appear in Phys. Rev.