Gallego Jara, Julia

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Gallego Jara, Julia

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Gallego Jara, Julia

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Universidad de Murcia. Departamento de Bioquímica y Biología Molecular"B" e Inmunología

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Open Access
Bacterial sirtuins overview: an open niche to explore
(Frontiers, 2021-10-26) Lozano Terol, Gema; Cánovas Díaz, Manuel; Diego Puente, Teresa de; Gallego Jara, Julia; Ortega Retuerta, Álvaro; Sola Martínez, Rosa Alba; Bioquímica y Biología Molecular B e Inmunología
Open Access
Bacterial sirtuins overview: an open niche to explore
(Frontiers Media, 2021-10-26) Lozano Terol, Gema; Cánovas Díaz, Manuel; Diego Puente, Teresa de; Gallego Jara, Julia; Ortega Retuerta, Álvaro; Sola Martínez, Rosa Alba; Bioquímica y Biología Molecular B e Inmunología
Sirtuins are deacetylase enzymes widely distributed in all domains of life. Although for decades they have been related only to histones deacetylation in eukaryotic organisms, today they are considered global regulators in both prokaryotes and eukaryotes. Despite the important role of sirtuins in humans, the knowledge about bacterial sirtuins is still limited. Several proteomics studies have shown that bacterial sirtuins deacetylate a large number of lysines in vivo, although the effect that this deacetylation causes in most of them remains unknown. To date, only the regulation of a few bacterial sirtuin substrates has been characterized, being their metabolic roles widely distributed: carbon and nitrogen metabolism, DNA transcription, protein translation, or virulence. One of the most current topics on acetylation and deacetylation focuses on studying stoichiometry using quantitative LC-MS/MS. The results suggest that prokaryotic sirtuins deacetylate at low stoichiometry sites, although more studies are needed to know if it is a common characteristic of bacterial sirtuins and its biological significance. Unlike eukaryotic organisms, bacteria usually have one or few sirtuins, which have been reported to have closer common ancestors with the human Sirt5 than with any other class. In this work, in addition to carrying out an in-depth review of the role of bacterial sirtuins in their physiology, a phylogenetic study has been performed that reveals the evolutionary differences between sirtuins of different bacterial species and even between homologous sirtuins.
Open Access
Engineering protein production by rationally choosing a carbon and nitrogen source using E. coli BL21 acetate metabolism knockout strains.
(Springer Nature, 2019-09-04) Lozano Terol, Gema; Cánovas Díaz, Manuel; Diego Puente, Teresa de; Gallego Jara, Julia; Sola Martínez, Rosa Alba; Bioquímica y Biología Molecular B e Inmunología
Background: Escherichia coli (E. coli) is a bacteria that is widely employed in many industries for the production of high interest bio-products such as recombinant proteins. Nevertheless, the use of E. coli for recombinant protein production may entail some disadvantages such as acetate overfow. Acetate is accumulated under some culture conditions, involves a decrease in biomass and recombinant protein production, and its metabolism is related to protein lysine acetylation. Thereby, the carbon and nitrogen sources employed are relevant factors in cell host metabolism, and the study of the central metabolism of E. coli and its regulation is essential for optimizing the production of biomass and recombinant proteins. In this study, our aim was to fnd the most favourable conditions for carrying out recombinant protein production in E. coli BL21 using two diferent approaches, namely, manipulation of the culture media composition and the deletion of genes involved in acetate metabolism and Nε-lysine acetylation. Results: We evaluated protein overexpression in E. coli BL21 wt and fve mutant strains involved in acetate metabolism (Δacs, ΔackA and Δpta) and lysine acetylation (ΔpatZ and ΔcobB) grown in minimal medium M9 (inorganic ammonium nitrogen source) and in complex TB7 medium (peptide-based nitrogen source) supplemented with glucose (PTS carbon source) or glycerol (non-PTS carbon source). We observed a dependence of recombinant protein production on acetate metabolism and the carbon and nitrogen source employed. The use of complex medium supplemented with glycerol as a carbon source entails an increase in protein production and an efcient use of resources, since is a sub-product of biodiesel synthesis. Furthermore, the deletion of the ackA gene results in a fvefold increase in protein production with respect to the wt strain and a reduction in acetate accumulation. Conclusion: The results showed that the use of diverse carbon and nitrogen sources and acetate metabolism knockout strains can redirect E. coli carbon fuxes to diferent pathways and afect the fnal yield of the recombinant protein bioprocess. Thereby, we obtained a fvefold increase in protein production and an efcient use of the resources employing the most suitable strain and culture conditions.
Open Access
Impact of the Expression System on Recombinant Protein Production in Escherichia coli BL21
(Frontiers Media, 2021-06-21) Lozano Terol, Gema; Cánovas Díaz, Manuel; Diego Puente, Teresa de; Gallego Jara, Julia; Martínez Vivancos, Adrián; Sola Martínez, Rosa Alba; Bioquímica y Biología Molecular B e Inmunología
Recombinant protein production for medical, academic, or industrial applications is essential for our current life. Recombinant proteins are obtained mainly through microbial fermentation, with Escherichia coli being the host most used. In spite of that, some problems are associated with the production of recombinant proteins in E. coli, such as the formation of inclusion bodies, the metabolic burden, or the inefficient translocation/transport system of expressed proteins. Optimizing transcription of heterologous genes is essential to avoid these drawbacks and develop competitive biotechnological processes. Here, expression of YFP reporter protein is evaluated under the control of four promoters of different strength (PT7lac, Ptrc, Ptac, and PBAD) and two different replication origins (high copy number pMB10 and low copy number p15A). In addition, the study has been carried out with the E. coli BL21 wt and the ackA mutant strain growing in a rich medium with glucose or glycerol as carbon sources. Results showed that metabolic burden associated with transcription and translation of foreign genes involves a decrease in recombinant protein expression. It is necessary to find a balance between plasmid copy number and promoter strength to maximize soluble recombinant protein expression. The results obtained represent an important advance on the most suitable expression system to improve both the quantity and quality of recombinant proteins in bioproduction engineering.
Open Access
An ideal spacing is required for the control of Class II CRP-dependent promoters by the status of CRP K100.
(Oxford Academic, 2020) Écija Conesa, Ana; Lozano Terol, Gema; Browning, Douglas F.; Busby, Steve J. W.; Wolfe, Alan J.; Cánovas Díaz, Manuel; Diego Puente, Teresa de; Gallego Jara, Julia; Bioquímica y Biología Molecular B e Inmunología
Transcription activation by the Escherichia coli CRP at Class II promoters is dependent on direct interactions between RNA polymerase and CRP, therefore the spatial proximity between both proteins plays a significant role in the ability of CRP to activate transcription. Using both in vivo and in vitro techniques, here we demonstrate that the CRP K100 positive charge, adjacent to AR2, is required for full promoter activity when CRP is optimally positioned. Accordingly, K100 mediated activation is very position-dependent and our data confirm that the largest impact of the K100 status on transcription activation occurs when the spacing between the CRP binding site and the A2 of the −10 element is 22 bp. From the results of this study and the progress in the understanding about open complex DNA scrunching, we propose that CRP-dependent promoters should now be numbered by the distance from the center of the DNA site for CRP and the most highly conserved base at position 2 of the −10 hexamer in bacterial promoters
Open Access
Bacterial Sirtuins Overview: An Open Niche to Explore
(Frontiers, 2021-09-26) Lozano Terol, Gema; Cánovas Díaz, Manuel; Diego Puente, Teresa de; Gallego Jara, Julia; Ortega Retuerta, Álvaro; Sola Martínez, Rosa Alba; Bioquímica y Biología Molecular B e Inmunología
Sirtuins are deacetylase enzymes widely distributed in all domains of life. Although for decades they have been related only to histones deacetylation in eukaryotic organisms, today they are considered global regulators in both prokaryotes and eukaryotes. Despite the important role of sirtuins in humans, the knowledge about bacterial sirtuins is still limited. Several proteomics studies have shown that bacterial sirtuins deacetylate a large number of lysines in vivo, although the effect that this deacetylation causes in most of them remains unknown. To date, only the regulation of a few bacterial sirtuin substrates has been characterized, being their metabolic roles widely distributed: carbon and nitrogen metabolism, DNA transcription, protein translation, or virulence. One of the most current topics on acetylation and deacetylation focuses on studying stoichiometry using quantitative LC-MS/MS. The results suggest that prokaryotic sirtuins deacetylate at low stoichiometry sites, although more studies are needed to know if it is a common characteristic of bacterial sirtuins and its biological significance. Unlike eukaryotic organisms, bacteria usually have one or few sirtuins, which have been reported to have closer phylogenetic similarity with the human Sirt5 than with any other human sirtuin. In this work, in addition to carrying out an in-depth review of the role of bacterial sirtuins in their physiology, a phylogenetic study has been performed that reveals the evolutionary differences between sirtuins of different bacterial species and even between homologous sirtuins.
Open Access
Influence of Home Indoor Dampness Exposure on Volatile Organic Compounds in Exhaled Breath of Mothers and Their Infants: The NELA Birth Cohort
(2022-07-07) Lozano Terol, Gema; Morales Bartolomé, Eva; García-Marcos Álvarez, Luis; Behalf of the NELA Study Group; Cánovas Díaz, Manuel; Diego Puente, Teresa de; Gallego Jara, Julia; Noguera Velasco, José Antonio; Sola Martínez, Rosa Alba; Bioquímica y Biología Molecular B e Inmunología
Currently, the effect of exposure to indoor air contaminants and the presence of dampness at home on respiratory/atopic health is of particular concern to physicians. The measurement of volatile organic compounds (VOCs) in exhaled breath is a useful approach for monitoring environmental exposures. A great advantage of this strategy is that it allows the study of the impact of pollutants on the metabolism through a non-invasive method. In this paper, the levels of nine VOCs (acetone, isoprene, toluene, p/m-xylene, o-xylene, styrene, benzaldehyde, naphthalene, and 2-ethyl-1-hexanol) in the exhaled breath of subjects exposed and not exposed to home dampness were assessed. Exhaled breath samples were collected from 337 mother–child pairs of a birth cohort and analysed by gaschromatography–mass-spectrometry. It was observed that the levels of 2-ethyl-1-hexanol in the exhaled breath of the mothers were significantly influenced by exposure to household humidity. In the case of the infants, differences in some of the VOC levels related to home dampness exposure; however, they did not reach statistical significance. In addition, it was also found that the eosinophil counts of the mothers exposed to home dampness were significantly elevated compared to those of the non-exposed mothers. To our knowledge, these findings show, for the first time, that exposure to home dampness may influence VOC patterns in exhaled breath.
Open Access
Characterization of acetyl-CoA synthetase kinetics and ATP-binding
(Elsevier, 2019) Lozano Terol, Gema; Écija Conesa, Ana; Zambelli, Barbara; Cánovas Díaz, Manuel; Diego Puente, Teresa de; Gallego Jara, Julia; Bioquímica y Biología Molecular B e Inmunología
Background The superfamily of adenylating enzymes is a large family of enzymes broadly distributed from bacteria to humans. Acetyl-CoA synthetase (Acs), member of this family, is a metabolic enzyme with an essential role in Escherichia coli (E. coli) acetate metabolism, whose catalytic activity is regulated by acetylation/deacetylation in vivo. Methods In this study, the kinetics and thermodynamic parameters of deacetylated and acetylated E. coli Acs were studied for the adenylating step. Moreover, the role of the T264, K270, D500 and K609 residues in catalysis and ATP-binding was also determined by Isothermal titration calorimetry. Results The results showed that native Acs enzyme binds ATP in an endothermic way. The dissociation constant has been determined and ATP-binding showed no significant differences between acetylated and deacetylated enzyme, although kcat was much higher for the deacetylated enzyme. However, K609 lysine mutation resulted in an increase in ATP-Acs-affinity and in a total loss of enzymatic activity, while T264 and D500 mutant proteins showed a total loss of ATP-binding ability and a decrease in catalytic activity. K609 site-specified acetylation induced a change in Acs conformation which resulted in an exothermic and more energetic ATP-binding. Conclusions The differences in ATP-binding could explain the broadly conserved inactivation of Acs when K609 is acetylated. General Significance The results presented in this study demonstrate the importance of the selected residues in Acs ATP-binding and represent an advance in our understanding of the adenylation step of the superfamily of adenylating enzymes and of their acetylation/deacetylation regulation.
Open Access
Impact of the Expression System on Recombinant Protein Production in Escherichia coli BL21
(2021-06-21) Lozano Terol, Gema; Cánovas Díaz, Manuel; Diego Puente, Teresa de; Gallego Jara, Julia; Martínez Vivancos, Adrián; Sola Martínez, Rosa Alba; Bioquímica y Biología Molecular B e Inmunología
Recombinant protein production for medical, academic, or industrial applications is essential for our current life. Recombinant proteins are obtained mainly through microbial fermentation, with Escherichia coli being the host most used. In spite of that, some problems are associated with the production of recombinant proteins in E. coli, such as the formation of inclusion bodies, the metabolic burden, or the inefficient translocation/transport system of expressed proteins. Optimizing transcription of heterologous genes is essential to avoid these drawbacks and develop competitive biotechnological processes. Here, expression of YFP reporter protein is evaluated under the control of four promoters of different strength (PT7lac, Ptrc, Ptac, and PBAD) and two different replication origins (high copy number pMB10 and low copy number p15A). In addition, the study has been carried out with the E. coli BL21 wt and the ackA mutant strain growing in a rich medium with glucose or glycerol as carbon sources. Results showed that metabolic burden associated with transcription and translation of foreign genes involves a decrease in recombinant protein expression. It is necessary to find a balance between plasmid copy number and promoter strength to maximize soluble recombinant protein expression. The results obtained represent an important advance on the most suitable expression system to improve both the quantity and quality of recombinant proteins in bioproduction engineering.
Open Access
Continuous intracellular pH measurement: Escherichia coli culture medium pH dependence
(2021-05-04) Lozano Terol, Gema; Cánovas Díaz, Manuel; Diego Puente, Teresa de; Gallego Jara, Julia; Bioquímica y Biología Molecular B e Inmunología
E. coli is the most used host microorganism to high-valuable compounds production in current industry. Cells pH homeostasis is a crucial factor to grow in different environment maintaining an intracellular pH control, however, to our knowledge, there is not a study about E. coli continuous intracellular pH growing in a culture. In this study the intracellular E. coli pH has been determined for cultures growing with glucose or glycerol as carbon sources. To determine intracellular E. coli pH, a novel, simple and accurate method based on pGFPR01 plasmid (kindly provided by Dr. Joan Slonczewski) was employed (1). This vector expressed ratiometric pHluorin. Thus, fluorescence ratio 410/470 showed a perfect lineal dependence with intracellular pH between 5.5 and 9. The results showed a surprising high intracellular pH for E. coli glucose cultures, which raised values of 8.5-9 at exponential growth phase. However, glycerol cultures showed a major regulation of intracellular pH, keeping the values at 6.5-7.5 in exponential and stationary stages. These differences show the dependence between the intracellular pH and the carbon source. In addition, the measurement method developed is a very useful tool for the control of bioprocesses.