Person: Gallego Jara, Julia
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The protein acetyltransferase PatZ from Escherichia coli is regulated by autoacetylation-induced oligomerization
Background: PatZ is the main Escherichia coli acetyltransferase and control acetyl-CoA synthetase (Acs) activity. Results: The kinetic and structural PatZ oligomer characteristics were determined. Conclusion: PatZ is a stable tetramer and forms an active octamer by autoacetylation to increase its stability. Significance: PTMs by acetylation have structural and functional roles in the cell.
Metabolismo del acetato en Escherichia coli : caracterización y regulación
Escherichia coli (E.coli) es una bacteria Gram-negativa que posee una serie de características que la han convertido en el principal modelo procariota en biología y biotecnología. El licopeno es un terpeno de gran interés industrial que posee diversas aplicaciones, aunque hasta el momento no se ha desarrollado un método de producción que pueda competir con la extracción a partir de tomates. A pesar de las muchas ventajas que presenta E. coli a nivel biotecnológico, también presenta ciertas desventajas. La principal de estas desventajas es la excreción de acetato en cultivos suplementados con glucosa como fuente de carbono. Por otro lado, las modificaciones post-traduccionales, como la N-ɛ acetilación de lisinas, son esenciales para comprender la regulación metabólica de todos los organismos. En E. coli, el metabolismo del acetato se encuentra íntimamente ligado a las modificaciones post-traduccionales por acetilación de lisinas a través de las concentraciones de acetil-CoA y acetil-fosfato, y de la regulación de la proteína acetil-CoA sintetasa (Acs). El objetivo principal de esta tesis doctoral fue avanzar en el conocimiento del metabolismo del acetato de E. coli y en su regulación por acetilación/desacetilación de lisinas A continuación se resumen los principales resultados obtenidos: En el capítulo 3 de esta tesis se ha llevado a cabo el desarrollo de un método de sobreproducción y extracción continua de licopeno con un sistema bifásico, empleando cepas de E. coli modificadas genéticamente. De esta manera, se ha conseguido una producción ampliamente superior a la obtenida con los métodos tradicionales. Por último, los resultados obtenidos en este capítulo corroboran el potencial de E. coli en el campo biotecnológico. El capítulo 4 de esta tesis se ha centrado en la caracterización bioquímica y biofísica de la acetiltransferasa PatZ. Se ha caracterizado cinéticamente la acetilación de la proteína Acs por PatZ por primera vez y se ha demostrado la autoacetilación reversible de PatZ La sirtuína CobB, principal desacetilasa de E.coli, ha sido caracterizada bioquímicamente en el capítulo 5 de esta tesis doctoral. Se ha estudiado cinéticamente la desacetilación de la proteína Acs por CobB y su inhibición por nicotinamida, estableciéndose un mecanismo de inhibición no competitivo. El capítulo 6 de esta tesis se centra en el estudio de la etapa de adenilación del mecanismo catalítico de la proteína Acs. Se ha llevado a cabo la caracterización cinética y termodinámica, respecto a la unión de ATP, de cuatro proteínas mutantes y de la proteína Acs nativa, desacetilada y acetilada. Los resultados han demostrado la importancia de los residuos estudiados en la capacidad catalítica y en la afinidad de Acs por ATP. En el capítulo 7 se ha estudiado la influencia del tipo de fuente de carbono y nitrógeno sobre distintos aspectos relacionados con el metabolismo del acetato en E. coli. Se han observado importantes diferencias en algunos de los parámetros fisiológicos determinados, así como en la excreción de acetato, y se ha determinado, por primera vez, el pH intracelular de forma continua para una bacteria en un cultivo. Finalmente, las principales conclusiones que podemos extraer de esta tesis doctoral son: 1) Se ha construido un sistema competitivo de producción de licopeno empleando cepas de E. coli modificadas genéticamente; 2) El estado oligomérico de PatZ está regulado por autoacetilación/desacetilación; 3) Acs es desacetilada por CobB en varias lisinas con una cinética monofásica; 4) La lisina 609 de Acs tiene un papel fundamental en la unión de la proteína a ATP y 5) La fuente de carbono y nitrógeno tiene una gran influencia en la fisiología y el control del pH intracelular de E. coli. Escherichia coli (E. coli) is a Gram-negative bacterium with several characteristics that have made it a global model in biology and biotechnology. Lycopene is an important terpenoid with several applications, although most of lycopene is obtained from tomato. In spite of the great advantages of E. coli in the biotechnology field, it has also some disadvantages. The main E. coli biotechnological drawback is the excretion of acetate in cultures supplemented with glucose as carbon source. For this, much effort has been put into deepening our knowledge of the acetate metabolism with the purpose of minimizing E. coli acetate overflow. By other way, post-translational modifications, such as N-ɛ lysine acetylation, are essential to know metabolism regulation in all the organisms. E. coli acetate metabolism is closely related to post-translational modifications by lysine acetylation through acetyl-phosphate and acetyl-CoA pools and acetyl-CoA synthetase (Acs) regulation by lysine acetylation. The main objective of this PhD thesis was to advance into the E. coli acetate metabolism knowledge and regulation by lysine acetylation/deacetylation. The main results of this PhD thesis are summarized below. In chapter 3 of this thesis a lycopene overproduction and continuous biphasic extraction system employing E. coli strains modified by genetic engineering has been developed. The results obtained in this chapter corroborate the potential role of E. coli in biotechnology field. Chapter 4 of this dissertation is focused on acetyltransferase PatZ biochemical characterization. The kinetics of Acs acetylation by PatZ have been characterized for the first time, and PatZ reversible autoacetylation has been described. In chapter 5, CobB sirtuin kinetics of Acs deacetylation have been characterized. Moreover, CobB inhibition by nicotinamide has been studied and a non-competitive inhibition mechanism was established. The chapter 6 of this dissertation is focused on the adenylating catalytic step of Acs protein. Kinetics and ATP binding calorimetric parameters were determined for the four mutant proteins and for Acs native protein, in the acetylated and deacetylated state. The results showed the important role of the selected residues in the catalytic and affinity Acs parameters. In chapter 7 of this thesis, the influence of carbon and nitrogen sources on E. coli acetate metabolism was evaluated. The E. coli acetate metabolism was studied for E. coli wt and five deficient strains. As regards physiological parameters and acetate excretion determined, several differences were observed depending on the culture medium and strains. Intracellular pH was determined for the first time in E. coli and important results were achieved. Finally, the main conclusions we can extract are: 1) A competitive lycopene production system using E. coli strains modified by genetic engineering has been developed; 2) PatZ oligomeric state is regulated by autoacetylation/deacetylation; 3) Acs is deacetylated by CobB in several lysines with monophasic kinetics; 4) Acs lysine 609 has an important role in Acs ATP-binding and 5) Carbon and nitrogen sources have a great influence on E. coli physiology and intracellular pH control.
Comunicaciones Orales.- Structural analysis with DSC and Fluorescence in different mutants in lysine 101 of Catabolite Regulator Protein from Escherichia coli.
Bacterial sirtuins overview: an open niche to explore
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.
Relationship between lung function and exhaled volatile organic compounds in healthy infants
Objective: The aim of this study is to assess, for the first time, the relationship between the volatilome and lung function in healthy infants, which may be of help for the early detection of certain respiratory diseases. Lung function tests are crucial in chronic respiratory diseases diagnosis. Moreover, volatile organic compounds (VOCs) analysis in exhaled breath is a noninvasive technique that enables the monitorization of oxidative stress, typical of some forms of airway inflammation. Methods: Lung function was studied in 50 healthy infants of 3–8 months of age and the following parameters were obtained: forced vital capacity (FVC), forced ex piratory volume at 0.5 s (FEV0.5), forced expiratory flow at 75% of FVC (FEF75), forced expiratory flow at 25%–75% of FVC (FEF25–75), and FEV0.5/FVC. Lung function was measured according to the raised volume rapid thoracoabdominal compression technique. In addition, a targeted analysis of six endogenous VOCs (acetone, isoprene, decane, undecane, tetradecane, and pentadecane) in the exhaled breath of the children was carried out by means of thermal desorption coupled gas chromatography‐single quadrupole mass spectrometry system. Results: A negatively significant relationship has been observed between levels of acetone, tetradecane, and pentadecane in exhaled breath and several of the lung function parameters. Levels of acetone (feature m/z = 58) were significantly nega tively associated with FVC and FVE0.5, levels of tetradecane (feature m/z = 71) with FEV0.5, and levels of pentadecane (feature m/z = 71) with FEV0.5 and FEF25–75. Conclusion: The findings of this study highlight a significant association between VOCs related to oxidative stress and lung function in healthy infants.
Engineering of microbial cell factories for production of plant-based natural products
Biotechnology has become a promising alternative to produce highly valuable products. Production using microorganisms competes with chemical synthesis and extraction from natural sources, leading to cheaper and more sustainable production. To produce natural plant products using microorganisms, the development of molecular biology techniques that allow us to genetically and metabolically modify host microorganisms is essential. Thus many biotechnological processes have been developed to obtain terpenes, alkaloids, or polyphenols with interesting applications in the pharmaceutical, food, or cosmetic industries. The increase in our knowledge regarding the metabolism of host organisms, together with the continuous development of genetic and metabolic engineering techniques, will allow, in the coming years, biotechnology to be positioned as the main way to obtain high-value plant natural products.
Regulation of the pyrimidine biosynthetic pathway by lysine acetylation of E. coli OPRTase
Data preprocessing workflow for exhaled breath analysis by GC/MS using open sources
The noninvasive diagnosis and monitoring of high prevalence diseases such as cardiovascular diseases, cancers and chronic respiratory diseases are currently priority objectives in the area of health. In this regard, the analysis of volatile organic compounds (VOCs) has been identified as a potential noninvasive tool for the diagnosis and surveillance of several diseases. Despite the advantages of this strategy, it is not yet a routine clinical tool. The lack of reproducible protocols for each step of the biomarker discovery phase is an obstacle of the current state. Specifically, this issue is present at the data preprocessing step. Thus, an open source workflow for preprocessing the data obtained by the analysis of exhaled breath samples using gas chromatography coupled with single quadrupole mass spectrometry (GC/MS) is presented in this paper. This workflow is based on the connection of two approaches to transform raw data into a useful matrix for statistical analysis. Moreover, this workflow includes matching compounds from breath samples with a spectral library. Three free packages (xcms, cliqueMS and eRah) written in the language R are used for this purpose. Furthermore, this paper presents a suitable protocol for exhaled breath sample collection from infants under 2 years of age for GC/MS.
Dynamic Lysine Acetylation Disrupts Isocitrate Lyase Function and Enables Metabolic Optimisation
Proteomic studies have suggested that Escherichia coli isocitrate lyase (ICL) undergoes multiple acetylation events, partially inhibiting its activity. However, the molecular basis of this regulation and the contribution of individual lysine residues had not been defined. This study demonstrates that acetylation of ICL in E. coli is acetyl-phosphate–dependent and reversible by the CobB deacetylase, establishing a key post-translational regulatory mechanism within the glyoxylate shunt. Site-specific acetylation at K13 and K308 inhibits ICL activity by destabilising the tetrameric assembly and rendering the protein more prone to degradation, whereas lysine-to-arginine substitutions at these positions alleviate this inhibition, enhancing carbon flux distribution, metabolic flexibility and biomass yield without the burden of plasmid-based overexpression. Leveraging this regulatory insight, a KR mutant bearing lysine-to-arginine substitutions at residues 13 and 308, engineered directly into the chromosomal aceA gene, maintained wild-type growth rates while reducing acetate overflow and improving metabolic balance during glucose depletion and acetate assimilation, leading to a 61% increase in lycopene production. These findings highlight regulatory-based metabolic engineering as a powerful strategy to optimise bioproduction and pave the way for extending this approach to other central metabolic enzymes to develop robust microbial cell factories for the sustainable synthesis of biofuels, biochemicals and high-value compounds.