Relative impact of three growth conditions on the Escherichia coli protein acetylome.

Nε-lysine acetylation is a common posttranslational modification observed in Escherichia coli. In the present study, integrative analysis of the proteome and acetylome was performed using label-free quantitative mass spectrometry to analyze the relative influence of three factors affecting growth. The results revealed differences in the proteome, mainly owing to the type of culture medium used (defined or complex). In the acetylome, 7482 unique acetylation sites were identified. Acetylation is directly related to the abundance of proteins, and the level of acetylation in each type of culture is associated with extracellular acetate concentration. Furthermore, most acetylated lysines in the exponential phase remained in the stationary phase without dynamic turnover. Interestingly, unique acetylation sites were detected in proteins whose presence or abundance was linked to the type of culture medium. Finally, the biological function of the acetylation changes was demonstrated for three central metabolic proteins (GapA, Mdh, and AceA).

Evolution of laboratory parameters in patients with lipid transfer protein syndrome after 3 years of immunotherapy.

Lipid transfer protein (LTP) syndrome is an increasingly prevailing disease, especially in the young population, with severely affected quality of life. Since 2013, a specific treatment, called sublingual immunotherapy (SLIT), with peach extract (SLIT-peach®) has been used, but with no long-term effectiveness studies. The main objective of the present study was to assess the long-term effectiveness of SLIT-peach® and to relate the clinical evolution of patients. This was an ambispective study conducted for 3 years. A total of 25 patients with LTP syndrome were selected and treated with SLIT-peach®. They underwent a provocation test in the first year with reintroduced foods that had produced symptoms in the past. Analytical determination of specific immunoglobulin E (IgE) and immunoglobulin G4 (IgG4) to peach (Pru p 3) was performed at the beginning of treatment, at the first year of initiation, and at the end of treatment. These data were compared with the control group comprising 14 patients with LTP syndrome without treatment. A statistically significant decrease in specific IgE to Pru p 3 at the end of the treatment and an increase in specific IgG4 to Pru p 3 1 year after treatment initiation were observed in the active group in relation to tolerance to foods with LTPs. These results indicate that food tolerance begins after the first year and is maintained after the end of 3 years of treatment. In conclusion, treatment with SLIT-peach® for 3 years is effective for patients with LTP syndrome, preventing the evolution of the disease, allowing patients to restart a diet with plant foods, and improving their quality of life.

Regulation of the pyrimidine biosynthetic pathway by lysine acetylation of E. coli OPRTase.

The de novo pyrimidine biosynthesis pathway is an important route due to the relevance of its products, its implications in health and its conservation among organisms. Here, we investigated the regulation by lysine acetylation of this pathway. To this aim, intracellular and extracellular metabolites of the route were quantified, revealing a possible blockage of the pathway by acetylation of the OPRTase enzyme (orotate phosphoribosyltransferase). Chemical acetylation of OPRTase by acetyl-P involved a decrease in enzymatic activity. To test the effect of acetylation in this enzyme, K26 and K103 residues were selected to generate site-specific acetylated proteins. Several differences were observed in kinetic parameters, emphasizing that the kcat of these mutants showed a strong decrease of 300 and 150-fold for OPRTase-103AcK and 19 and 6.3-fold for OPRTase-26AcK, for forward and reverse reactions. In vivo studies suggested acetylation of this enzyme by a nonenzymatic acetyl-P-dependent mechanism and a reversion of this process by the CobB deacetylase. A complementation assay of a deficient strain in the pyrE gene with OPRTase-26AcK and OPRTase-103AcK was performed, and curli formation, stoichiometric parameters and orotate excretion were measured. Complementation with acetylated enzymes entailed a profile very similar to that of the ∆pyrE strain, especially in the case of complementation with OPRTase-103AcK. These results suggest regulation of the de novo pyrimidine biosynthesis pathway by lysine acetylation of OPRTase in Escherichia coli. This finding is of great relevance due to the essential role of this route and the OPRTase enzyme as a target for antimicrobial, antiviral and cancer treatments.

Impact of the Expression System on Recombinant Protein Production in Escherichia coli BL21.

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 (P T7 lac , Ptrc, Ptac, and PBAD) and two different replication origins (high copy number pMB1' 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.

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.

A Compressive Review about Taxol®: History and Future Challenges.

Taxol®, which is also known as paclitaxel, is a chemotherapeutic agent widely used to treat different cancers. Since the discovery of its antitumoral activity, Taxol® has been used to treat over one million patients, making it one of the most widely employed antitumoral drugs. Taxol® was the first microtubule targeting agent described in the literature, with its main mechanism of action consisting of the disruption of microtubule dynamics, thus inducing mitotic arrest and cell death. However, secondary mechanisms for achieving apoptosis have also been demonstrated. Despite its wide use, Taxol® has certain disadvantages. The main challenges facing Taxol® are the need to find an environmentally sustainable production method based on the use of microorganisms, increase its bioavailability without exerting adverse effects on the health of patients and minimize the resistance presented by a high percentage of cells treated with paclitaxel. This review details, in a succinct manner, the main aspects of this important drug, from its discovery to the present day. We highlight the main challenges that must be faced in the coming years, in order to increase the effectiveness of Taxol® as an anticancer agent.

Engineering protein production by rationally choosing a carbon and nitrogen source using E. coli BL21 acetate metabolism knockout strains.

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 overflow. 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 find the most favourable conditions for carrying out recombinant protein production in E. coli BL21 using two different 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 five 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 efficient use of resources, since is a sub-product of biodiesel synthesis. Furthermore, the deletion of the ackA gene results in a fivefold 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 fluxes to different pathways and affect the final yield of the recombinant protein bioprocess. Thereby, we obtained a fivefold increase in protein production and an efficient use of the resources employing the most suitable strain and culture conditions.