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![Examples of relevant curated pathways in C. salexigens. a Pathways related with compatible solutes metabolism. b Entner–Doudoroff (ED) and partial glycolysis (EM) pathways of central metabolism. Names of reactions and associated genes are shown in red. A discontinuous arrow means a new reaction or a re-annotated gen-associated reaction in iFP764, with respect to iOA548. A red-truncated arrow represents a gene-associated reaction non-included in iFP764, respect to iOA548. a glc-d[c]: d-glucose; f6p[c]: beta-d-fructose-6-phosphate; tre6p[c]: alpha, alpha-trehalose 6-phosphate; tre[c]: alpha, alpha, trehalose; chol[c]: Choline; dmgly[c]: N,N-dimethylglycine; sarc[c]: Sarcosine; gly[c]: Glycine; thr-l[c]: l-Threonine; phom[c]: O-phospho-l-homoserine; hom-l[c]: l-homoserine; asp-L[c]: l-aspartate; aspsa[c]: l-aspartate-4-semialdehyde; 4pasp[c]: 4-phospho-l-aspartate; 24abtn[c]: l-2-4-diamino-butanoate; ect-l[c]: ectoine; 2mdi6car[c]: 2-ethyl-4,5-dihydropyrimidine-6-carboxylate; hdect[c]: 5-Hydroxyectoine; nA4aL24dab[c]: N-alpha-acetyl-l-2,4-diaminobutyrate; nG4aL24dab[c]: N-4-Acetyl-l-2,4-diaminobutanoate. b glc-d[c]: d-glucose; f6p[c]: beta-d-fructose-6-phosphate; fdp[c]: beta-d-fructose-1,6-bisphosphate; g6p[c]: d-glucose 6-phosphate; g3p[c]: glyceraldehyde-3-phosphate; gl[c]: d-glucono-1,5-lactone; 6pgl[c]: d-glucono-1,5-lactone 6-phosphate; glcn[c]: d-gluconic-acid; 6pgc[c]: 6-phospho-d-gluconate; 2ddg6p[c]: 2-Dehydro-3-deoxy-6-phospho-d-gluconate; 2-ketgluc[c]: 2-keto-d-gluconic acid; 2dhdglu6[c]: 6-phospho-2-dehydro-d-gluconate](publication/322341446/figure/fig2/AS:962852890365970@1606573426181/Examples-of-relevant-curated-pathways-in-C-salexigens-a-Pathways-related-with.gif)
Examples of relevant curated pathways in C. salexigens. a Pathways related with compatible solutes metabolism. b Entner–Doudoroff (ED) and partial glycolysis (EM) pathways of central metabolism. Names of reactions and associated genes are shown in red. A discontinuous arrow means a new reaction or a re-annotated gen-associated reaction in iFP764, with respect to iOA548. A red-truncated arrow represents a gene-associated reaction non-included in iFP764, respect to iOA548. a glc-d[c]: d-glucose; f6p[c]: beta-d-fructose-6-phosphate; tre6p[c]: alpha, alpha-trehalose 6-phosphate; tre[c]: alpha, alpha, trehalose; chol[c]: Choline; dmgly[c]: N,N-dimethylglycine; sarc[c]: Sarcosine; gly[c]: Glycine; thr-l[c]: l-Threonine; phom[c]: O-phospho-l-homoserine; hom-l[c]: l-homoserine; asp-L[c]: l-aspartate; aspsa[c]: l-aspartate-4-semialdehyde; 4pasp[c]: 4-phospho-l-aspartate; 24abtn[c]: l-2-4-diamino-butanoate; ect-l[c]: ectoine; 2mdi6car[c]: 2-ethyl-4,5-dihydropyrimidine-6-carboxylate; hdect[c]: 5-Hydroxyectoine; nA4aL24dab[c]: N-alpha-acetyl-l-2,4-diaminobutyrate; nG4aL24dab[c]: N-4-Acetyl-l-2,4-diaminobutanoate. b glc-d[c]: d-glucose; f6p[c]: beta-d-fructose-6-phosphate; fdp[c]: beta-d-fructose-1,6-bisphosphate; g6p[c]: d-glucose 6-phosphate; g3p[c]: glyceraldehyde-3-phosphate; gl[c]: d-glucono-1,5-lactone; 6pgl[c]: d-glucono-1,5-lactone 6-phosphate; glcn[c]: d-gluconic-acid; 6pgc[c]: 6-phospho-d-gluconate; 2ddg6p[c]: 2-Dehydro-3-deoxy-6-phospho-d-gluconate; 2-ketgluc[c]: 2-keto-d-gluconic acid; 2dhdglu6[c]: 6-phospho-2-dehydro-d-gluconate
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Background
The halophilic bacterium Chromohalobacter salexigens is a natural producer of ectoines, compatible solutes with current and potential biotechnological applications. As production of ectoines is an osmoregulated process that draws away TCA intermediates, bacterial metabolism needs to be adapted to cope with salinity changes. To explore an...
Citations
... To tackle this obstacle, MetaDraft uses previously built high-quality GEMs as templates for metabolic network reconstruction . For this task, a GEM from Chromohalobacter salexigens (Piubeli et al. 2018), a member of the Halomonacedeae family, was chosen as a template. This model was chosen since this species is phylogenetically close, and more importantly, the former has a higher quality than other model alternatives, as demonstrated by the increased number of mapped reactions and compounds as well as metabolic identifiers employed (Supplementary Information Table S6). ...
... E Essential genes subsystems salinity (2.5 M). For this task, the biomass reaction equation from iFP764 under low and high salt concentrations was employed (Piubeli et al. 2018). The biomass reaction under medium salt concentration (1 M) was estimated by interpolating the stoichiometric coefficients for the main biomass precursor macromolecules and fitting the growth-associated energy requirement (GAM) to experimental data from this study (Supplementary Data Table S20). ...
Poly-hydroxybutyrate (PHB) is an environmentally friendly alternative for conventional fossil fuel-based plastics that is produced by various microorganisms. Large-scale PHB production is challenging due to the comparatively higher biomanufacturing costs. A PHB overproducer is the haloalkaliphilic bacterium Halomonas campaniensis, which has low nutritional requirements and can grow in cultures with high salt concentrations, rendering it resistant to contamination. Despite its virtues, the metabolic capabilities of H. campaniensis as well as the limitations hindering higher PHB production remain poorly studied. To address this limitation, we present HaloGEM, the first high-quality genome-scale metabolic network reconstruction, which encompasses 888 genes, 1528 reactions (1257 gene-associated), and 1274 metabolites. HaloGEM not only displays excellent agreement with previous growth data and experiments from this study, but it also revealed nitrogen as a limiting nutrient when growing aerobically under high salt concentrations using glucose as carbon source. Among different nitrogen source mixtures for optimal growth, HaloGEM predicted glutamate and arginine as a promising mixture producing increases of 54.2% and 153.4% in the biomass yield and PHB titer, respectively. Furthermore, the model was used to predict genetic interventions for increasing PHB yield, which were consistent with the rationale of previously reported strategies. Overall, the presented reconstruction advances our understanding of the metabolic capabilities of H. campaniensis for rationally engineering this next-generation industrial biotechnology platform.
Key points
A comprehensive genome-scale metabolic reconstruction of H. campaniensis was developed.
Experiments and simulations predict N limitation in minimal media under aerobiosis.
In silico media design increased experimental biomass yield and PHB titer.
... In this research, we explored the adaptive mechanisms of microbial halophiles, which are primarily characterized by the regulation of genes associated with sodium efflux and potassium uptake components of the 'salt-in' strategy and the biosynthesis and transport of organic compatible solutes, either through uptake or de novo synthesis [22,23]. Building upon this foundation, our study utilized KEGG gene annotations to delve into the transport and the biosynthesis pathways of microorganisms in DT, a hypersaline salt lake, and XT, a moderately saline counterpart. ...
The Dong Taijinar (DT) and Xi Taijinar (XT) Salt Lakes have been extensively researched for their mineral richness. However, the composition and distribution of their microbial communities are still poorly known. In this study, we employed metagenomic sequencing to explore the diversity and potential functions of the microbial populations in DT and XT. Our findings indicate that the salinity levels in DT (332.18–358.30 g/L) were tenfold higher than in XT (20.09–36.83 g/L). Notably, archaea dominated the DT domain at 96.16%, while bacteria prevailed in XT at 93.09%. In DT, the bacterial community comprised 33 phyla and 1717 genera, with Marinobacter emerging as the dominant genus, showing a positive correlation with the total phosphorus content. The archaeal community in DT included four main phyla and 153 genera. The most abundant genera were Natronomonas (24.61%) and Halorubrum (23.69%), which had a strong positive correlation with the concentrations of Na+, Ca2+, and Cl−. Conversely, XT hosted 33 phyla and 1906 bacterial genera, with Loktanella as the dominant genus. The archaeal taxonomy in XT encompassed four phyla and 149 genera. In both salt lakes, Proteobacteria and Euryarchaeota were the most abundant bacterial and archaeal phyla, respectively. Our analysis of the halophilic mechanisms of these microorganisms suggests that the bacteria in XT tend to synthesize compatible solutes, whereas the archaea in DT adopt a ‘salt-in’ strategy, integrating salt into their cellular machinery to cope with the high-salinity environment.
... The mechanisms of microbial halophiles mainly included 1) the regulation of Na + e ux-and K + uptake-related genes (part of the so-called "salt-in" strategy), and 2) organic compatible solute biosynthesis and transport (accumulation of compatible solutes by uptake or de novo synthesis) (22,23). Han et al. ...
The Dong Taijinar Salt Lake (DT) and Xi Taijinar Salt Lake (XT) have been widely studied as mineral-rich areas. However, little is known about the composition and distribution of the microbial communities in these two lakes. In this study, metagenomics sequencing was used to analyze the diversity and potential functions of the microbial communities in DT and XT. According to our report, the salinity of DT (332.18–358.30 g/L) was 10 times higher than that of XT (20.09–36.83 g/L). Interestingly, the dominant domain in DT was Archaea (96.16%), while that in XT was Bacteria (93.09%). The distribution of Bacteria in the DT revealed 33 phyla and 1717 genera. The dominant genus in DT was Marinobacillus , which was positively correlated with total phosphorus content. There were four main phyla and 153 genera identified in the Archaea of DT. The most abundant Archaea genera in DT were Natronomonas (24.61%) and Halorubrum (23.69%), which were mainly positively correlated with the Na ⁺ , Ca ²⁺ , and Cl ⁻ contents. Similarly, there were 33 phyla and 1906 genera of Bacteria in XT, and Loktanella was the dominant genus. The archaeal taxonomy in XT mainly included four phyla and 149 genera. Proteobacteria and Euryarchaeota were the most abundant bacterial and archaeal phyla in the two salt lakes. Analysis of the halophilic mechanisms of the microorganisms identified in these two salt lakes revealed that the Bacteria in XT preferred to synthesize compatible solutes, whereas the Archaea in DT preferred a "salt-in" adaptation strategy in salt-stressed environments.
... Ectoine production yields from fermentation primarily depend on the salt concentration of the medium, carbon, and nitrogen ratios involved in metabolic overflow, strain growth rates, and cellular densities. Moreover, these factors are affected by culture conditions including temperature and pH (León et al. 2018;Piubeli et al. 2018;Weinisch et al. 2018;Dong et al. 2021;Jiang et al. 2022). Optimal ectoineproducing strains have been suggested to require rapid cellular proliferation (e.g., an OD 600 value of approximately 1.2 after 8 h), wide salinity tolerance ranges (e.g., 0-3.0 M NaCl), and maximal extracellular ectoine release rates. ...
Ectoine is a natural amino acid derivative and one of the most widely used compatible solutes produced by Halomonas species that affects both cellular growth and osmotic equilibrium. The positive effects of UV mutagenesis on both biomass and ectoine content production in ectoine-producing strains have yet to be reported. In this study, the wild-type H. campaniensis strain XH26 (CCTCCM2019776) was subjected to UV mutagenesis to increase ectoine production. Eight rounds of mutagenesis were used to generate mutated XH26 strains with different UV-irradiation exposure times. Ectoine extract concentrations were then evaluated among all strains using high-performance liquid chromatography analysis, alongside whole genome sequencing with the PacBio RS II platform and comparison of the wild-type strain XH26 and the mutant strain G8-52 genomes. The mutant strain G8-52 (CCTCCM2019777) exhibited the highest cell growth rate and ectoine yields among mutated strains in comparison with strain XH26. Further, ectoine levels in the aforementioned strain significantly increased to 1.51 ± 0.01 g L⁻¹ (0.65 g g⁻¹ of cell dry weight), representing a twofold increase compared to wild-type cells (0.51 ± 0.01 g L⁻¹) when grown in culture medium for ectoine accumulation. Concomitantly, electron microscopy revealed that mutated strain G8-52 cells were obviously shorter than wild-type strain XH26 cells. Moreover, strain G8-52 produced a relatively stable ectoine yield (1.50 g L⁻¹) after 40 days of continuous subculture. Comparative genomics analysis suggested that strain XH26 harbored 24 mutations, including 10 nucleotide insertions, 10 nucleotide deletions, and unique single nucleotide polymorphisms. Notably, the genes orf00723 and orf02403 (lipA) of the wild-type strain mutated to davT and gabD in strain G8-52 that encoded for 4-aminobutyrate-2-oxoglutarate transaminase and NAD-dependent succinate-semialdehyde dehydrogenase, respectively. Consequently, these genes may be involved in increased ectoine yields. These results suggest that continuous multiple rounds of UV mutation represent a successful strategy for increasing ectoine production, and that the mutant strain G8-52 is suitable for large-scale fermentation applications.
... Thus, the system can present a large solution space, and that is why the fluxes are limited to using upper and lower constraints on each reaction individually (v i ): v i lower ≤ v i ≤ v i upper These bounds allow the reconstruction to be used to simulate specific conditions. It is noteworthy that the model generated initially may differ, both in scope and limits, from the model obtained at the end, which is due to the multiple validation and refinement performed to obtain a robust model capable of simulating the phenotypic behavior with a high degree of correspondence with that obtained in vivo [96]. The fourth and final step of the process of obtaining the reconstruction consists of the evaluation followed by the validation of the network. ...
Over the past few decades, antimicrobial resistance (AMR) has emerged as an important threat to public health, resulting from the global propagation of multidrug-resistant strains of various bacterial species. Knowledge of the intrinsic factors leading to this resistance is necessary to overcome these new strains. This has contributed to the increased use of omics technologies and their extrapolation to the system level. Understanding the mechanisms involved in antimicrobial resistance acquired by microorganisms at the system level is essential to obtain answers and explore options to combat this resistance. Therefore, the use of robust whole-genome sequencing approaches and other omics techniques such as transcriptomics, proteomics, and metabolomics provide fundamental insights into the physiology of antimicrobial resistance. To improve the efficiency of data obtained through omics approaches, and thus gain a predictive understanding of bacterial responses to antibiotics, the integration of mathematical models with genome-scale metabolic models (GEMs) is essential. In this context, here we outline recent efforts that have demonstrated that the use of omics technology and systems biology, as quantitative and robust hypothesis-generating frameworks, can improve the understanding of antibiotic resistance, and it is hoped that this emerging field can provide support for these new efforts.
... GEMs also provide a mechanistic framework to integrate other omics data to understand context-specific metabolism. For example, integrating proteomic profiles obtained during microbial oxidation (Barco et al., 2015) or exposure to space into GEMs can enable the understanding, modeling, and prediction of organism metabolism in such specific conditions Piubeli et al., 2018). These could serve as mathematical modeling platforms for extreme environments on earth and space missions (Milojevic and Weckwerth, 2020). ...
Extremophilic bacteria have numerous uncovered biotechnological potentials. Acidophilic bacteria are important iron oxidizers that are valuable in bioleaching and in studying extreme environments on earth and in space. Despite their obvious potential, little is known about the genetic traits that underpin their metabolic functions, which are equally poorly understood from a mechanistic perspective. Novel bioinformatics and computational biology pipelines can be used to analyze whole genomes to obtain insights into the phenotypic potential of organisms as well as develop a mathematical model representation of metabolism. Whole-genome sequence analysis and a genome-scale metabolic network model was curated for an iron-oxidizing bacterium initially isolated from an acid mine drainage in Turkey, previously identified as Alicyclobacillus tolerans. The genome contained a high proportion of genes for energy generation from carbohydrates, amino acids synthesis and conversion, nucleic acid metabolism and repair which contribute to robust adaption to their extreme environments. Several candidate genes for pyrite metabolism, iron uptake, regulation and storage, as well as genes for resistance to important heavy metals were annotated. A curated genome-scale metabolic network analysis accurately predicted facultative anaerobic growth, heterotrophic characteristics, and growth on a wide variety of carbon sources. This is the first in-depth in silico analysis of A. tolerans to the best of our knowledge which is expected to lay the groundwork for future research and drive innovations in environmental microbiology and biotechnological applications. The genomic data and mechanistic framework will have applications in biomining, synthetic geomicrobiology on earth, as well as for space exploration and settlement.
... For example, Nayak and colleagues used CEG as a reference database to screen potential drug targets for pathogens causing bacterial pneumonia (7). It was also used to determine the essentiality of metabolism-associated enzymes in Chromohalobacter salexigens (8). The first release of CEG included a total of 2861 clusters covered by 16 prokaryotic gene datasets. ...
Essential genes are key elements for organisms to maintain their living. Building databases that store essential genes in the form of homologous clusters, rather than storing them as a singleton, can provide more enlightening information such as the general essentiality of homologous genes in multiple organisms. In 2013, the first database to store prokaryotic essential genes in clusters, CEG (Clusters of Essential Genes), was constructed. Afterward, the amount of available data for essential genes increased by a factor >3 since the last revision. Herein, we updated CEG to version 2, including more prokaryotic essential genes (from 16 gene datasets to 29 gene datasets) and newly added eukaryotic essential genes (nine species), specifically the human essential genes of 12 cancer cell lines. For prokaryotes, information associated with drug targets, such as protein structure, ligand–protein interaction, virulence factor and matched drugs, is also provided. Finally, we provided the service of essential gene prediction for both prokaryotes and eukaryotes. We hope our updated database will benefit more researchers in drug targets and evolutionary genomics.
Database URL: http://cefg.uestc.cn/ceg
... El muestreo uniforme ha sido utilizado exitosamente en un sin número de modelos metabólicos a escala genómica para explorar y explicar fenotipos de adaptación metabólica en patógenos (47) , células animales (46) , plantas (48) y organismos de interés industrial (49) . ...
El metabolismo representa el nivel biológico que más se relaciona con los fenotipos de la célula y, las alteraciones o reprogramaciones de éste pueden, entre otras, (i) afectar la producción de metabolitos primarios o secundarios en microorganismos de interés biotecnológico, (ii) favorecer o no la inhibición del crecimiento en organismos patógenos y (iii) desarrollar desórdenes metabólicos como la obesidad o la diabetes. Es por ello, que el estudio del metabolismo, el rediseño, y el redireccionamiento de fluxes metabólicos se ha convertido en un área importante de investigación (también conocida como Ingeniería Metabólica), ya que ha permitido el desarrollo y diseño de procesos biológicos mejorados, la identificación de blancos terapéuticos, el diseño de estrategias terapéuticas para curar desordenes metabólicos y la identificación de biomarcadores en cáncer, entre otros. Actualmente, se han desarrollado metodologías computacionales que permiten estudiar el metabolismo celular a diferentes condiciones medioambientales, dirigiendo la experimentación con las predicciones del modelo. El propósito de esta revisión es resaltar la importancia del análisis de fluxes metabólicos como una metodología general para estudiar la reprogramación metabólica en distintos organismos de interés biotecnológico, médico, y terapéutico. Este trabajo condensa las bases teóricas y los conceptos claves para entender el análisis de fluxes metabólicos, lo cual será un insumo fundamental para aquellos que se están adentrando al mundo de la biología de sistemas o áreas afines.
... Osmolytes and compatible solutes such as ectoine ((S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid, THP(B)) and hydroxyectoine ((4S,5S)-5-hydroxy-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid, THP(A)) ( Fig. 1) are produced by some halophilic microorganisms to survive in environments with high salinity, such as salt lakes in desert regions. 1 These microorganisms achieve osmoadaptation to high external salt concentrations by the regulation of osmotic pressure via the accumulation of molar concentrations of ectoine. [2][3][4] In this context, an interesting property of ectoine is the compensating effect on water dynamics as opposed to that of sodium chloride. 5 Ectoine has a zwitterionic structure in the solid state 6 and in aqueous solution 7,8 at neutral pH. ...
... Due to its versatile properties, ectoine is currently used in a multitude of biotechnological, cosmetic and medical applications. 2,3,[22][23][24][25] Most prominently, it is used in sunscreens, despite evidence from recent studies concerning its influence on UV irradiation of biological targets, showing Janus-faced behaviour. 17,20,[26][27][28] Bünger et al. irradiated human keratinocytes with UV-A (340-400 nm). ...
Ectoine is a small zwitterionic osmolyte and compatible solute, which does not interfere with cell metabolism even at molar concentrations. Plasmid DNA (pUC19) was irradiated with ultraviolet radiation (UV-C at 266 nm) under quasi physiological conditions (PBS) and in pure water in the presence and absence of ectoine ( THP(B) ) and hydroxyectoine ( THP(A) ). Different types of UV induced DNA damage were analysed: DNA single-strand breaks (SSB), abasic sites and cyclobutane pyrimidine dimers (CPD). The complex interplay between these factors was observed with respect to the nature and occurrence of the DNA damage with 266 nm photons. In PBS, the cosolutes show an efficient protection against base damage, whilst in pure water a dramatic shift from SSB damage to base damage was observed when cosolutes were added. To test whether these effects are caused from ectoine binding to DNA, further experiments were conducted: Small-angle X-ray scattering (SAXS), surface-plasmon resonance (SPR) measurements and Raman spectroscopy. The results show, for the first time, close interaction between ectoine and DNA. This is in stark contrast to the assumption made by preferential exclusion models, which are often used to interpret the behaviour of compatible solutes within cells and with biomolecules. It is tentatively proposed, that the alterations of UV damage to DNA is attributed to ectoine influence on nucleobases through the direct interaction between ectoine and DNA.
... Furthermore, the same study found C. salexigens to have a higher ectoine yield at lower culturing temperatures compared to a selection of other ectoine producers, including H. elongata [14]. A metabolic model for osmoadaptation in C. salexigens has also been published [34]. Put together, C. salexigens is emerging as one of the key organisms to study osmoadaptation, which increases our need to understand the basic biochemical and structural aspects of its ectoine-synthesizing enzymes. ...
l‐2,4‐diaminobutyric acid (DABA) aminotransferases can catalyze the formation of amines at the distal ω‐position of substrates, and is the intial and rate‐limiting enzyme in the biosynthesis pathway of the cytoprotecting molecule (S)‐2‐methyl‐1,4,5,6‐tetrahydro‐4‐pyrimidine carboxylic acid (ectoine). Although there is an industrial interest in the biosynthesis of ectoine, the DABA aminotransferases remain poorly characterized. Herein, we present the crystal structure of EctB (2.45 Å), a DABA aminotransferase from Chromohalobacter salexigens DSM 3043, a well‐studied organism with respect to osmoadaptation by ectoine biosynthesis. We investigate the enzyme’s oligomeric state to show that EctB from C. salexigens is a tetramer of two functional dimers, and suggest conserved recognition sites for dimerization that also includes the characteristic gating loop that helps shape the active site of the neighboring monomer. Although ω‐transaminases are known to have two binding pockets to accommodate for their dual substrate specificity, we herein provide the first description of two binding pockets in the active site that may account for the catalytic character of DABA aminotransferases. Furthermore, our biochemical data reveal that the EctB enzyme from C. salexigens is a thermostable, halotolerant enzyme with a broad pH tolerance which may be linked to its tetrameric state. Put together, this study creates a solid foundation for a deeper structural understanding of DABA aminotransferases and opening up for future downstream studies of EctB’s catalytic character and its redesign as a better catalyst for ectoine biosynthesis. In summary, we believe that the EctB enzyme from C. salexigens can serve as a benchmark enzyme for characterization of DABA aminotransferases.
Database
Structural data are available in PDB database under the accession number 6RL5.
