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The production of lactic acid from agroindustry waste products, such as whey, heavily relies on microorganisms within the genusLactobacillus. In this work, a genome-scale metabolic model was implemented from Vinay-Lara (iLca334_548), improved adding some enzymatic reactions and used to analyse metabolic fluxes ofLacticaseibacillus paracasei, which is aLactobacillusstrain isolated from whey used in the large-scale production of lactic acid. Overall, the highest rate of lactic acid productivity was 2.9 g l-1h-1, which equates to a dilution rate of 0.125 h-1, when continuous culture conditions were established. Restrictions on lactic acid production caused by exchange reactions, complex culture medium and intracellular metabolite concentrations were considered and included in the model. In total, theiLca334_548 model consisted of 1046 reactions and 959 metabolites, and flow balance analysis better predicted lactate flux than biomass. The distribution of fluxes exhibited an increase in lactate formation as biomass decreased. This finding is supported by the reactions carried out by glyceraldehyde 3-phosphate dehydrogenase, pyruvate formate lyase and ribose-5-phosphate isomerase, corroborating the modelled phenotype with experimental data. In conclusion, there is potential for the improvement of lactate production in a complex media by amino acid catabolism, especially when lactate is derived from pyruvate.
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Ácido Láctico , Soro do Leite , Aminoácidos/metabolismo , Biomassa , Fermentação , Ácido Láctico/metabolismo , Piruvatos , Soro do Leite/metabolismoRESUMO
Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb), leading to pulmonary and extrapulmonary TB, whereby Mtb is disseminated to many other organs and tissues. Dissemination occurs early during the disease, and bacteria can be found first in the lymph nodes adjacent to the lungs and then later in the extrapulmonary organs, including the spleen. The early global gene expression response of human tissue macrophages and intracellular clinical isolates of Mtb has been poorly studied. Using dual RNA-seq, we have explored the mRNA profiles of two closely related clinical strains of the Latin American and Mediterranean (LAM) family of Mtb in infected human splenic macrophages (hSMs). This work shows that these pathogens mediate a distinct host response despite their genetic similarity. Using a genome-scale host-pathogen metabolic reconstruction to analyze the data further, we highlight that the infecting Mtb strain also determines the metabolic response of both the host and pathogen. Thus, macrophage ontogeny and the genetic-derived program of Mtb direct the host-pathogen interaction.
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Mycobacterium tuberculosis , Tuberculose , Interações Hospedeiro-Patógeno/genética , Humanos , Macrófagos/metabolismo , Mycobacterium tuberculosis/genética , RNA-Seq , Tuberculose/microbiologiaRESUMO
Efficient approaches to increase plant lipid production are necessary to meet current industrial demands for this important resource. While Jatropha curcas cell culture can be used for in vitro lipid production, scaling up the system for industrial applications requires an understanding of how growth conditions affect lipid metabolism and yield. Here we present a bottom-up metabolic reconstruction of J. curcas supported with labeling experiments and biomass characterization under three growth conditions. We show that the metabolic model can accurately predict growth and distribution of fluxes in cell cultures and use these findings to pinpoint energy expenditures that affect lipid biosynthesis and metabolism. In addition, by using constraint-based modeling approaches we identify network reactions whose joint manipulation optimizes lipid production. The proposed model and computational analyses provide a stepping stone for future rational optimization of other agronomically relevant traits in J. curcas.
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Jatropha/metabolismo , Metabolismo dos Lipídeos , Engenharia Metabólica , Biomassa , Células Cultivadas , Lipídeos/biossíntese , Engenharia Metabólica/métodos , Redes e Vias Metabólicas , Modelos BiológicosRESUMO
Metabolism underpins the pathogenic strategy of the causative agent of TB, Mycobacterium tuberculosis (Mtb), and therefore metabolic pathways have recently re-emerged as attractive drug targets. A powerful approach to study Mtb metabolism as a whole, rather than just individual enzymatic components, is to use a systems biology framework, such as a Genome-Scale Metabolic Network (GSMN) that allows the dynamic interactions of all the components of metabolism to be interrogated together. Several GSMNs networks have been constructed for Mtb and used to study the complex relationship between the Mtb genotype and its phenotype. However, the utility of this approach is hampered by the existence of multiple models, each with varying properties and performances. Here we systematically evaluate eight recently published metabolic models of Mtb-H37Rv to facilitate model choice. The best performing models, sMtb2018 and iEK1011, were refined and improved for use in future studies by the TB research community.
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Genoma Bacteriano , Redes e Vias Metabólicas , Mycobacterium tuberculosis/genética , Teorema de Bayes , Biomassa , Carbono/metabolismo , Colesterol/metabolismo , Meios de Cultura , Reações Falso-Positivas , Genótipo , Glicerol/metabolismo , Modelos Biológicos , Mycobacterium tuberculosis/metabolismo , Fenótipo , Valor Preditivo dos Testes , Software , Biologia de Sistemas , TermodinâmicaRESUMO
Chitin is an aminopolysaccharide of industrial interest commonly obtained from shrimp processing waste through chemical or biotechnological means. Current environmental concerns offer a stimulating perspective for chitin bioextraction with lactic acid bacteria since a considerable reduction in the use of corrosive and pollutant products is possible. Nevertheless, the efficiency of this bioprocess is still a matter of discussion. In this work, the experimental studies of chitin bioextraction from Pacific white shrimp (Litopenaeus vannamei) waste with a mixed culture of Lactobacillus plantarum, Lactobacillus bulgaricus and Streptococcus thermophilus are used in process simulation using Aspen Plus software for the analysis of the potential application of a bioprocess on plant scale. The experimental results of characterization in shake flasks and 1-litre bioreactor indicated that 50 h of fermentation with the mixed culture of lactic acid bacteria was enough to extract more than 90% of minerals and proteins from the shrimp waste. The use of experimental parameters in the simulation allowed a reliable representation of the bioprocess yielding normalized root mean square values below 10%. Simulation was used for the assessment of the impact of the raw material variability on the production costs and gross margin. In this regard, the gross margin of the operation ranged from 42 to 52% depending on the raw material composition and product yield.
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Clavulanic acid (CA) is produced by Streptomyces clavuligerus (S. clavuligerus) as a secondary metabolite. Knowledge about the carbon flux distribution along the various routes that supply CA precursors would certainly provide insights about metabolic performance. In order to evaluate metabolic patterns and the possible accumulation of tricarboxylic acid (TCA) cycle intermediates during CA biosynthesis, batch and subsequent continuous cultures with steadily declining feed rates were performed with glycerol as the main substrate. The data were used to in silico explore the metabolic capabilities and the accumulation of metabolic intermediates in S. clavuligerus. While clavulanic acid accumulated at glycerol excess, it steadily decreased at declining dilution rates; CA synthesis stopped when glycerol became the limiting substrate. A strong association of succinate, oxaloacetate, malate, and acetate accumulation with CA production in S. clavuligerus was observed, and flux balance analysis (FBA) was used to describe the carbon flux distribution in the network. This combined experimental and numerical approach also identified bottlenecks during the synthesis of CA in a batch and subsequent continuous cultivation and demonstrated the importance of this type of methodologies for a more advanced understanding of metabolism; this potentially derives valuable insights for future successful metabolic engineering studies in S. clavuligerus.
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Ciclo do Ácido Cítrico , Ácido Clavulânico/biossíntese , Streptomyces/metabolismo , Glicerol , Engenharia Metabólica , Streptomyces/genéticaRESUMO
In this work, we expanded and updated a genome-scale metabolic model of Streptomyces clavuligerus. The model includes 1021 genes and 1494 biochemical reactions; genome-reaction information was curated and new features related to clavam metabolism and to the biomass synthesis equation were incorporated. The model was validated using experimental data from the literature and simulations were performed to predict cellular growth and clavulanic acid biosynthesis. Flux balance analysis (FBA) showed that limiting concentrations of phosphate and an excess of ammonia accumulation are unfavorable for growth and clavulanic acid biosynthesis. The evaluation of different objective functions for FBA showed that maximization of ATP yields the best predictions for cellular behavior in continuous cultures, while the maximization of growth rate provides better predictions for batch cultures. Through gene essentiality analysis, 130 essential genes were found using a limited in silico media, while 100 essential genes were identified in amino acid-supplemented media. Finally, a strain design was carried out to identify candidate genes to be overexpressed or knocked out so as to maximize antibiotic biosynthesis. Interestingly, potential metabolic engineering targets, identified in this study, have not been tested experimentally.
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Antibacterianos/biossíntese , Genoma Bacteriano , Streptomyces/genética , Streptomyces/metabolismoRESUMO
Clavulanic acid (CA), a potent inhibitor of ß-lactamase enzymes, is produced by Streptomyces clavuligerus (Sc) cultivation processes, for which low yields are commonly obtained. Improved knowledge of the clavam biosynthetic pathway, especially the steps involved in the inversion of 3S-5S into 3R-5R stereochemical configuration, would help to eventually identify bottlenecks in the pathway. In this work, we studied the role of acetate in CA biosynthesis by a combined continuous culture and computational simulation approach. From this we derived a new model for the synthesis of N-acetyl-glycyl-clavaminic acid (NAG-clavam) by Sc. Acetylated compounds, such as NAG-clavam and N-acetyl-clavaminic acid, have been reported in the clavam pathway. Although the acetyl group is present in the ß-lactam intermediate NAG-clavam, it is unknown how this group is incorporated. Hence, under the consideration of the experimentally proven accumulation of acetate during CA biosynthesis, and the fact that an acetyl group is present in the NAG-clavam structure, a computational evaluation of the tentative formation of NAG-clavam was performed for the purpose of providing further understanding. The proposed reaction mechanism consists of two steps: first, acetate reacts with ATP to produce a reactive acylphosphate intermediate; second, a direct nucleophilic attack of the terminal amino group of N-glycyl-clavaminic on the carbonyl carbon of the acylphosphate intermediate leads to a tetrahydral intermediate, which collapses and produces ADP and N-acetyl-glycyl-clavaminic acid. The calculations suggest that for the proposed reaction mechanism, the reaction proceeds until completion of the first step, without the direct action of an enzyme, where acetate and ATP are involved. For this step, the computed activation energy was â 2.82kcal/mol while the reaction energy was â 2.38kcal/mol. As this is an endothermic chemical process with a relatively small activation energy, the reaction rate should be considerably high. The calculations offered in this work should not be considered as a definite characterization of the potential energy surface for the reaction between acetate and ATP, but rather as a first approximation that provides valuable insight about the reaction mechanism. Finally, a complete route for the inversion of the stereochemical configuration from (3S, 5S)-clavaminic acid into (3R, 5R)-clavulanic acid is proposed, including a novel alternative for the double epimerization using proline racemase and NAG-clavam formation.
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Compostos Aza/química , Modelos Químicos , Estrutura Molecular , EstereoisomerismoRESUMO
Chitosan has gained agro-industrial interest due to its potential applications in food preservation. In this work, chitosan applications for exotic fruit coating, using feijoa as a case of study, were evaluated. For this, we synthetized and characterized chitosan from shrimp shells and tested its performance. Chemical formulations for coating preparation using chitosan were proposed and tested. Mechanical properties, porosity, permeability, and fungal and bactericidal characteristics were used to verify the potential application of the film in the protection of fruits. The results indicated that synthetized chitosan has comparable properties to commercial chitosan (deacetylation degree > 82%), and, for the case of feijoa, the chitosan coating achieved significant reduction of microorganisms and fungal growth (0 UFC/mL for sample 3). Further, membrane permeability allowed oxygen exchange suitable for fruit freshness and natural physiological weight loss, thus delaying oxidative degradation and prolonging shelf-life. Chitosan's characteristic of a permeable film proved to be a promising alternative for the protection and extension of the freshness of post-harvest exotic fruits.
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Sigma factors and sigma factor-related mechanisms control antibiotic production in Streptomyces. In this contribution, the orf21 gene was overexpressed in the wild-type strain of Streptomyces clavuligerus ATCC2764, yielding S. clavuligerus/pIORF21, to further evaluate its regulatory effect on clavulanic acid (CA) biosynthesis under different culture medium conditions. The orf21 overexpression, regulated under the constitutive promoter ermE*, led to 2.6-fold increase in CA production in GSPG medium, and a 1.8-fold decrease using ISP medium. As for GYM and MYM media, S. clavuligerus/pIORF21 strain showed higher aerial mycelium production compared to control. Glycerol uptake rate profile was affected by orf21 overexpression. Furthermore, in GSPG, S. clavuligerus/pIORF21 slightly increased the expression of adpA and gcas genes, whilst, in ISP, the claR gene expression was drastically reduced, which is consistent with a decreased CA production, observed in this medium. These findings suggest the protein encoded by the orf21 gene plays a role in the regulation of CA biosynthesis as a response to the nutritional composition of the medium.
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We have previously reported the use of a combination of computational simulations and targeted experiments to build a first generation mathematical model of peppermint (Menthaxpiperita) essential oil biosynthesis. Here, we report on the expansion of this approach to identify the key factors controlling monoterpenoid essential oil biosynthesis under adverse environmental conditions. We also investigated determinants of essential oil biosynthesis in transgenic peppermint lines with modulated essential oil profiles. A computational perturbation analysis, which was implemented to identify the variables that exert prominent control over the outputs of the model, indicated that the essential oil composition should be highly dependent on certain biosynthetic enzyme concentrations [(+)-pulegone reductase and (+)-menthofuran synthase], whereas oil yield should be particularly sensitive to the density and/or distribution of leaf glandular trichomes, the specialized anatomical structures responsible for the synthesis and storage of essential oils. A microscopic evaluation of leaf surfaces demonstrated that the final mature size of glandular trichomes was the same across all experiments. However, as predicted by the perturbation analysis, differences in the size distribution and the total number of glandular trichomes strongly correlated with differences in monoterpenoid essential oil yield. Building on various experimental data sets, appropriate mathematical functions were selected to approximate the dynamics of glandular trichome distribution/density and enzyme concentrations in our kinetic model. Based on a chi2 statistical analysis, simulated and measured essential oil profiles were in very good agreement, indicating that modeling is a valuable tool for guiding metabolic engineering efforts aimed at improving essential oil quality and quantity.
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Mentha piperita/química , Modelos Teóricos , Óleos Voláteis/química , Folhas de Planta/química , Genes de Plantas , Genótipo , Mentha piperita/genética , Plantas Geneticamente ModificadasRESUMO
The integration of mathematical modeling and experimental testing is emerging as a powerful approach for improving our understanding of the regulation of metabolic pathways. In this study, we report on the development of a kinetic mathematical model that accurately simulates the developmental patterns of monoterpenoid essential oil accumulation in peppermint (Mentha x piperita). This model was then used to evaluate the biochemical processes underlying experimentally determined changes in the monoterpene pathway under low ambient-light intensities, which led to an accumulation of the branchpoint intermediate (+)-pulegone and the side product (+)-menthofuran. Our simulations indicated that the environmentally regulated changes in monoterpene profiles could only be explained when, in addition to effects on biosynthetic enzyme activities, as yet unidentified inhibitory effects of (+)-menthofuran on the branchpoint enzyme pulegone reductase (PR) were assumed. Subsequent in vitro analyses with recombinant protein confirmed that (+)-menthofuran acts as a weak competitive inhibitor of PR (K(i) = 300 muM). To evaluate whether the intracellular concentration of (+)-menthofuran was high enough for PR inhibition in vivo, we isolated essential oil-synthesizing secretory cells from peppermint leaves and subjected them to steam distillations. When peppermint plants were grown under low-light conditions, (+)-menthofuran was selectively retained in secretory cells and accumulated to very high levels (up to 20 mM), whereas under regular growth conditions, (+)-menthofuran levels remained very low (<400 muM). These results illustrate the utility of iterative cycles of mathematical modeling and experimental testing to elucidate the mechanisms controlling flux through metabolic pathways.
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Vias Biossintéticas/fisiologia , Mentha piperita/química , Modelos Teóricos , Monoterpenos/metabolismo , Óleos de Plantas/química , Biologia de Sistemas/métodos , Simulação por Computador , Monoterpenos Cicloexânicos , Cinética , Estrutura Molecular , Monoterpenos/análise , Oxirredutases/antagonistas & inibidores , Oxirredutases/metabolismoRESUMO
Species of the genus Streptomyces are known for their ability to produce multiple secondary metabolites; their genomes have been extensively explored to discover new bioactive compounds. The richness of genomic data currently available allows filtering for high quality genomes, which in turn permits reliable comparative genomics studies and an improved prediction of biosynthetic gene clusters (BGCs) through genome mining approaches. In this work, we used 121 genome sequences of the genus Streptomyces in a comparative genomics study with the aim of estimating the genomic diversity by protein domains content, sequence similarity of proteins and conservation of Intergenic Regions (IGRs). We also searched for BGCs but prioritizing those with potential antibiotic activity. Our analysis revealed that the pan-genome of the genus Streptomyces is clearly open, with a high quantity of unique gene families across the different species and that the IGRs are rarely conserved. We also described the phylogenetic relationships of the analyzed genomes using multiple markers, obtaining a trustworthy tree whose relationships were further validated by Average Nucleotide Identity (ANI) calculations. Finally, 33 biosynthetic gene clusters were detected to have potential antibiotic activity and a predicted mode of action, which might serve up as a guide to formulation of related experimental studies.
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Streptomyces clavuligerus (S. clavuligerus) has been widely studied for its ability to produce clavulanic acid (CA), a potent inhibitor of ß-lactamase enzymes. In this study, S. clavuligerus cultivated in 2D rocking bioreactor in fed-batch operation produced CA at comparable rates to those observed in stirred tank bioreactors. A reduced model of S. clavuligerus metabolism was constructed by using a bottom-up approach and validated using experimental data. The reduced model was implemented for in silico studies of the metabolic scenarios arisen during the cultivations. Constraint-based analysis confirmed the interrelations between succinate, oxaloacetate, malate, pyruvate, and acetate accumulations at high CA synthesis rates in submerged cultures of S. clavuligerus. Further analysis using shadow prices provided a first view of the metabolites positive and negatively associated with the scenarios of low and high CA production.
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Microalgae biomass exploitation as a carbon-neutral energy source is currently limited by several factors, productivity being one of the most relevant. Due to the high absorption properties of light-harvesting antenna, photosynthetic cells tend to capture an excessive amount of energy that cannot be entirely channeled through the electron transfer chain that ends up dissipated as heat and fluorescence, reducing the overall light use efficiency. Aiming to minimize this hurdle, in this work we studied the effect of decreasing concentrations of Magnesium (Mg2+) on the chlorophyll a content, photosynthetic performance, biomass and lipid production of autotrophic cultures of Botryococcus braunii LB 572. We also performed, for the first time, a comparative lipidomic analysis to identify the influence of limited Mg2+ supply on the lipid profile of this algae. The results indicated that a level of 0.0037 g L-1 MgSO4 caused a significant decline on chlorophyll a content with a concomitant 2.3-fold reduction in the biomass absorption coefficient. In addition, the Mg2+ limitation caused a decrease in the total carbohydrate content and triggered lipid accumulation, achieving levels of up to 53% DCW, whereas the biomass productivity remained similar for all tested conditions. The lipidome analysis revealed that the lowest Mg2+ concentrations also caused a differential lipid profile distribution, with an enrichment of neutral lipids and an increase of structural lipids. In that sense, we showed that Mg2+ limitation represents an alternative optimization approach that not only enhances accumulation of neutral lipids in B. braunii cells but also may potentially lead to a better areal biomass productivity due to the reduction in the cellular light absorption properties of the cells.
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Streptomyces clavuligerus is a filamentous Gram-positive bacterial producer of the ß-lactamase inhibitor clavulanic acid. Antibiotics biosynthesis in the Streptomyces genus is usually triggered by nutritional and environmental perturbations. In this work, a new genome scale metabolic network of Streptomyces clavuligerus was reconstructed and used to study the experimentally observed effect of oxygen and phosphate concentrations on clavulanic acid biosynthesis under high and low shear stress. A flux balance analysis based on experimental evidence revealed that clavulanic acid biosynthetic reaction fluxes are favored in conditions of phosphate limitation, and this is correlated with enhanced activity of central and amino acid metabolism, as well as with enhanced oxygen uptake. In silico and experimental results show a possible slowing down of tricarboxylic acid (TCA) due to reduced oxygen availability in low shear stress conditions. In contrast, high shear stress conditions are connected with high intracellular oxygen availability favoring TCA activity, precursors availability and clavulanic acid (CA) production.
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Clavulanic acid (CA) is a ß-lactam antibiotic with a strong inhibitory effect on ß-lactamase enzymes. CA is produced in submerged cultures by the filamentous Gram-positive bacterium Streptomyces clavuligerus (S. clavuligerus). CA is an unstable molecule in aqueous solution and its stability depends strongly on temperature and concentration. In this contribution, the experimental data of CA stability, produced in chemically defined media and exposed to temperatures between -80 and 25 °C, are presented. The chromophore clavulanate-imidazole (CAI) is commonly used for analysis and quantification of CA samples by High Performance Liquid Chromatography (HPLC); nevertheless, this molecule is also susceptible to suffer degradation in aqueous solution, potentially affecting the quantification of CA. Data of CAI concentration for samples conserved at 4 °C and 25 °C are also presented. A reversible-irreversible kinetic model was applied to estimate the degradation rate of CA. Data from numerical simulations of CA degradation using the proposed kinetic model are also graphically presented. The data show the clavulanic acid instability in fermentation broths, in a range of temperatures of interest for bioprocess operation, downstream processing, samples quantification, conservation and storage.
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Background: Clavulanic acid (CA), a ß-lactamase inhibitor, is industrially produced by the fermentation of Streptomyces clavuligerus. The efficiency of CA production is associated with media composition, culture conditions and physiological and genetic strain characteristics. However, the molecular pathways that govern CA regulation in S. clavuligerus remain unknown. Methods and Results: Here we used RNA-seq to perform a comparative transcriptome analysis of S. clavuligerus ATCC 27064 wild-type strain grown in both a favorable soybean-based medium and in limited media conditions to further contribute to the understanding of S. clavuligerus metabolism and its regulation. A total of 350 genes were found to be differentially expressed between conditions; 245 genes were up-regulated in favorable conditions compared to unfavorable. Conclusion: The up-regulated expression of many regulatory and biosynthetic CA genes was positively associated with the favorable complex media condition along with pleiotropic regulators, including proteases and some genes whose biological function have not been previously reported. Knowledge from differences between transcriptomes from complex/defined media represents an advance in the understanding of regulatory paths involved in S. clavuligerus' metabolic response, enabling the rational design of future experiments.
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Clavulanic acid (CA) is a ß-lactam antibiotic inhibitor of ß-lactamase enzymes, which confers resistance to bacteria against several antibiotics. CA is produced in submerged cultures by the filamentous Gram-positive bacterium Streptomyces clavuligerus; yield and downstream process are compromised by a degradation phenomenon, which is not yet completely elucidated. In this contribution, a study of degradation kinetics of CA at low temperatures (-80, -20, 4, and 25 °C) and pH 6.8 in chemically-defined fermentation broths is presented. Samples of CA in the fermentation broths showed a fast decline of concentration during the first 5 h followed by a slower, but stable, reaction rate in the subsequent hours. A reversible-irreversible kinetic model was applied to explain the degradation rate of CA, its dependence on temperature and concentration. Kinetic parameters for the equilibrium and irreversible reactions were calculated and the proposed kinetic model was validated with experimental data of CA degradation ranging 16.3 mg/L to 127.0 mg/L. Degradation of the chromophore CA-imidazole, which is commonly used for quantifications by High Performance Liquid Chromatography, was also studied at 4 °C and 25 °C, showing a rapid rate of degradation according to irreversible first-order kinetics. A hydrolysis reaction mechanism is proposed as the cause of CA-imidazole loss in aqueous solutions.
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The performance of software tools for de novo transcriptome assembly greatly depends on the selection of software parameters. Up to now, the development of de novo transcriptome assembly for prokaryotes has not been as remarkable as that for eukaryotes. In this contribution, Rockhopper2 was used to perform a comparative transcriptome analysis of Streptomyces clavuligerus exposed to diverse environmental conditions. The study focused on assessing the incidence of software parameters on software performance for the identification of differentially expressed genes as a final goal. For this, a statistical optimization was performed using the Transrate Assembly Score (TAS). TAS was also used for evaluating the software performance and for comparing it with related tools, e.g., Trinity. Transcriptome redundancy and completeness were also considered for this analysis. Rockhopper2 and Trinity reached a TAS value of 0.55092 and 0.58337, respectively. Trinity assembles transcriptomes with high redundancy, with 55.6% of transcripts having some duplicates. Additionally, we observed that the total number of differentially expressed genes (DEG) and their annotation greatly depends on the method used for removing redundancy and the tools used for transcript quantification. To our knowledge, this is the first work aimed at assessing de novo assembly software for prokaryotic organisms.