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Background: Weissella confusa is a member of the lactic acid bacterium group commonly found in many salt-fermented foods. Strains of W. confusa isolated from high-salinity environments have been shown to tolerate salt stress to some extent. However, the specific responses and mechanisms of W. confusa under salt stress are not fully understood. Methods: To study the effect of NaCl stress on W. confusa, growth performance and metabolite profiles of the strains were compared between a NaCl-free group and a 35% NaCl-treated group. Growth performance was assessed by measuring viable cell counts and examining the cells using scanning electron microscopy (SEM). Intracellular and extracellular metabolites were analyzed by non-targeted metabolomics based on liquid chromatography-mass spectrometry (LC-MS). Results: It was found that the viable cell count of W. confusa decreased with increasing salinity, and cells could survive even in saturated saline (35%) medium for 24 h. When exposed to 35% NaCl, W. confusa cells exhibited surface pores and protein leakage. Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, 42 different metabolites were identified in the cells and 18 different metabolites in the culture medium. These different metabolites were mainly involved in amino acid metabolism, carbohydrate metabolism, and nucleotide metabolism. In addition, salt-exposed cells exhibited higher levels of intracellular ectoine and lactose, whose precursors, such as aspartate, L-2,4-diaminobutanoate, and galactinol, were reduced in the culture medium. Conclusions: This study provides insight into the metabolic responses of W. confusa under salt stress, revealing its ability to maintain viability and alter metabolism in response to high NaCl concentrations. Key metabolites such as ectoine and lactose, as well as changes in amino acid and nucleotide metabolism, may contribute to its tolerance to salt. These findings may improve our understanding of the bacterium's survival mechanisms and have potential applications in food fermentation and biotechnology. [ABSTRACT FROM AUTHOR]

Frozen dough technology is important in modern bakery operations, facilitating the transportation of dough at low temperatures to downstream sales points. However, the freeze–thaw process imposes significant stress on baker's yeast, resulting in diminished viability and fermentation capacity. Understanding the mechanisms underlying freeze–thaw stress is essential for mitigating its adverse effects on yeast performance. This review delves into the intricate mechanisms underlying freeze–thaw stress, focusing specifically on Saccharomyces cerevisiae, the primary yeast used in baking, and presents a wide range of biotechnological approaches to enhance freeze–thaw resistance in S. cerevisiae. Strategies include manipulating intracellular metabolites, altering membrane composition, managing antioxidant defenses, mediating aquaporin expression, and employing adaptive evolutionary and breeding techniques. Addressing challenges and strategies associated with freeze–thaw stress, this review provides valuable insights for future research endeavors, aiming to enhance the freeze–thaw tolerance of baker's yeast and contribute to the advancement of bakery science. [ABSTRACT FROM AUTHOR]

Metabolomics has proven to be a sensitive tool for monitoring biochemical processes in cell culture. It enables multi-analysis, clarifying the correlation between numerous metabolic pathways. Together with other analysis, it thus provides a global view of a cell's physiological state. A comprehensive analysis of molecular changes is also required in the case of mesenchymal stem cells (MSCs), which currently represent an essential portion of cells used in regenerative medicine. Reproducibility and correct measurement are closely connected to careful metabolite extraction, and sample preparation is always a critical point. Our study aimed to compare the efficiencies of four harvesting and six extraction methods. Several organic reagents (methanol, ethanol, acetonitrile, methanol–chloroform, MTBE) and harvesting approaches (trypsinization vs. scraping) were tested. We used untargeted nuclear magnetic resonance spectroscopy (NMR) to determine the most efficient method for the extraction of metabolites from human adherent cells, specifically human dermal fibroblasts adult (HDFa) and dental pulp stem cells (DPSCs). A comprehensive dataset of 29 identified and quantified metabolites were determined to possess statistically significant differences in the abundances of several metabolites when the cells were detached mechanically to organic solvent compared to when applying enzymes mainly in the classes of amino acids and peptides for both types of cells. Direct scraping to organic solvent is a method that yields higher abundances of determined metabolites. Extraction with the use of different polar reagents, 50% and 80% methanol, or acetonitrile, mostly showed the same quality. For both HDFa and DPSC cells, the MTBE method, methanol–chloroform, and 80% ethanol extractions showed higher extraction efficiency for the most identified and quantified metabolites Thus, preparation procedures provided a cell sample processing protocol that focuses on maximizing extraction yield. Our approach may be useful for large-scale comparative metabolomic studies of human mesenchymal stem cell samples. [ABSTRACT FROM AUTHOR]

Background: Weissella confusa is a member of the lactic acid bacterium group commonly found in many salt-fermented foods. Strains of W. confusa isolated from high-salinity environments have been shown to tolerate salt stress to some extent. However, the specific responses and mechanisms of W. confusa under salt stress are not fully understood. Methods: To study the effect of NaCl stress on W. confusa, growth performance and metabolite profiles of the strains were compared between a NaCl-free group and a 35% NaCl-treated group. Growth performance was assessed by measuring viable cell counts and examining the cells using scanning electron microscopy (SEM). Intracellular and extracellular metabolites were analyzed by non-targeted metabolomics based on liquid chromatography-mass spectrometry (LC-MS). Results: It was found that the viable cell count of W. confusa decreased with increasing salinity, and cells could survive even in saturated saline (35%) medium for 24 h. When exposed to 35% NaCl, W. confusa cells exhibited surface pores and protein leakage. Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, 42 different metabolites were identified in the cells and 18 different metabolites in the culture medium. These different metabolites were mainly involved in amino acid metabolism, carbohydrate metabolism, and nucleotide metabolism. In addition, salt-exposed cells exhibited higher levels of intracellular ectoine and lactose, whose precursors, such as aspartate, L-2,4-diaminobutanoate, and galactinol, were reduced in the culture medium. Conclusions: This study provides insight into the metabolic responses of W. confusa under salt stress, revealing its ability to maintain viability and alter metabolism in response to high NaCl concentrations. Key metabolites such as ectoine and lactose, as well as changes in amino acid and nucleotide metabolism, may contribute to its tolerance to salt. These findings may improve our understanding of the bacterium’s survival mechanisms and have potential applications in food fermentation and biotechnology.

Many studies have focused on the influence of dietary supplements on gut microbiota composition, but limited research have reported their effects on specific bacterial species in the gut. Lactiplantibacillus plantarum is one of the most widely studied probiotics, with a wide range of sources and good environmental adaptability. In this study, in order to elucidate the adaptation strategies of L. plantarum to the gut of mice supplemented with carbohydrates, peptides and minerals, whole genome resequencing and intracellular metabolites detection were performed, and high-frequency mutant genes and differential metabolites were screened. The results suggested different types of dietary supplements do have different effects on L. plantarum from the gut of mice. Additionally, KEGG annotation unveiled that the effects of these dietary supplements on the gene level of L. plantarum primarily pertained to environmental information processing, while the differential metabolites were predominantly associated with metabolism. This study provided new perspectives on the adaptive mechanism of L. plantarum in response to the host’s gut environment, suggesting that the diversity of the genome and metabolome of L. plantarum was correlated with dietary supplements. Furthermore, this study offered useful guidance in the effective utilization of dietary supplements. [ABSTRACT FROM AUTHOR]

Microbes commonly use metabolites produced by other organisms to compete effectively with others in their environment. As a result, microbial communities are composed of networks of metabolically interdependent organisms. How these networks evolve and shape population diversity, stability, and community function is a subject of active research. But how did these metabolic interactions develop initially? In particular, how and why are metabolites such as amino acids, cofactors, and nucleobases released for the benefit of others when there apparently is no incentive to do so? Here, we discuss the hypothesis that metabolite provisioning is not itself adaptive but rather can be a natural consequence of other evolved biological functions. We outline two examples of metabolite provisioning as a by-product of other functions by considering cell lysis and regulated metabolite efflux outside their canonical roles and explore their potential to facilitate the emergence of interdependent metabolite sharing.

Context. The clinical value of human sperm metabolites has not been established due to the technical complexity in detecting these metabolites when sperm numbers are low. Aims. To detect endogenous intracellular metabolites in fresh and post-thaw human spermatozoa using 800 MHz nuclear magnetic resonance (NMR) spectroscopy equipped with a 1.7-mm cryo-probe. Methods. Processed spermatozoa from 25 normozoospermic ejaculates were subjected to extraction of intracellular metabolites and then profiled by sensitivity-enhanced NMR spectroscopy equipped with a 1.7-mm cryogenically cooled micro-coil probe. In parallel, some of the processed sperm fractions were subjected to freeze-thawing and were then analysed for intracellular metabolites. Key results. Twenty-three metabolites were profiled from only 1.25 million sperm cells. Comparison of the metabolomic signature of pre-freeze and post-thaw sperm cells did not show significant changes in the levels of metabolites. Conclusions. Sensitivity-enhanced NMR spectroscopy equipped with a 1.7-mm cryogenically cooled micro-coil probe is a potential tool for identifying intracellular metabolites when sperm number is low. Implications. Use of sensitivity-enhanced NMR spectroscopy opens up the opportunity to test for endogenous metabolites in samples with a limited number of spermatozoa, to understand the patho-physiology of infertility. [ABSTRACT FROM AUTHOR]

Metabolomics has proven to be a sensitive tool for monitoring biochemical processes in cell culture. It enables multi-analysis, clarifying the correlation between numerous metabolic pathways. Together with other analysis, it thus provides a global view of a cell’s physiological state. A comprehensive analysis of molecular changes is also required in the case of mesenchymal stem cells (MSCs), which currently represent an essential portion of cells used in regenerative medicine. Reproducibility and correct measurement are closely connected to careful metabolite extraction, and sample preparation is always a critical point. Our study aimed to compare the efficiencies of four harvesting and six extraction methods. Several organic reagents (methanol, ethanol, acetonitrile, methanol–chloroform, MTBE) and harvesting approaches (trypsinization vs. scraping) were tested. We used untargeted nuclear magnetic resonance spectroscopy (NMR) to determine the most efficient method for the extraction of metabolites from human adherent cells, specifically human dermal fibroblasts adult (HDFa) and dental pulp stem cells (DPSCs). A comprehensive dataset of 29 identified and quantified metabolites were determined to possess statistically significant differences in the abundances of several metabolites when the cells were detached mechanically to organic solvent compared to when applying enzymes mainly in the classes of amino acids and peptides for both types of cells. Direct scraping to organic solvent is a method that yields higher abundances of determined metabolites. Extraction with the use of different polar reagents, 50% and 80% methanol, or acetonitrile, mostly showed the same quality. For both HDFa and DPSC cells, the MTBE method, methanol–chloroform, and 80% ethanol extractions showed higher extraction efficiency for the most identified and quantified metabolites Thus, preparation procedures provided a cell sample processing protocol that focuses on maximizing extraction yield. Our approach may be useful for large-scale comparative metabolomic studies of human mesenchymal stem cell samples.

BACKGROUND: Zygosaccharomyces rouxii plays an irreplaceable role in the manufacture of traditional fermented foods, which are produced in a high‐salt environment. However, there is little research on strategies for improving salt tolerance of Z. rouxii. RESULTS: In this study, metabolomics was used to reveal the changes in intracellular metabolites under salt stress, and the results show that most of the carbohydrate contents decreased, the contents of xanthohumol and glycerol increased (fold change 4.07 and 5.35, respectively), while the contents of galactinol, xylitol and d‐threitol decreased (fold change −9.43, −5.83 and −3.59, respectively). In addition, the content of four amino acids and six organic acids decreased, while that of the ten nucleotides increased. Notably, except for stearic acid (C18:0), all fatty acid contents increased. Guided by the metabolomics results, the effect of addition of seven exogenous fatty acids (C12:0, C14:0, C16:0, C18:0, C16:1, C18:1, and C18:2) on the salt tolerance of Z. rouxii was analyzed, and the results suggested that four exogenous fatty acids (C12:0, C16:0, C16:1, and C18:1) can increase the biomass yield and maximum growth rate. Physiological analyses demonstrated that exogenous fatty acids could regulate the distribution of fatty acids in the cell membrane, increase the degree of unsaturation, improve membrane fluidity, and maintain cell integrity, morphology and surface roughness. CONCLUSION: These results are applicable to revealing the metabolic mechanisms of Z. rouxii under salt stress and screening potential protective agents to improve stress resistance by adding exogenous fatty acids. © 2022 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

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ABSTRACT All organisms rely on complex metabolites such as amino acids, nucleotides, and cofactors for essential metabolic processes. Some microbes synthesize these fundamental ingredients of life de novo, while others rely on uptake to fulfill their metabolic needs. Although certain metabolic processes are inherently “leaky,” the mechanisms enabling stable metabolite provisioning among microbes in the absence of a host remain largely unclear. In particular, how can metabolite provisioning among free-living bacteria be maintained under the evolutionary pressure to economize resources? Salvaging, the process of “recycling and reusing,” can be a metabolically efficient route to obtain access to required resources. Here, we show experimentally how precursor salvaging in engineered Escherichia coli populations can lead to stable, long-term metabolite provisioning. We find that salvaged cobamides (vitamin B12 and related enzyme cofactors) are readily made available to nonproducing population members, yet salvagers are strongly protected from overexploitation. We also describe a previously unnoted benefit of precursor salvaging, namely, the removal of the nonfunctional, proliferation-inhibiting precursor. As long as compatible precursors are present, any microbe possessing the terminal steps of a biosynthetic process can, in principle, forgo de novo biosynthesis in favor of salvaging. Consequently, precursor salvaging likely represents a potent, yet overlooked, alternative to de novo biosynthesis for the acquisition and provisioning of metabolites in free-living bacterial populations. IMPORTANCE Recycling gives new life to old things. Bacteria have the ability to recycle and reuse complex molecules they encounter in their environment to fulfill their basic metabolic needs in a resource-efficient way. By studying the salvaging (recycling and reusing) of vitamin B12 precursors, we found that metabolite salvaging can benefit others and provide stability to a bacterial community at the same time. Salvagers of vitamin B12 precursors freely share the result of their labor yet cannot be outcompeted by freeloaders, likely because salvagers retain preferential access to the salvaging products. Thus, salvaging may represent an effective, yet overlooked, mechanism of acquiring and provisioning nutrients in microbial populations.

Studies of intracellular metabolites of Corynebacterium glutamicum mostly were done at levels of biomass concentrations below 10 g L−1. In this work we could show that it is also possible up to 32 g L−1 biomass concentration. Hereby we could point towards a faster filtration process with a modification of the already established automated fast filtration. We also could quantify the influence of a genetic modification of the lysine feedback inhibited asparto kinase in the aspartate metabolism with the automated fast filtration at higher biomass concentrations. [ABSTRACT FROM AUTHOR]

Evolution facilitates emergence of fitter phenotypes by efficient allocation of cellular resources in conjunction with beneficial mutations. However, system-wide pleiotropic effects that redress the perturbations to the apex node of the transcriptional regulatory networks remain unclear. Here, we elucidate that absence of global transcriptional regulator CRP in Escherichia coli results in alterations in key metabolic pathways under glucose respiratory conditions, favouring stress- or hedging-related functions over growth-enhancing functions. Further, we disentangle the growth-mediated effects from the CRP regulation-specific effects on these metabolic pathways. We quantitatively illustrate that the loss of CRP perturbs proteome efficiency, as evident from metabolic as well as ribosomal proteome fractions, that corroborated with intracellular metabolite profiles. To address how E. coli copes with such systemic defect, we evolved Δcrp mutant in the presence of glucose. Besides acquiring mutations in the promoter of glucose transporter ptsG, the evolved populations recovered the metabolic pathways to their pre-perturbed state coupled with metabolite re-adjustments, which altogether enabled increased growth. By contrast to Δcrp mutant, the evolved strains remodelled their proteome efficiency towards biomass synthesis, albeit at the expense of carbon efficiency. Overall, we comprehensively illustrate the genetic and metabolic basis of pleiotropic effects, fundamental for understanding the growth physiology.

Rosa roxburghii Tratt. is widely applied in food, cosmetics, and traditional medicine, and has been demonstrated to possess diverse bioactivities. Plant endophytic fungi are important microbial resources with great potential for application in many fields. They not only establish mutualistic symbiosis with host plants but also produce a variety of bioactive compounds. Therefore, in the present study, endophytic fungi were isolated from R. roxburghii, the diversity and antimicrobial activities were evaluated. As a result, 242 strains of endophytic Sordariomycetes were successfully isolated. Multigene phylogenetic analyses showed that these isolates included eight orders, 19 families, 33 genera. The dominant genera were Diaporthe (31.4%), Fusarium (14.4%), Chaetomium (7.9%), Dactylonectria (7.0%), Graphium (4.5%), Colletotrichum (4.1%), and Clonostachys (4.1%). For different tissues of R. roxburghii, alpha diversity analysis revealed that the diversity of fungal communities decreased in the order of root, fruit, stem, flower, leaf, and seed, and Clonostachys and Dactylonectria exhibited obvious tissue specificity. Meanwhile, functional annotation of 33 genera indicated that some fungi have multitrophic lifestyles combining endophytic, pathogenic, and saprophytic behavior. Additionally, antimicrobial activities of endophytic Sordariomycetes against Lasiodiplodia theobromae, Botryosphaeria dothidea, Colletotrichum capsici, Pyricularia oryzae, Rhizoctonia solani, Fusarium oxysporum, Pseudomonas syringae, Pantoea agglomerans, Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa were screened. Dual culture test assays showed that there were 40 different endophytic species with strong inhibition of at least one or moderate inhibition of two or more against the 12 tested strains. The results from the filter paper diffusion method suggested that extracellular metabolites may be more advantageous than intracellular metabolites in the development of antimicrobial agents. Eleven isolates with good activities were screened. In particular, Hypomontagnella monticulosa HGUP194009 and Nigrospora sphaerica HGUP191020 have shown promise in both broad-spectrum and intensity. Finally, some fungi that commonly cause disease have been observed to have beneficial biological activities as endophytic fungi. In conclusion, this study showed the species composition, alpha diversity, and lifestyle diversity of endophytic Sordariomycetes from R. roxburghii and demonstrated these isolates are potential sources for exploring antimicrobial agents.

Rosa roxburghii Tratt. is widely applied in food, cosmetics, and traditional medicine, and has been demonstrated to possess diverse bioactivities. Plant endophytic fungi are important microbial resources with great potential for application in many fields. They not only establish mutualistic symbiosis with host plants but also produce a variety of bioactive compounds. Therefore, in the present study, endophytic fungi were isolated from R. roxburghii , the diversity and antimicrobial activities were evaluated. As a result, 242 strains of endophytic Sordariomycetes were successfully isolated. Multigene phylogenetic analyses showed that these isolates included eight orders, 19 families, 33 genera. The dominant genera were Diaporthe (31.4%), Fusarium (14.4%), Chaetomium (7.9%), Dactylonectria (7.0%), Graphium (4.5%), Colletotrichum (4.1%), and Clonostachys (4.1%). For different tissues of R. roxburghii , alpha diversity analysis revealed that the diversity of fungal communities decreased in the order of root, fruit, stem, flower, leaf, and seed, and Clonostachys and Dactylonectria exhibited obvious tissue specificity. Meanwhile, functional annotation of 33 genera indicated that some fungi have multitrophic lifestyles combining endophytic, pathogenic, and saprophytic behavior. Additionally, antimicrobial activities of endophytic Sordariomycetes against Lasiodiplodia theobromae , Botryosphaeria dothidea , Colletotrichum capsici , Pyricularia oryzae , Rhizoctonia solani , Fusarium oxysporum , Pseudomonas syringae , Pantoea agglomerans , Staphylococcus aureus , Bacillus subtilis , Escherichia coli , and Pseudomonas aeruginosa were screened. Dual culture test assays showed that there were 40 different endophytic species with strong inhibition of at least one or moderate inhibition of two or more against the 12 tested strains. The results from the filter paper diffusion method suggested that extracellular metabolites may be more advantageous than intracellular metabolites in the development of antimicrobial agents. Eleven isolates with good activities were screened. In particular, Hypomontagnella monticulosa HGUP194009 and Nigrospora sphaerica HGUP191020 have shown promise in both broad-spectrum and intensity. Finally, some fungi that commonly cause disease have been observed to have beneficial biological activities as endophytic fungi. In conclusion, this study showed the species composition, alpha diversity, and lifestyle diversity of endophytic Sordariomycetes from R. roxburghii and demonstrated these isolates are potential sources for exploring antimicrobial agents. [ABSTRACT FROM AUTHOR]

Abstract Microorganisms generally encounter a fluctuating environment in their natural habitat and similar conditions also happen in large-scale bioreactors. In this work, the dynamic response of intracellular and extracellular metabolites of Aspergillus niger was investigated after sudden exposure to high and low excess glucose concentrations in chemostats. It was found that the steady-state pathway turnover time of the carbon flux through the central carbon metabolism (CCM) was PP pathway 50 s, EMP pathway 20 s, and TCA cycle 189 s, and an upper limit for individual metabolite concentrations in the CCM was estimated. Regardless of the glucose pulse size, little changes of amino acids levels were observed except for aspartate, which showed a significant decrease. The ATP paradox, known from other organisms, was also observed in the studied A. niger strain. However, a different response of the NAD+/NADH ratio to the glucose pulses was found in A. niger compared to previously published observations on Penicillium chrysogenum and Saccharomyces cerevisiae. These findings are valuable for better understanding A. niger culture performance in large-scale bioreactors.

ABSTRACT Global transcriptional regulators coordinate complex genetic interactions that bestow better adaptability for an organism against external and internal perturbations. These transcriptional regulators are known to control an enormous array of genes with diverse functionalities. However, regulator-driven molecular mechanisms that underpin precisely tuned translational and metabolic processes conducive for rapid exponential growth remain obscure. Here, we comprehensively reveal the fundamental role of global transcriptional regulators FNR, ArcA, and IHF in sustaining translational and metabolic efficiency under glucose fermentative conditions in Escherichia coli. By integrating high-throughput gene expression profiles and absolute intracellular metabolite concentrations, we illustrate that these regulators are crucial in maintaining nitrogen homeostasis, govern expression of otherwise unnecessary or hedging genes, and exert tight control on metabolic bottleneck steps. Furthermore, we characterize changes in expression and activity profiles of other coregulators associated with these dysregulated metabolic pathways, determining the regulatory interactions within the transcriptional regulatory network. Such systematic findings emphasize their importance in optimizing the proteome allocation toward metabolic enzymes as well as ribosomes, facilitating condition-specific phenotypic outcomes. Consequentially, we reveal that disruption of this inherent trade-off between ribosome and metabolic proteome economy due to the loss of regulators resulted in lowered growth rates. Moreover, our findings reinforce that the accumulations of intracellular metabolites in the event of proteome repartitions negatively affects the glucose uptake. Overall, by extending the three-partition proteome allocation theory concordant with multi-omics measurements, we elucidate the physiological consequences of loss of global regulators on central carbon metabolism restraining the organism to attain maximal biomass synthesis. IMPORTANCE Cellular proteome allocation in response to environmental or internal perturbations governs their eventual phenotypic outcome. This entails striking an effective balance between amino acid biosynthesis by metabolic proteins and its consumption by ribosomes. However, the global transcriptional regulator-driven molecular mechanisms that underpin their coordination remains unexplored. Here, we emphasize that global transcriptional regulators, known to control expression of a myriad of genes, are fundamental for precisely tuning the translational and metabolic efficiencies that define the growth optimality. Towards this, we systematically characterized the single deletion effect of FNR, ArcA, and IHF regulators of Escherichia coli on exponential growth under anaerobic glucose fermentative conditions. Their absence disrupts the stringency of proteome allocation, which manifests as impairment in key metabolic processes and an accumulation of intracellular metabolites. Furthermore, by incorporating an extension to the empirical growth laws, we quantitatively demonstrate the general design principles underlying the existence of these regulators in E. coli.

ABSTRACT Microbes commonly use metabolites produced by other organisms to compete effectively with others in their environment. As a result, microbial communities are composed of networks of metabolically interdependent organisms. How these networks evolve and shape population diversity, stability, and community function is a subject of active research. But how did these metabolic interactions develop initially? In particular, how and why are metabolites such as amino acids, cofactors, and nucleobases released for the benefit of others when there apparently is no incentive to do so? Here, we discuss the hypothesis that metabolite provisioning is not itself adaptive but rather can be a natural consequence of other evolved biological functions. We outline two examples of metabolite provisioning as a by-product of other functions by considering cell lysis and regulated metabolite efflux outside their canonical roles and explore their potential to facilitate the emergence of interdependent metabolite sharing.

Abstract Absolute metabolite concentrations are essential information for quantitative metabolomics studies, as concentrations are closely related to metabolic reactions, enzyme kinetics and other important biological activities. A well-performed metabolites extraction procedure, a reliable detection method, and a robust quantitative approach are all critical factors for obtaining absolute metabolite concentrations. Here, we used a HPLC-MS/MS based platform to successfully develop a 13C-labeled quantitative metabolomics approach, and applied this novel method to quantify bacterial metabolite concentrations in three different domains (i.e., intracellularly, extracellularly and to the whole culture), with high accuracy for a model Escherichia coli bacteria. The bacterial culture was grown in universal 13C-labeled medium and the metabolites were extracted by 40/40/20/methanol/ACN/H 2 O with 0.1% formic acid. One hundred and twenty-five metabolites were initially screened and one hundred and six 13C metabolites were confidently detected from the model bacteria grown in 13C-labeled medium. A subset of twenty-one metabolites was subsequently quantified using 12C-metabolite chemical standards to assist the calculation of 13C metabolite concentration. This rigorous 13C-labled quantitative method was then applied to characterize the metabolic profile changes in three domains of E. coli going through antibiotic treatment. Our results demonstrated that metabolites from all three domains can be used to significantly differentiate the ampicillin treatment group and control group (without ampicillin). In conclusion, our work demonstrated that the quantitative metabolomics approach can be used as a valuable tool to study bacterial metabolism in different domains and to understand their response to environmental perturbations. Graphical abstract Image 1 Highlights • Development of a 13C-based quantitative targeted metabolic profiling approach. • Metabolite quantitation using cost-effective and largely available 12C-based standards. • Investigation of bacterial metabolome intracellularly, extracellularly and to the whole culture. • Detection of bacterial metabolic changes during antibiotic treatment in three different domains. [ABSTRACT FROM AUTHOR]

Metabolomics is a tool with immense potential for providing insight into the impact of biological processes on the environment. Here, we used metabolomics methods to characterize intracellular metabolites within marine microorganisms during a manipulation experiment that was designed to test the impact of two sources of microbial mortality, protozoan grazing and viral lysis. Intracellular metabolites were analyzed with targeted and untargeted mass spectrometry methods. The treatment with reduced viral mortality showed the largest changes in metabolite concentrations, although there were organic compounds that shifted when the impact of protozoan grazers was reduced. Intracellular concentrations of guanine, phenylalanine, glutamic acid, and ectoine presented significant responses to changes in the source of mortality. Unexpectedly, variability in metabolite concentrations were not accompanied by increases in microbial abundance which indicates that marine microorganisms altered their internal organic carbon stores without changes in biomass or microbial growth. We used Weighted Correlation Network Analysis (WGCNA) to identify correlations between the targeted and untargeted mass spectrometry data. This analysis revealed multiple unknown organic compounds were correlated with compatible solutes, also called osmolytes or chemical chaperones, which emphasizes the dominant role of compatible solutes in marine microorganisms.

Lab-on-a-chip sensing technologies have changed how cell biology research is conducted. This review summarises the progress in the lab-on-a-chip devices implemented for the detection of cellular metabolites. The review is divided into two subsections according to the methods used for the metabolite detection. Each section includes a table which summarises the relevant literature and also elaborates the advantages of, and the challenges faced with that particular method. The review continues with a section discussing the achievements attained due to using lab-on-a-chip devices within the specific context. Finally, a concluding section summarises what is to be resolved and discusses the future perspectives.

Exploring the dynamic behavior of cellular metabolism requires a standard laboratory method that guarantees rapid sampling and extraction of the cellular content. We propose a versatile sampling technique applicable to cells with different cell wall and cell membrane properties. The technique is based on irreversible electroporation with simultaneous quenching and extraction by using a microfluidic device. By application of electric pulses in the millisecond range, permanent lethal pores are formed in the cell membrane of Escherichia coli and Saccharomyces cerevisiae, facilitating the release of the cellular contents; here demonstrated by the measurement of glucose-6-phosphate and the activity of the enzyme glucose-6-phosphate dehydrogenase. The successful application of this device was demonstrated by pulsed electric field treatment in a flow-through configuration of the microfluidic chip in combination with sampling, inactivation, and extraction of the intracellular content in a few seconds. Minimum electric field strengths of 10 kV/cm for E. coli and 7.5 kV/cm for yeast S. cerevisiae were required for successful cell lysis. The results are discussed in the context of applications in industrial biotechnology, where metabolomics analyses are important.

Single-cell biology provides insights into some of the most fundamental processes in biology and promotes the understanding of life's mysteries. As the technologies to study single-cells expand, they will require sophisticated analytical tools to make sense of various behaviors and components of single-cells as well as their relations in the adherent tissue culture. In this paper, we revealed cell heterogeneity and uncovered the connections between cell adhesion strength and cell viability at single-cell resolution by extracting single adherent cells of interest from a standard tissue culture by using a microfluidic chip-based live single-cell extractor (LSCE). We believe that this method will provide a valuable new tool for single-cell biology. [ABSTRACT FROM AUTHOR]

Sample preparation is one of the most important steps in metabolome analysis. The challenges of determining microbial metabolome have been well discussed within the research community and many improvements have already been achieved in last decade. The analysis of intracellular metabolites is particularly challenging. Environmental perturbations may considerably affect microbial metabolism, which results in intracellular metabolites being rapidly degraded or metabolized by enzymatic reactions. Therefore, quenching or the complete stop of cell metabolism is a pre-requisite for accurate intracellular metabolite analysis. After quenching, metabolites need to be extracted from the intracellular compartment. The choice of the most suitable metabolite extraction method/s is another crucial step. The literature indicates that specific classes of metabolites are better extracted by different extraction protocols. In this review, we discuss the technical aspects and advancements of quenching and extraction of intracellular metabolite analysis from microbial cells. [ABSTRACT FROM AUTHOR]

Microorganisms produce and secrete many primary and secondary metabolites to the surrounding environment during their growth. Therefore, extracellular metabolites provide important information about the changes in microbial metabolism due to different environmental cues. The determination of these metabolites is also comparatively easier than the extraction and analysis of intracellular metabolites as there is no need for cell rupture. Many analytical methods are already available and have been used for the analysis of extracellular metabolites from microorganisms over the last two decades. Here, we review the applications and benefits of extracellular metabolite analysis. We also discuss different sample preparation protocols available in the literature for both types (e.g., metabolites in solution and in gas) of extracellular microbial metabolites. Lastly, we evaluate the authenticity of using extracellular metabolomics data in the metabolic modelling of different industrially important microorganisms. [ABSTRACT FROM AUTHOR]

Metabolomics analysis is extremely essential to explore the metabolism characteristics of Saccharopolyspora erythraea. The lack of suitable methods for the determination of intracellular metabolites, however, hinders the application of metabolomics analysis for S. erythraea. Acyl-CoAs are important precursors of erythromycin; phosphorylated sugars are intermediate metabolites in EMP pathway or PPP pathway; organic acids are intermediate metabolites in TCA cycle. Reliable determination methods for intracellular acyl-CoAs, phosphorylated sugars, and organic acids of S. erythraea were designed and validated in this study. Using the optimized determination methods, the pool sizes of intracellular metabolites during an erythromycin fermentation process were precisely quantified by isotope dilution mass spectroscopy method. The quantification results showed that the specific erythromycin production rate was positively correlated with the pool sizes of propionyl-CoA as well as many other intracellular metabolites. The experiment under the condition without propanol, which is a precursor of propionyl-CoA and an important substrate in industrial erythromycin production process, also corroborated the correlation between specific erythromycin production rate and intracellular propionyl-CoA pool size. As far as we know, this is the first paper to conduct the metabolomics analysis of S. erythraea, which makes the metabolomics analysis of S. erythraea in the industrial erythromycin production process possible. [ABSTRACT FROM AUTHOR]

Engineering of enzymes and pathways is generally required for the development of efficient strains for bioproduction processes. To this end, quantitative and reliable data of intracellular metabolites are highly desired, but often not available, especially for conditions more close to industrial applications, i.e. at high cell density and product concentration. Here, we investigated the intracellular metabolite profiles of an engineered l-lysine-producing Corynebacterium glutamicum strain and the corresponding wild-type strain to assess the impacts of deregulation of product inhibition of the key enzymes aspartate kinase and phosphoenolpyruvate carboxylase and to identify potentials for their further improvement. A bioreactor system with automated fast-sampling, filtration and on-filter quenching of the metabolism was used for a more reliable determination of intracellular metabolites in batch cultures with optical cell density (OD660) up to 40. The l-lysine-producing strain showed substantially different metabolite profiles in the amino acid metabolism, including increased intracellular pool sizes in the l-lysine-, l-homoserine- and l-threonine pathways and decreased intracellular pool sizes for all other determined amino acids. By comparing data of in vitro inhibition of the engineered enzymes and determined intracellular concentrations of the inhibitors it was found that the inferred in vivo activities of these enzymes are still significantly below their in vitro maximums. This work demonstrates the usefulness of metabolic analysis for assessing the impact of engineered enzymes and identifying targets for further strain development. [ABSTRACT FROM AUTHOR]

Model-based analyses have become an integral part of modern metabolic engineering and systems biology in order to gain knowledge about complex and not directly observable cellular processes. For quantitative analyses, not only experimental data, but also measurement errors, play a crucial role. The total measurement error of any analytical protocol is the result of an accumulation of single errors introduced by several processing steps. Here, we present a framework for the quantification of intracellular metabolites, including error propagation during metabolome sample processing. Focusing on one specific protocol, we comprehensively investigate all currently known and accessible factors that ultimately impact the accuracy of intracellular metabolite concentration data. All intermediate steps are modeled, and their uncertainty with respect to the final concentration data is rigorously quantified. Finally, on the basis of a comprehensive metabolome dataset of Corynebacterium glutamicum, an integrated error propagation analysis for all parts of the model is conducted, and the most critical steps for intracellular metabolite quantification are detected.

Limosilactobacillus reuteri is a resident probiotic in the intestines of animals with strong environmental adaptability. In the current study, to elucidate the adaptation strategies of L. reuteri to the intestine of mice supplemented with three dietary components (oil, peptide and cellulose), whole genome resequencing and determination of intracellular metabolites were carried out. The resequencing results showed that L. reuteri TO60 (LR-TO) from the intestines of mice supplemented with high docosahexaenoic acid (DHA) tuna oil, or fermented Benincasa hispida culture (L. reuteri FB151, abbreviated as LR-FB) had relatively consistent single nucleotide polymorphisms (SNPs) and insertions and deletions (InDels), and those of L. reuteri from the intestines of mice supplemented with Rhopilema esculentum polypeptides (L. reuteri RE225, abbreviated as LR-RE) were quite different from them. The KEGG functional annotation of resequencing and the intracellular metabolites suggested that in all of the L. reuteri adapted to the intestinal environment mainly by regulating carbohydrate metabolism and membrane transport metabolic pathways. However, there were still some differences in their genes and metabolites, which may be related to the changes in the intestinal environment caused by three different types of dietary interventions. Our study analyzed the adaptation mechanism of L. reuteri to different isolated sources at the genetic level and metabolic level, which is helpful to clarify the adaptation strategies of probiotics to the intestinal environment, and provide supplementary information for the use of dietary nutritional supplements. [ABSTRACT FROM AUTHOR]

Diffusion-tensor imaging and single voxel diffusion-weighted magnetic resonance spectroscopy were used at 7T to explore in vivo age-related microstructural changes in the corpus callosum. Sixteen healthy elderly (age range 60–71 years) and 13 healthy younger controls (age range 23–32 years) were included in the study. In healthy elderly, we found lower water fractional anisotropy and higher water mean diffusivity and radial diffusivity in the corpus callosum, indicating the onset of demyelination processes with healthy aging. These changes were not associated with a concomitant significant difference in the cytosolic diffusivity of the intra-axonal metabolite N-acetylaspartate ( p = 0.12), the latter representing a pure measure of intra-axonal integrity. It was concluded that the possible intra-axonal changes associated with normal aging processes are below the detection level of diffusion-weighted magnetic resonance spectroscopy in our experiment (e.g., smaller than 10%) in the age range investigated. Lower axial diffusivity of total creatine was observed in the elderly group ( p = 0.058), possibly linked to a dysfunction in the energy metabolism associated with a deficit in myelin synthesis. [ABSTRACT FROM AUTHOR]

Sample preparation is one of the most important steps in metabolome analysis. The challenges of determining microbial metabolome have been well discussed within the research community and many improvements have already been achieved in last decade. The analysis of intracellular metabolites is particularly challenging. Environmental perturbations may considerably affect microbial metabolism, which results in intracellular metabolites being rapidly degraded or metabolized by enzymatic reactions. Therefore, quenching or the complete stop of cell metabolism is a pre-requisite for accurate intracellular metabolite analysis. After quenching, metabolites need to be extracted from the intracellular compartment. The choice of the most suitable metabolite extraction method/s is another crucial step. The literature indicates that specific classes of metabolites are better extracted by different extraction protocols. In this review, we discuss the technical aspects and advancements of quenching and extraction of intracellular metabolite analysis from microbial cells.

Microorganisms produce and secrete many primary and secondary metabolites to the surrounding environment during their growth. Therefore, extracellular metabolites provide important information about the changes in microbial metabolism due to different environmental cues. The determination of these metabolites is also comparatively easier than the extraction and analysis of intracellular metabolites as there is no need for cell rupture. Many analytical methods are already available and have been used for the analysis of extracellular metabolites from microorganisms over the last two decades. Here, we review the applications and benefits of extracellular metabolite analysis. We also discuss different sample preparation protocols available in the literature for both types (e.g., metabolites in solution and in gas) of extracellular microbial metabolites. Lastly, we evaluate the authenticity of using extracellular metabolomics data in the metabolic modelling of different industrially important microorganisms.

In Saccharomyces cerevisiae ethanol dissimilation is initiated by its oxidation and activation to cytosolic acetyl-CoA. The associated consumption of ATP strongly limits yields of biomass and acetyl-CoA-derived products. Here, we explore the implementation of an ATP-independent pathway for acetyl-CoA synthesis from ethanol that, in theory, enables biomass yield on ethanol that is up to 40% higher. To this end, all native yeast acetaldehyde dehydrogenases (ALDs) were replaced by heterologous acetylating acetaldehyde dehydrogenase (A-ALD). Engineered Ald- strains expressing different A-ALDs did not immediately grow on ethanol, but serial transfer in ethanol-grown batch cultures yielded growth rates of up to 70% of the wild-type value. Mutations in ACS1 were identified in all independently evolved strains and deletion of ACS1 enabled slow growth of non-evolved Ald- A-ALD strains on ethanol. Acquired mutations in A-ALD genes improved affinity-VmaxKm for acetaldehyde. One of five evolved strains showed a significant 5% increase of its biomass yield in ethanol-limited chemostat cultures. Increased production of acetaldehyde and other by-products was identified as possible cause for lower than theoretically predicted biomass yields. This study proves that the native yeast pathway for conversion of ethanol to acetyl-CoA can be replaced by an engineered pathway with the potential to improve biomass and product yields. [ABSTRACT FROM AUTHOR]

Monitoring amino acids and vitamins is important for understanding human health, food nutrition and the culture of mammalian cells used to produce therapeutic proteins in biotechnology. A method including ion pairing reversed-phase liquid chromatography with tandem mass spectrometry was developed and optimized to quantify 21 amino acids and 9 water-soluble vitamins in Chinese hamster ovary (CHO) cells and culture media. By optimizing the chromatographic separation, scan time, monitoring time window, and sample preparation procedure, and using isotopically labeled 13 C, 15 N and 2 H internal standards, low limits of quantitation (≤0.054 mg/L), good precision (<10%) and good accuracy (100 ± 10%) were achieved for nearly all the 30 compounds. Applying this method to CHO cell extracts, statistically significant differences in the metabolite levels were measured between two cell lines originated from the same host, indicating differences in genetic makeup or metabolic activities and nutrient supply levels in the culture media. In a fed-batch process of manufacturing scale bioreactors, two distinguished trends for changes in amino acid concentrations were identified in response to feeding. Ten essential amino acids showed a zigzag pattern with maxima at the feeding days, and 9 non-essential amino acids displayed a smoothly changing profile as they were mainly products of cellular metabolism. Five of 9 vitamins accumulated continuously during the culture period, suggesting that they were fed in access. The method serves as an effective tool for the development and optimization of mammalian cell cultures. [ABSTRACT FROM AUTHOR]

Understanding the mechanism of metabolic network response to extracellular changes has become one of the primary objectives of systems biology. Quantitative metabolic network profiling of model organisms such as E. coli is the principal approach. Well-designed analytical protocols for the determination of intracellular metabolites may provide a crucial premise to quantitative profiling of cellular metabolism. This article reviews and evaluates the existing methods for determining intracellular metabolites of E. coli in terms of sample preparation and mass spectrometry (MS) analysis. Critical issues including the variation of metabolic status and interference of external pools during cell sampling, sample loss during quenching procedures, degradation of metabolites and efficiency of extraction, and matrix effects in separation and quantification are discussed. Moreover, suggestions are provided to solve the analytical problems based on the results from the recent studies in order to establish a precise and reliable analytical platform for the intracellular metabolites. [ABSTRACT FROM AUTHOR]

For quantification of intracellular metabolites, mass spectrometry combined with liquid chromatography, capillary electrophoresis, or gas chromatography is currently the method of choice, especially when combined with stable isotope-labeled internal standards (SIL-ISs). Due to the difficulties in finding a biological matrix free of intracellular metabolites, a standard addition based validation is needed. Here, we present an alternative by producing a matrix with minimal signal interferences on both the analytes and their SIL-ISs. The matrix was obtained by cultivating Saccharomyces cerevisiae in [ 13 C 6 ]glucose/nonlabeled glucose (50:50, w/w) growth medium. The areas of both 12 C 6 and 13 C 6 fractions of ATP in the matrix were measured to be 2% of the sum of the areas of all ATP isotopes detected. The matrix allowed for spiking of both the nonlabeled and SIL-ISs and more straightforward validation. The intra- and inter-day accuracy and precision were ⩾80% and ⩽20%, respectively. The methodology was used for quantification of nucleotides, coenzymes, and redox compounds from Saccharomyces cerevisiae . The determined energy charge ratio was 0.9, whereas the Mal-CoA/Ac-CoA ratio was 0.04. The analysis of the redox compounds was challenging due to the oxidation of NADH and NADPH, when dissolved in water or tributylamine. The oxidation was reduced by dissolving them in ammonium acetate solution (pH 8.0). [ABSTRACT FROM AUTHOR]

Production of intracellular metabolites or biofuels from algae involves various processing steps, and extensive work on laboratory- and pilot-scale algae cultivation, harvesting and processing has been reported. As algal drying and cell disruption are integral processes of the unit operations, this review examines recent advances in algal drying and disruption for nutrition or biofuel production. Challenges and prospects of the processing are also outlined. Engineering improvements in addressing the challenges of energy efficiency and cost-effective and rigorous techno-economic analyses for a clearer prospect comparison between different processing methods are highlighted. Holistic life cycle assessments need to be conducted in assessing the energy balance and the potential environmental impacts of algal processing. The review aims to provide useful information for future development of efficient and commercially viable algal food products and biofuels production. [ABSTRACT FROM AUTHOR]

Central metabolite profiles from glucose in microbial communities during methanogenic process were compared between a stable methanogenic reactor (MR) and a deteriorated reactor (DR). The concentrations of intracellular metabolites related to the Embden–Meyerhof and pentose phosphate pathways, with the exception of pyruvate, remained high in the MR, showing increased carbon flux in the glycolysis pathway during stable methanogenesis. Extracellular acetate temporarily accumulated in the MR, consistent with higher ATP level in the MR. Intracellular concentrations of the intermediates in the reductive branch of tricarboxylic acid cycle, malate, fumarate, and succinate were higher in the DR. Low NADH/NAD + ratio both in the MR and DR would suggest NADH consumption during acetate and lactate/succinate production in the MR and DR, respectively. Intracellular glutamate levels were higher in the MR, correlating with lower NADPH/NADP + ratio concentrations in the MR. These findings contribute to a better understanding of the metabolic state during stable methanogenesis. [ABSTRACT FROM AUTHOR]

Highlights: [•] Quantitative metabolomics is a crucial ingredient for systems biology of microorganism. [•] Measurement protocols for intracellular metabolite quantification are highly complex and error-prone. [•] Thorough analyses of systematic and random errors are essential to increase both, accuracy and precision, of in vivo measurement data. [•] At current state of the art many sources of systematic errors are known and can be corrected. [•] However, establishing a gold standard fails due to fundamental limitations. [Copyright &y& Elsevier]