Spermidine is essential for fasting-mediated autophagy and longevity.

Caloric restriction and intermittent fasting prolong the lifespan and healthspan of model organisms and improve human health. The natural polyamine spermidine has been similarly linked to autophagy enhancement, geroprotection and reduced incidence of cardiovascular and neurodegenerative diseases across species borders. Here, we asked whether the cellular and physiological consequences of caloric restriction and fasting depend on polyamine metabolism. We report that spermidine levels increased upon distinct regimens of fasting or caloric restriction in yeast, flies, mice and human volunteers. Genetic or pharmacological blockade of endogenous spermidine synthesis reduced fasting-induced autophagy in yeast, nematodes and human cells. Furthermore, perturbing the polyamine pathway in vivo abrogated the lifespan- and healthspan-extending effects, as well as the cardioprotective and anti-arthritic consequences of fasting. Mechanistically, spermidine mediated these effects via autophagy induction and hypusination of the translation regulator eIF5A. In summary, the polyamine-hypusination axis emerges as a phylogenetically conserved metabolic control hub for fasting-mediated autophagy enhancement and longevity.

Inhibiting Hippo pathway kinases releases WWC1 to promote AMPAR-dependent synaptic plasticity and long-term memory in mice.

The localization, number, and function of postsynaptic AMPA-type glutamate receptors (AMPARs) are crucial for synaptic plasticity, a cellular correlate for learning and memory. The Hippo pathway member WWC1 is an important component of AMPAR-containing protein complexes. However, the availability of WWC1 is constrained by its interaction with the Hippo pathway kinases LATS1 and LATS2 (LATS1/2). Here, we explored the biochemical regulation of this interaction and found that it is pharmacologically targetable in vivo. In primary hippocampal neurons, phosphorylation of LATS1/2 by the upstream kinases MST1 and MST2 (MST1/2) enhanced the interaction between WWC1 and LATS1/2, which sequestered WWC1. Pharmacologically inhibiting MST1/2 in male mice and in human brain-derived organoids promoted the dissociation of WWC1 from LATS1/2, leading to an increase in WWC1 in AMPAR-containing complexes. MST1/2 inhibition enhanced synaptic transmission in mouse hippocampal brain slices and improved cognition in healthy male mice and in male mouse models of Alzheimer's disease and aging. Thus, compounds that disrupt the interaction between WWC1 and LATS1/2 might be explored for development as cognitive enhancers.

Time-resolved proteomic analyses of senescence highlight metabolic rewiring of mitochondria.

Mitochondrial dysfunction and cellular senescence are hallmarks of aging. However, the relationship between these two phenomena remains incompletely understood. In this study, we investigated the rewiring of mitochondria upon development of the senescent state in human IMR90 fibroblasts. Determining the bioenergetic activities and abundance of mitochondria, we demonstrate that senescent cells accumulate mitochondria with reduced OXPHOS activity, resulting in an overall increase of mitochondrial activities in senescent cells. Time-resolved proteomic analyses revealed extensive reprogramming of the mitochondrial proteome upon senescence development and allowed the identification of metabolic pathways that are rewired with different kinetics upon establishment of the senescent state. Among the early responding pathways, the degradation of branched-chain amino acid was increased, whereas the one carbon folate metabolism was decreased. Late-responding pathways include lipid metabolism and mitochondrial translation. These signatures were confirmed by metabolic flux analyses, highlighting metabolic rewiring as a central feature of mitochondria in cellular senescence. Together, our data provide a comprehensive view on the changes in mitochondrial proteome in senescent cells and reveal how the mitochondrial metabolism is rewired in senescent cells.

Hippo-released WWC1 facilitates AMPA receptor regulatory complexes for hippocampal learning.

Learning and memory rely on changes in postsynaptic glutamergic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type receptor (AMPAR) number, spatial organization, and function. The Hippo pathway component WW and C2 domain-containing protein 1 (WWC1) regulates AMPAR surface expression and impacts on memory performance. However, synaptic binding partners of WWC1 and its hierarchical position in AMPAR complexes are largely unclear. Using cell-surface proteomics in hippocampal tissue of Wwc1-deficient mice and by generating a hippocampus-specific interactome, we show that WWC1 is a major regulatory platform in AMPAR signaling networks. Under basal conditions, the Hippo pathway members WWC1 and large tumor-suppressor kinase (LATS) are associated, which might prevent WWC1 effects on synaptic proteins. Reduction of WWC1/LATS binding through a point mutation at WWC1 elevates the abundance of WWC1 in AMPAR complexes and improves hippocampal-dependent learning and memory. Thus, uncoupling of WWC1 from the Hippo pathway to AMPAR-regulatory complexes provides an innovative strategy to enhance synaptic transmission.

Mediobasal hypothalamic FKBP51 acts as a molecular switch linking autophagy to whole-body metabolism.

The mediobasal hypothalamus (MBH) is the central region in the physiological response to metabolic stress. The FK506-binding protein 51 (FKBP51) is a major modulator of the stress response and has recently emerged as a scaffolder regulating metabolic and autophagy pathways. However, the detailed protein-protein interactions linking FKBP51 to autophagy upon metabolic challenges remain elusive. We performed mass spectrometry-based metabolomics of FKBP51 knockout (KO) cells revealing an increased amino acid and polyamine metabolism. We identified FKBP51 as a central nexus for the recruitment of the LKB1/AMPK complex to WIPI4 and TSC2 to WIPI3, thereby regulating the balance between autophagy and mTOR signaling in response to metabolic challenges. Furthermore, we demonstrated that MBH FKBP51 deletion strongly induces obesity, while its overexpression protects against high-fat diet (HFD)-induced obesity. Our study provides an important novel regulatory function of MBH FKBP51 within the stress-adapted autophagy response to metabolic challenges.

Myo-Inositol Levels in the Dorsal Hippocampus Serve as Glial Prognostic Marker of Mild Cognitive Impairment in Mice.

Dementia is a devastating age-related disorder. Its therapy would largely benefit from the identification of susceptible subjects at early, prodromal stages of the disease. To search for such prognostic markers of cognitive impairment, we studied spatial navigation in male BALBc vs. B6N mice in combination with in vivo magnetic resonance spectroscopy (1H-MRS). BALBc mice consistently showed higher escape latencies than B6N mice, both in the Water Cross Maze (WCM) and the Morris water maze (MWM). These performance deficits coincided with higher levels of myo-inositol (mIns) in the dorsal hippocampus before and after training. Subsequent biochemical analyses of hippocampal specimens by capillary immunodetection and liquid chromatography mass spectrometry-based (LC/MS) metabolomics revealed a higher abundance of glial markers (IBA-1, S100B, and GFAP) as well as distinct alterations in metabolites including a decrease in vitamins (pantothenic acid and nicotinamide), neurotransmitters (acetylcholine), their metabolites (glutamine), and acetyl-L-carnitine. Supplementation of low abundant acetyl-L-carnitine via the drinking water, however, failed to revert the behavioral deficits shown by BALBc mice. Based on our data we suggest (i) BALBc mice as an animal model and (ii) hippocampal mIns levels as a prognostic marker of mild cognitive impairment (MCI), due to (iii) local changes in microglia and astrocyte activity, which may (iv) result in decreased concentrations of promnesic molecules.

Stress-primed secretory autophagy promotes extracellular BDNF maturation by enhancing MMP9 secretion.

The stress response is an essential mechanism for maintaining homeostasis, and its disruption is implicated in several psychiatric disorders. On the cellular level, stress activates, among other mechanisms, autophagy that regulates homeostasis through protein degradation and recycling. Secretory autophagy is a recently described pathway in which autophagosomes fuse with the plasma membrane rather than with lysosomes. Here, we demonstrate that glucocorticoid-mediated stress enhances secretory autophagy via the stress-responsive co-chaperone FK506-binding protein 51. We identify the matrix metalloproteinase 9 (MMP9) as one of the proteins secreted in response to stress. Using cellular assays and in vivo microdialysis, we further find that stress-enhanced MMP9 secretion increases the cleavage of pro-brain-derived neurotrophic factor (proBDNF) to its mature form (mBDNF). BDNF is essential for adult synaptic plasticity and its pathway is associated with major depression and posttraumatic stress disorder. These findings unravel a cellular stress adaptation mechanism that bears the potential of opening avenues for the understanding of the pathophysiology of stress-related disorders.

SARS-CoV-2-mediated dysregulation of metabolism and autophagy uncovers host-targeting antivirals.

Viruses manipulate cellular metabolism and macromolecule recycling processes like autophagy. Dysregulated metabolism might lead to excessive inflammatory and autoimmune responses as observed in severe and long COVID-19 patients. Here we show that SARS-CoV-2 modulates cellular metabolism and reduces autophagy. Accordingly, compound-driven induction of autophagy limits SARS-CoV-2 propagation. In detail, SARS-CoV-2-infected cells show accumulation of key metabolites, activation of autophagy inhibitors (AKT1, SKP2) and reduction of proteins responsible for autophagy initiation (AMPK, TSC2, ULK1), membrane nucleation, and phagophore formation (BECN1, VPS34, ATG14), as well as autophagosome-lysosome fusion (BECN1, ATG14 oligomers). Consequently, phagophore-incorporated autophagy markers LC3B-II and P62 accumulate, which we confirm in a hamster model and lung samples of COVID-19 patients. Single-nucleus and single-cell sequencing of patient-derived lung and mucosal samples show differential transcriptional regulation of autophagy and immune genes depending on cell type, disease duration, and SARS-CoV-2 replication levels. Targeting of autophagic pathways by exogenous administration of the polyamines spermidine and spermine, the selective AKT1 inhibitor MK-2206, and the BECN1-stabilizing anthelmintic drug niclosamide inhibit SARS-CoV-2 propagation in vitro with IC50 values of 136.7, 7.67, 0.11, and 0.13 µM, respectively. Autophagy-inducing compounds reduce SARS-CoV-2 propagation in primary human lung cells and intestinal organoids emphasizing their potential as treatment options against COVID-19.

Lipid metabolism is associated with temperament, corticosteroid, and hematological measures in infant rhesus monkeys ( Macaca mulatta).

Individuals can differ in how their behavioral and physiological systems are organized. The fact that these individual differences persist across life suggests they are supported by physical structures that may co-vary. Here, we explored three datasets associated with health and behavioral outcomes, which were obtained from infant rhesus monkeys during standardized assessment of biobehavioral organization. Variation in biobehavioral measures was related to variation in molecular pathways, as assessed by metabolomics. Plasma from infant male rhesus monkeys ( Macaca mulatta) ( n=52) was subjected to metabolite profiling. Principal component analyses identified multiple factors that explained 60%-80% of the variance in the metabolite measures. Correlational and regression analyses of corticosteroid, hematological, and temperament measures revealed significant relationships with indicators of lipid metabolism. Significant relationships were found for cortisol responses to stress and adrenocorticotropin (ACTH) stimulation, indicators of innate immunity (monocytes and natural killer (NK) cells), hemoglobin/hematocrit, and three measures of temperament. It will be important to replicate this first-of-a-kind study to determine whether the relationship between measures of biobehavioral organization and lipid metabolism are a general result, or one that is specific to early development.

Multiplexed Profiling and Data Processing Methods to Identify Temperature-Regulated Primary Metabolites Using Gas Chromatography Coupled to Mass Spectrometry.

This book chapter describes the analytical procedures required for the profiling of a metabolite fraction enriched for primary metabolites. The profiling is based on routine gas chromatography coupled to mass spectrometry (GC-MS). The generic profiling method is adapted to plant material, specifically to the analysis of plant material that was exposed to temperature stress. The method can be combined with stable isotope labeling and tracing experiments and is equally applicable to preparations of plant material and microbial photosynthetic organisms. The described methods are modular and can be multiplexed, that is, the same sample or a paired identical backup sample can be analyzed sequentially by more than one of the described procedures. The modules include rapid sampling and metabolic inactivation protocols for samples in a wide weight range, sample extraction procedures, chemical derivatization steps that are required to make the metabolite fraction amenable to gas chromatographic analysis, routine GC-MS methods, and procedures of data processing and data mining. A basic and extendable set of standardizations for metabolite recovery and retention index alignment of the resulting GC-MS chromatograms is included. The methods have two applications: (1) The rapid screening for changes of relative metabolite pools sizes under temperature stress and (2) the verification by exact quantification using GC-MS protocols that are extended by internal and external standardization.

Plasma Metabolite Profiles in First Episode Psychosis: Exploring Symptoms Heterogeneity/Severity in Schizophrenia and Bipolar Disorder Cohorts.

INTRODUCTION: The first symptoms of psychosis are frequently shared amongst several neuropsychiatry disorders, which makes the differentiation by clinical diagnosis challenging. Early recognition of symptoms is important in the management of psychosis. Therefore, the implementation of molecular biomarkers will be crucial for transforming the currently used diagnostic and therapeutic approach, improving insights into the underlying biological processes and clinical management. OBJECTIVES: To define a set of metabolites that supports diagnosis or prognosis of schizophrenia (SCZ) and bipolar disorder (BD) at first onset psychosis. METHODS: Plasma samples from 55 drug-naïve patients, 28 SCZ and 27 BD, and 42 healthy controls (HC). All participants underwent a seminaturalistic treatment regimen, clinically evaluated on a weekly basis until achieving clinical remission. All clinical or sociodemographic aspects considered for this study were equivalent between the groups at first-onset psychosis time point. The plasma samples were analyzed by untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) using reversed-phase and hydrophilic interaction chromatography. The acquired molecular features were analyzed with MetaboAnalyst. RESULTS: We identified two patient groups with different metabolite profiles. Both groups are composed of SCZ and BD patients. We found differences between these two groups regarding general symptoms of PANSS score after remission (p = 0.008), and the improvement of general symptoms (delta of the score at remission minus the baseline) (-0.50 vs. -0.33, p = 0.019). CONCLUSION: Our results suggest that plasma metabolite profiles cluster clinical remission phenotypes based on PANSS general psychopathology scores.

Paroxetine Administration Affects Microbiota and Bile Acid Levels in Mice.

Recent interest in the role of microbiota in health and disease has implicated gut microbiota dysbiosis in psychiatric disorders including major depressive disorder. Several antidepressant drugs that belong to the class of selective serotonin reuptake inhibitors have been found to display antimicrobial activities. In fact, one of the first antidepressants discovered serendipitously in the 1950s, the monoamine-oxidase inhibitor Iproniazid, was a drug used for the treatment of tuberculosis. In the current study we chronically treated DBA/2J mice for 2 weeks with paroxetine, a selective serotonin reuptake inhibitor, and collected fecal pellets as a proxy for the gut microbiota from the animals after 7 and 14 days. Behavioral testing with the forced swim test revealed significant differences between paroxetine- and vehicle-treated mice. Untargeted mass spectrometry and 16S rRNA profiling of fecal pellet extracts showed several primary and secondary bile acid level, and microbiota alpha diversity differences, respectively between paroxetine- and vehicle-treated mice, suggesting that microbiota functions are altered by the drug. In addition to their lipid absorbing activities bile acids have important signaling activities and have been associated with gastrointestinal diseases and colorectal cancer. Antidepressant drugs like paroxetine should therefore be used with caution to prevent undesirable side effects.

Exposure-induced changes of plasma metabolome and gene expression in patients with panic disorder.

BACKGROUND: Anxiety disorders including panic disorder (PD) are the most prevalent psychiatric diseases leading to high disability and burden in the general population. Acute panic attacks are distinctive for PD but also frequent in other anxiety disorders. The neurobiology or specific molecular changes leading to and present during panic attacks are insufficiently known so far. METHODS: In the present pilot study, we investigated dynamic metabolomic and gene expression changes in peripheral blood of patients with PD (n = 25) during two exposure-induced acute panic attacks. RESULTS: The results show that the metabolite glyoxylate was dynamically regulated in peripheral blood. Additionally, glyoxylate levels were associated with basal anxiety levels and showed gender-related differences at baseline. As glyoxylate is part of the degradation circuit of cholecystokinin, this suggests that this neuropeptide might be directly involved in exposure-induced panic attacks. Only gene expression changes of very small magnitude were observed in this experimental setting. CONCLUSIONS: From this first metabolome and gene expression study in exposure-induced acute panic attacks in PD we conclude that metabolites can potentially serve as dynamic markers for different anxiety states. However, these findings have to be replicated in cohorts with greater sample sizes.

Ketamine's Effects on the Glutamatergic and GABAergic Systems: A Proteomics and Metabolomics Study in Mice.

Ketamine, a noncompetitive, voltage-dependent N-Methyl-D-aspartate receptor (NMDAR) antagonist, has been shown to have a rapid antidepressant effect and is used for patients experiencing treatment-resistant depression. We carried out a time-dependent targeted mass spectrometry-based metabolomics profiling analysis combined with a quantitative based on in vivo 15N metabolic labeling proteome comparison of ketamine- and vehicle-treated mice. The metabolomics and proteomics datasets were used to further elucidate ketamine's mode of action on the gamma-aminobutyric acid (GABA)ergic and glutamatergic systems. In addition, myelin basic protein levels were analyzed by Western Blot. We found altered GABA, glutamate and glutamine metabolite levels and ratios as well as increased levels of putrescine and serine - 2 positive modulators of the NMDAR. In addition, GABA receptor (GABAR) protein levels were reduced, whereas the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) subunit Gria2 protein levels were increased upon ketamine treatment. The significantly altered metabolite and protein levels further significantly correlated with the antidepressant-like behavior, which was assessed using the forced swim test. In conclusion and in line with previous research, our data indicate that ketamine impacts the AMPAR subunit Gria2 and results in decreased GABAergic inhibitory neurotransmission leading to increased excitatory neuronal activity.

Partially 13C-labeled mouse tissue as reference for LC-MS based untargeted metabolomics.

The inclusion of stable isotope-labeled reference standards in the sample is an established method for the detection and relative quantification of metabolic features in untargeted metabolomics. In order to quantify as many metabolites as possible, the reference should ideally include the same metabolites in their stable isotope-labeled form as the sample under investigation. We present here an attempt to use partially 13C-labeled mouse material as internal standard for relative metabolite quantification of mouse and human samples in untargeted metabolomics. We fed mice for 14 days with a13C-labeled Ralstonia eutropha based diet. Tissue and blood amino acids from these mice showed 13C enrichment levels that ranged from 6% to 75%. We used MetExtract II software to automatically detect native and labeled peak pairs in an untargeted manner. In a dilution series and with the implementation of a correction factor, partially 13C-labeled mouse plasma resulted in accurate relative quantification of human plasma amino acids using liquid chromatography coupled to mass spectrometry, The coefficient of variation for the relative quantification is reduced from 27% without internal standard to 10% with inclusion of partially 13C-labeled internal standard. We anticipate the method to be of general use for the relative metabolite quantification of human specimens.

Ketamine's antidepressant effect is mediated by energy metabolism and antioxidant defense system.

Fewer than 50% of all patients with major depressive disorder (MDD) treated with currently available antidepressants (ADs) show full remission. Moreover, about one third of the patients suffering from MDD does not respond to conventional ADs and develop treatment-resistant depression (TRD). Ketamine, a non-competitive, voltage-dependent N-Methyl-D-aspartate receptor (NMDAR) antagonist, has been shown to have a rapid antidepressant effect, especially in patients suffering from TRD. Hippocampi of ketamine-treated mice were analysed by metabolome and proteome profiling to delineate ketamine treatment-affected molecular pathways and biosignatures. Our data implicate mitochondrial energy metabolism and the antioxidant defense system as downstream effectors of the ketamine response. Specifically, ketamine tended to downregulate the adenosine triphosphate (ATP)/adenosine diphosphate (ADP) metabolite ratio which strongly correlated with forced swim test (FST) floating time. Furthermore, we found increased levels of enzymes that are part of the 'oxidative phosphorylation' (OXPHOS) pathway. Our study also suggests that ketamine causes less protein damage by rapidly decreasing reactive oxygen species (ROS) production and lend further support to the hypothesis that mitochondria have a critical role for mediating antidepressant action including the rapid ketamine response.

Rapid in situ 13C tracing of sucrose utilization in Arabidopsis sink and source leaves.

BACKGROUND: Conventional metabolomics approaches face the problem of hidden metabolic phenotypes where only fluxes are altered but pool sizes stay constant. Metabolic flux experiments are used to detect such hidden flux phenotypes. These experiments are, however, time consuming, may be cost intensive, and involve specialists for modeling. We fill the gap between conventional metabolomics and flux modeling. We present rapid stable isotope tracing assays and analysis strategies of 13C labeling data. For this purpose, we combine the conventional metabolomics approach that detects significant relative changes of metabolite pool sizes with analyses of differential utilization of 13C labeled carbon. As a test case, we use uniformly labeled 13C-sucrose. RESULTS: We present petiole and hypocotyl feeding assays for the rapid in situ feeding (≤ 4 h) of isotopically labeled metabolic precursor to whole Arabidopsis thaliana rosettes. The assays are assessed by conventional gas chromatography-mass spectrometry based metabolite profiling that was extended by joined differential analysis of 13C-labeled sub-pools and of 13C enrichment of metabolites relative to the enrichment of 13C-sucrose within each sample. We apply these analyses to the sink to source transition continuum of leaves from single A. thaliana rosettes and characterize the associated relative changes of metabolite pools, as well as previously hidden changes of sucrose-derived carbon partitioning. We compared the contribution of sucrose as a carbon source in predominantly sink to predominantly source leaves and identified a set of primary metabolites with differential carbon utilization during sink to source transition. CONCLUSION: The presented feeding assays and data evaluation strategies represent a rapid and easy-to-use tool box for enhanced metabolomics studies that combine differential pool size analysis with screening for differential carbon utilization from defined stable isotope labeled metabolic precursors.

Systems analysis of ethanol production in the genetically engineered cyanobacterium Synechococcus sp. PCC 7002.

BACKGROUND: Future sustainable energy production can be achieved using mass cultures of photoautotrophic microorganisms, which are engineered to synthesize valuable products directly from CO2 and sunlight. As cyanobacteria can be cultivated in large scale on non-arable land, these phototrophic bacteria have become attractive organisms for production of biofuels. Synechococcus sp. PCC 7002, one of the cyanobacterial model organisms, provides many attractive properties for biofuel production such as tolerance of seawater and high light intensities. RESULTS: Here, we performed a systems analysis of an engineered ethanol-producing strain of the cyanobacterium Synechococcus sp. PCC 7002, which was grown in artificial seawater medium over 30 days applying a 12:12 h day-night cycle. Biosynthesis of ethanol resulted in a final accumulation of 0.25% (v/v) ethanol, including ethanol lost due to evaporation. The cultivation experiment revealed three production phases. The highest production rate was observed in the initial phase when cells were actively growing. In phase II growth of the producer strain stopped, but ethanol production rate was still high. Phase III was characterized by a decrease of both ethanol production and optical density of the culture. Metabolomics revealed that the carbon drain due to ethanol diffusion from the cell resulted in the expected reduction of pyruvate-based intermediates. Carbon-saving strategies successfully compensated the decrease of central intermediates of carbon metabolism during the first phase of fermentation. However, during long-term ethanol production the producer strain showed clear indications of intracellular carbon limitation. Despite the decreased levels of glycolytic and tricarboxylic acid cycle intermediates, soluble sugars and even glycogen accumulated in the producer strain. The changes in carbon assimilation patterns are partly supported by proteome analysis, which detected decreased levels of many enzymes and also revealed the stress phenotype of ethanol-producing cells. Strategies towards improved ethanol production are discussed. CONCLUSIONS: Systems analysis of ethanol production in Synechococcus sp. PCC 7002 revealed initial compensation followed by increasing metabolic limitation due to excessive carbon drain from primary metabolism.

Insights into isoprene production using the cyanobacterium Synechocystis sp. PCC 6803.

BACKGROUND: Cyanobacteria are phototrophic prokaryotes that convert inorganic carbon as CO2 into organic compounds at the expense of light energy. They need only inorganic nutrients and can be cultivated to high densities using non-arable land and seawater. This has made cyanobacteria attractive organisms for the production of biofuels and chemical feedstock. Synechocystis sp. PCC 6803 is one of the most widely used cyanobacterial model strains. Based on its available genome sequence and genetic tools, Synechocystis has been genetically modified to produce different biotechnological products. Efficient isoprene production is an attractive goal because this compound is widely used as chemical feedstock. RESULTS: Here, we report on our attempts to generate isoprene-producing strains of Synechocystis using a plasmid-based strategy. As previously reported, a codon-optimized plant isoprene synthase (IspS) was expressed under the control of different Synechocystis promoters that ensure strong constitutive or light-regulated ispS expression. The expression of the ispS gene was quantified by qPCR and Western blotting, while the amount of isoprene was quantified using GC-MS. In addition to isoprene measurements in the headspace of closed culture vessels, single photon ionization time-of-flight mass spectrometry (SPI-MS) was applied, which allowed online measurements of isoprene production in open-cultivation systems under various conditions. Under standard conditions, a good correlation existed between ispS expression and isoprene production rate. The cultivation of isoprene production strains under NaCl-supplemented conditions decreased isoprene production despite enhanced ispS mRNA levels. The characterization of the metabolome of isoprene-producing strains indicated that isoprene production might be limited by insufficient precursor levels. Transcriptomic analysis revealed the upregulation of mRNA and regulatory RNAs characteristic of acclimation to metabolic stress. CONCLUSIONS: Our best production strains produced twofold higher isoprene amounts in the presence of low NaCl concentrations than previously reported strains. These results will guide future attempts to establish isoprene production in cyanobacterial hosts.

Profiling methods to identify cold-regulated primary metabolites using gas chromatography coupled to mass spectrometry.

This book chapter describes the analytical procedures required for the profiling of a metabolite fraction enriched for primary metabolites. The profiling is based on routine gas chromatography coupled to mass spectrometry (GC-MS). The generic profiling method is adapted to plant material, specifically to the analysis of single leaves from plants that were exposed to temperature stress experiments. The described method is modular. The modules include a rapid sampling and metabolic inactivation protocol for samples in a wide size range, a sample extraction procedure, a chemical derivatization step that is required to make the metabolite fraction amenable to gas chromatographic analysis, a routine GC-MS method, and finally the procedures of data processing and data mining. A basic and extendable set of standardizations for metabolite recovery and retention index alignment of the resulting GC-MS chromatograms is included. The method has two applications: (1) the rapid screening for changes of relative metabolite pools sizes under temperature stress and (2) the verification of cold-regulated metabolites by exact quantification using a GC-MS protocol with extended internal and external standardization.