SOS genes are rapidly induced while translesion synthesis polymerase activity is temporally regulated.

The DNA damage inducible SOS response in bacteria serves to increase survival of the species at the cost of mutagenesis. The SOS response first initiates error-free repair followed by error-prone repair. Here, we have employed a multi-omics approach to elucidate the temporal coordination of the SOS response. Escherichia coli was grown in batch cultivation in bioreactors to ensure highly controlled conditions, and a low dose of the antibiotic ciprofloxacin was used to activate the SOS response while avoiding extensive cell death. Our results show that expression of genes involved in error-free and error-prone repair were both induced shortly after DNA damage, thus, challenging the established perception that the expression of error-prone repair genes is delayed. By combining transcriptomics and a sub-proteomics approach termed signalomics, we found that the temporal segregation of error-free and error-prone repair is primarily regulated after transcription, supporting the current literature. Furthermore, the heterology index (i.e., the binding affinity of LexA to the SOS box) was correlated to the maximum increase in gene expression and not to the time of induction of SOS genes. Finally, quantification of metabolites revealed increasing pyrimidine pools as a late feature of the SOS response. Our results elucidate how the SOS response is coordinated, showing a rapid transcriptional response and temporal regulation of mutagenesis on the protein and metabolite levels.

Changes in Plasma Pyruvate and TCA Cycle Metabolites upon Increased Hepatic Fatty Acid Oxidation and Ketogenesis in Male Wistar Rats.

Altered hepatic mitochondrial fatty acid ß-oxidation and associated tricarboxylic acid (TCA) cycle activity contributes to lifestyle-related diseases, and circulating biomarkers reflecting these changes could have disease prognostic value. This study aimed to determine hepatic and systemic changes in TCA-cycle-related metabolites upon the selective pharmacologic enhancement of mitochondrial fatty acid ß-oxidation in the liver, and to elucidate the mechanisms and potential markers of hepatic mitochondrial activity. Male Wistar rats were treated with 3-thia fatty acids (e.g., tetradecylthioacetic acid (TTA)), which target mitochondrial biogenesis, mitochondrial fatty acid ß-oxidation, and ketogenesis predominantly in the liver. Hepatic and plasma concentrations of TCA cycle intermediates and anaplerotic substrates (LC-MS/MS), plasma ketones (colorimetric assay), and acylcarnitines (HPLC-MS/MS), along with associated TCA-cycle-related gene expression (qPCR) and enzyme activities, were determined. TTA-induced hepatic fatty acid ß-oxidation resulted in an increased ratio of plasma ketone bodies/nonesterified fatty acid (NEFA), lower plasma malonyl-CoA levels, and a higher ratio of plasma acetylcarnitine/palmitoylcarnitine (C2/C16). These changes were associated with decreased hepatic and increased plasma pyruvate concentrations, and increased plasma concentrations of succinate, malate, and 2-hydroxyglutarate. Expression of several genes encoding TCA cycle enzymes and the malate-oxoglutarate carrier (Slc25a11), glutamate dehydrogenase (Gdh), and malic enzyme (Mdh1 and Mdh2) were significantly increased. In conclusion, the induction of hepatic mitochondrial fatty acid ß-oxidation by 3-thia fatty acids lowered hepatic pyruvate while increasing plasma pyruvate, as well as succinate, malate, and 2-hydroxyglutarate.

Activation of multiple stress responses in Staphylococcus aureus substantially lowers the minimal inhibitory concentration when combining two novel antibiotic drug candidates.

The past few decades have been plagued by an increasing number of infections caused by antibiotic resistant bacteria. To mitigate the rise in untreatable infections, we need new antibiotics with novel targets and drug combinations that reduce resistance development. The novel ß-clamp targeting antimicrobial peptide BTP-001 was recently shown to have a strong additive effect in combination with the halogenated pyrrolopyrimidine JK-274. In this study, the molecular basis for this effect was examined by a comprehensive proteomic and metabolomic study of the individual and combined effects on Staphylococcus aureus. We found that JK-274 reduced activation of several TCA cycle enzymes, likely via increasing the cellular nitric oxide stress, and BTP-001 induced oxidative stress in addition to inhibiting replication, translation, and DNA repair processes. Analysis indicated that several proteins linked to stress were only activated in the combination and not in the single treatments. These results suggest that the strong additive effect is due to the activation of multiple stress responses that can only be triggered by the combined effect of the individual mechanisms. Importantly, the combination dose required to eradicate S. aureus was well tolerated and did not affect cell viability of immortalized human keratinocyte cells, suggesting a species-specific response. Our findings demonstrate the potential of JK-274 and BTP-001 as antibiotic drug candidates and warrant further studies.

Serum TCA cycle metabolites in Lewy bodies dementia and Alzheimer's disease: Network analysis and cognitive prognosis.

Abnormalities in the Tri-Carboxylic-Acid (TCA) cycle have been documented in dementia. Through network analysis, TCA cycle metabolites could indirectly reflect known dementia-related abnormalities in biochemical pathways, and key metabolites might be associated with prognosis. This study analyzed TCA cycle metabolites as predictors of cognitive decline in a mild dementia cohort and explored potential interactions with the diagnosis of Lewy Body Dementia (LBD) or Alzheimer's Disease (AD) and APOE-ε4 genotype. We included 145 mild dementia patients (LBD = 59; AD = 86). Serum TCA cycle metabolites were analyzed at baseline, and partial correlation networks were conducted. Cognitive performance was measured annually over 5-years with the Mini-mental State Examination. Longitudinal mixed-effects Tobit models evaluated each baseline metabolite as a predictor of 5-years cognitive decline. APOE-ε4 and diagnosis interactions were explored. Results showed comparable metabolite concentrations in LBD and AD. Multiple testing corrected networks showed larger coefficients for a negative correlation between pyruvate - succinate and positive correlations between fumarate - malate and citrate - Isocitrate in both LBD and AD. In the total sample, adjusted mixed models showed significant associations between baseline citrate concentration and longitudinal MMSE scores. In APOE-ε4 carriers, baseline isocitrate predicted MMSE scores. We conclude that, in mild dementia, serum citrate concentrations could be associated with subsequent cognitive decline, as well as isocitrate concentrations in APOE-ε4 carriers. Downregulation of enzymatic activity in the first half of the TCA cycle (decarboxylating dehydrogenases), with upregulation in the latter half (dehydrogenases only), might be indirectly reflected in serum TCA cycle metabolites' networks.

Serum-free alginate-C2C12 cells microcapsule as a model of alternative animal protein source.

Due to the climate change crisis, and environmental impacts of the traditional meat sector, the production of artificial animal protein based on in vitro cell culture technology is proposed as an alternative. Furthermore, since traditional animal serum-supplemented cultures pose scientific challenges such as batch variation and contamination risks, artificial animal protein cultures are currently in urgent need of not only serum-free cultures, but also microcarrier culture systems for scalability. However, serum-free microcarrier-based culture system for the differentiation of muscle cells is not available to date. Therefore, we established an edible alginate microcapsules culture system for the differentiation of C2C12 cells in serum-free conditions. Furthermore, metabolites related to central carbon metabolism were profiled based on targeted metabolomics using mass spectrometry. The C2C12 cells cultured in alginate microcapsules displayed high viability throughout 7 days and successfully differentiated within 4 days in serum and serum-free cultures except for AIM-V cultures, which was confirmed by CK activity and MHC immunostaining. Lastly, to the best of our knowledge, this is the first report to compare metabolite profiles between monolayer and alginate microcapsule culture systems. Alginate microcapsule culture showed higher levels of intracellular glycolysis and TCA cycle intermediates, lactate, and the contribution of essential amino acids compared to the monolayer culture. We believe our serum-free alginate microcapsule culture system is adaptable to different species of muscle cells and contributes to future food technology as a proof of concept for the scalability of alternative animal protein source production.

Integrative omics-analysis of lipid metabolism regulation by peroxisome proliferator-activated receptor a and b agonists in male Atlantic cod.

Lipid metabolism is essential in maintaining energy homeostasis in multicellular organisms. In vertebrates, the peroxisome proliferator-activated receptors (PPARs, NR1C) regulate the expression of many genes involved in these processes. Atlantic cod (Gadus morhua) is an important fish species in the North Atlantic ecosystem and in human nutrition, with a highly fatty liver. Here we study the involvement of Atlantic cod Ppar a and b subtypes in systemic regulation of lipid metabolism using two model agonists after in vivo exposure. WY-14,643, a specific PPARA ligand in mammals, activated cod Ppara1 and Ppara2 in vitro. In vivo, WY-14,643 caused a shift in lipid transport both at transcriptional and translational level in cod. However, WY-14,643 induced fewer genes in the fatty acid beta-oxidation pathway compared to that observed in rodents. Although GW501516 serves as a specific PPARB/D ligand in mammals, this compound activated cod Ppara1 and Ppara2 as well as Pparb in vitro. In vivo, it further induced transcription of Ppar target genes and caused changes in lipid composition of liver and plasma. The integrative approach provide a foundation for understanding how Ppars are engaged in regulating lipid metabolism in Atlantic cod physiology. We have shown that WY-14,643 and GW501516 activate Atlantic cod Ppara and Pparb, affect genes in lipid metabolism pathways, and induce changes in the lipid composition in plasma and liver microsomal membranes. Particularly, the combined transcriptomic, proteomics and lipidomics analyses revealed that effects of WY-14,643 on lipid metabolism are similar to what is known in mammalian studies, suggesting conservation of Ppara functions in mediating lipid metabolic processes in fish. The alterations in the lipid profiles observed after Ppar agonist exposure suggest that other chemicals with similar Ppar receptor affinities may cause disturbances in the lipid regulation of fish. Model organism: Atlantic cod (Gadus morhua). LSID: urn:lsid:zoobank.org:act:389BE401-2718-4CF2-BBAE-2E13A97A5E7B. COL Identifier: 6K72F.

Survival of Escherichia coli after high-antibiotic stress is dependent on both the pregrown physiological state and incubation conditions.

Introduction: The survival of bacterial cells exposed to antibiotics depends on the mode of action, the antibiotics concentration, and the duration of treatment. However, it also depends on the physiological state of the cells and the environmental conditions. In addition, bacterial cultures contain sub-populations that can survive high antibiotic concentrations, so-called persisters. Research on persisters is challenging due to multiple mechanisms for their formation and low fractions, down to and below one millionth of the total cell population. Here, we present an improved version of the persister assay used to enumerate the amount of persisters in a cell population. Methods: The persister assay with high antibiotic stress exposure was performed at both growth supporting and non-supporting conditions. Escherichia coli cells were pregrown to various growth stages in shake flasks and bench-top bioreactors. In addition, the physiological state of E. coli before antibiotic treatment was determined by quantitative mass spectrometry-based metabolite profiling. Results: Survival of E. coli strongly depended on whether the persister assay medium supported growth or not. The results were also highly dependent on the type of antibiotic and pregrown physiological state of the cells. Therefore, applying the same conditions is critical for consistent and comparable results. No direct connection was observed between antibiotic efficacy to the metabolic state. This also includes the energetic state (i.e., the intracellular concentration of ATP and the adenylate energy charge), which has earlier been hypothesized to be decisive for persister formation. Discussion: The study provides guides and suggestions for the design of future experimentation in the research fields of persisters and antibiotic tolerance.

Optimized Fast Filtration-Based Sampling and Extraction Enables Precise and Absolute Quantification of the Escherichia coli Central Carbon Metabolome.

Precise and accurate quantification is a prerequisite for interpretation of targeted metabolomics data, but this task is challenged by the inherent instability of the analytes. The sampling, quenching, extraction, and sample purification conditions required to recover and stabilize metabolites in representative extracts have also been proven highly dependent on species-specific properties. For Escherichia coli, unspecific leakage has been demonstrated for conventional microbial metabolomics sampling protocols. We herein present a fast filtration-based sampling protocol for this widely applied model organism, focusing on pitfalls such as inefficient filtration, selective loss of biomass, matrix contamination, and membrane permeabilization and leakage. We evaluate the effect of and need for removal of extracellular components and demonstrate how residual salts can challenge analytical accuracy of hyphenated mass spectrometric analyses, even when sophisticated correction strategies are applied. Laborious extraction procedures are bypassed by direct extraction in cold acetonitrile:water:methanol (3:5:2, v/v%), ensuring compatibility with sample concentration and thus, any downstream analysis. By applying this protocol, we achieve and demonstrate high precision and low metabolite turnover, and, followingly, minimal perturbation of the inherent metabolic state. This allows us to herein report absolute intracellular concentrations in E. coli and explore its central carbon metabolome at several commonly applied cultivation conditions.

PCNA regulates primary metabolism by scaffolding metabolic enzymes.

The essential roles of proliferating cell nuclear antigen (PCNA) as a scaffold protein in DNA replication and repair are well established, while its cytosolic roles are less explored. Two metabolic enzymes, alpha-enolase (ENO1) and 6-phosphogluconate dehydrogenase (6PGD), both contain PCNA interacting motifs. Mutation of the PCNA interacting motif APIM in ENO1 (F423A) impaired its binding to PCNA and resulted in reduced cellular levels of ENO1 protein, reduced growth rate, reduced glucose consumption, and reduced activation of AKT. Metabolome and signalome analysis reveal large consequences of impairing the direct interaction between PCNA and ENO1. Metabolites above ENO1 in glycolysis accumulated while lower glycolytic and TCA cycle metabolite pools decreased in the APIM-mutated cells; however, their overall energetic status were similar to parental cells. Treating haematological cancer cells or activated primary monocytes with a PCNA targeting peptide drug containing APIM (ATX-101) also lead to a metabolic shift characterized by reduced glycolytic rate. In addition, we show that ATX-101 treatments reduced the ENO1 - PCNA interaction, the ENO1, GAPDH and 6PGD protein levels, as well as the 6PGD activity. Here we report for the first time that PCNA acts as a scaffold for metabolic enzymes, and thereby act as a direct regulator of primary metabolism.

SMUG1 regulates fat homeostasis leading to a fatty liver phenotype in mice.

Fatty liver diseases are a major health threat across the western world, leading to cirrhosis and premature morbidity and mortality. Recently, a correlation between the base excision repair enzyme SMUG1 and metabolic homeostasis was identified. As the molecular mechanisms remain unknown, we exploited a SMUG1-knockout mouse model to gain insights into this association by characterizing the liver phenotype in young vs old SMUG1-null mice. We observed increased weight and fat content in one-year old animals, with altered activity of enzymes important for fatty acids influx and uptake. Consistently, lipidomic profiling showed accumulation of free fatty acids and triglycerides in SMUG1-null livers. Old SMUG1-knockout mice also displayed increased hepatocyte senescence and DNA damage at telomeres. Interestingly, RNA sequencing revealed widespread changes in the expression of lipid metabolic genes already in three months old animals. In summary, SMUG1 modulates fat metabolism favouring net lipogenesis and resulting in development of a fatty liver phenotype.

The impact of serum-free culture on HEK293 cells: From the establishment of suspension and adherent serum-free adaptation cultures to the investigation of growth and metabolic profiles.

Serum-free cultures are preferred for application in clinical cell therapy and facilitate the purification processes of bioproducts, such as vaccines and recombinant proteins. It can replace traditional cell culture - eliminating potential issues posed by animal-derived serum supplementation, such as lot to lot variation and risks of pathogen infection from the host animal. However, adapting cells to serum-free conditions can be challenging and time-consuming, and is cell line and medium dependent. In addition, the knowledge of the impact of serum-free culture on cellular metabolism is limited. Herein, we successfully established serum-free suspension and adherent cultures through two adaptation procedures for HEK293 cells in serum-free Freestyle 293 medium. Furthermore, growth kinetics and intracellular metabolic profiles related to central carbon metabolism were investigated. The entire adaptation procedure took 1 month, and high cell viability (>90%) was maintained throughout. The serum-free adherent culture showed the best growth performance, measured as the highest cell density and growth rate. The largest differences in metabolic profiles were observed between culture modes (adherent vs. suspension), followed by culture medium condition (control growth medium vs. serum-free medium). Metabolic differences related to the adaptation procedures were only seen in suspension cultures. Interestingly, the intracellular itaconate concentration was significantly higher in suspension cells compared to adherent cells. Furthermore, when the cells back-adapted from serum-free to serum-supplemented control medium, their metabolic profiles were immediately reversed, highlighting the effect of extracellular components on metabolic phenotype. This study provides strategies for efficient serum-free cultivation and deeper insights into the cellular responses related to growth and metabolism responses to diverse culture conditions.

Halogenated Pyrrolopyrimidines with Low MIC on Staphylococcus aureus and Synergistic Effects with an Antimicrobial Peptide.

Currently, there is a world-wide rise in antibiotic resistance causing burdens to individuals and public healthcare systems. At the same time drug development is lagging behind. Therefore, finding new ways of treating bacterial infections either by identifying new agents or combinations of drugs is of utmost importance. Additionally, if combination therapy is based on agents with different modes of action, resistance is less likely to develop. The synthesis of 21 fused pyrimidines and a structure-activity relationship study identified two 6-aryl-7H-pyrrolo [2,3-d] pyrimidin-4-amines with potent activity towards Staphylococcus aureus. The MIC-value was found to be highly dependent on a bromo or iodo substitution in the 4-benzylamine group and a hydroxyl in the meta or para position of the 6-aryl unit. The most active bromo and iodo derivatives had MIC of 8 mg/L. Interestingly, the most potent compounds experienced a four-fold lower MIC-value when they were combined with the antimicrobial peptide betatide giving MIC of 1-2 mg/L. The front runner bromo derivative also has a low activity towards 50 human kinases, including thymidylate monophosphate kinase, a putative antibacterial target.

Pyruvate Supports RET-Dependent Mitochondrial ROS Production to Control Mycobacterium avium Infection in Human Primary Macrophages.

Macrophages deploy a variety of antimicrobial programs to contain mycobacterial infection. Upon activation, they undergo extensive metabolic reprogramming to meet an increase in energy demand, but also to support immune effector functions such as secretion of cytokines and antimicrobial activities. Here, we report that mitochondrial import of pyruvate is linked to production of mitochondrial ROS and control of Mycobacterium avium (M. avium) infection in human primary macrophages. Using chemical inhibition, targeted mass spectrometry and single cell image analysis, we showed that macrophages infected with M. avium switch to aerobic glycolysis without any major imbalances in the tricarboxylic acid cycle volume or changes in the energy charge. Instead, we found that pyruvate import contributes to hyperpolarization of mitochondria in infected cells and increases production of mitochondrial reactive oxygen species by the complex I via reverse electron transport, which reduces the macrophage burden of M. avium. While mycobacterial infections are extremely difficult to treat and notoriously resistant to antibiotics, this work stresses out that compounds specifically inducing mitochondrial reactive oxygen species could present themself as valuable adjunct treatments.

Nutrient-depended metabolic switching during batch cultivation of Streptomyces coelicolor explored with absolute quantitative mass spectrometry-based metabolite profiling.

The well-known secondary metabolite-producing bacterium Streptomyces coelicolor is a natural choice for the development of super-hosts optimized for the heterologous expression of antibiotic biosynthetic gene clusters (BGCs). In this study, we used S. coelicolor M145 and its derivative strain M1146 where all active BGCs have been deleted and generated high-resolution quantitative time series metabolite profiles under two cultivation conditions (phosphate and nitrogen limitation to cease growth and trigger secondary metabolism). Five targeted LC-MS/MS-based methods were used to quantify intracellular primary metabolites covering phosphorylated metabolites, amino acids, organic acids, (deoxy) nucleoside/sugar phosphates, Nicotinamide adenine dinucleotide (NAD), and Coenzyme A (CoA). The nitrogen limitation resulted in a sharp decline in respiration and an immediate drop in the cell mass concentration. Intracellularly, a reduction in the level of the metabolites next to α-ketoglutarate in the tricarboxylic acid cycle and a decrease in the NADH pool were among the most prominent adaptation to this nutrient limitation. Phosphate limitation evoked a different adaptation of the metabolite pools as most of the phosphorylated metabolite pools except 6-phosphogluconic acid (6PG) pool were downregulated. 13C-isotope-labeling experiments revealed the simultaneous activity of both glycolysis and gluconeogenesis during the co-utilization of glucose and glutamate. The S. coelicolor M1146 strain had similar time-series metabolite profile dynamics as the parent M145 strain, except for a visibly increased 6PG pool in the stationary phase. In general, the nutrient limitation had a larger effect on the metabolite pool levels than the absence of secondary metabolite production in M1146. This study provides new insight into the primary carbon metabolism and its link to the secondary metabolism which is needed for further optimization of both super-host genotype and cultivation conditions. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-022-03146-x.

Overexpression of Two New Acyl-CoA:Diacylglycerol Acyltransferase 2-Like Acyl-CoA:Sterol Acyltransferases Enhanced Squalene Accumulation in Aurantiochytrium limacinum.

Thraustochytrids are heterotrophic marine eukaryotes known to accumulate large amounts of triacylglycerols, and they also synthesize terpenoids like carotenoids and squalene, which all have an increasing market demand. However, a more extensive knowledge of the lipid metabolism is needed to develop thraustochytrids for profitable biomanufacturing. In this study, two putative type-2 Acyl-CoA:diacylglycerol acyltransferases (DGAT2) genes of Aurantiochytrium sp. T66, T66ASATa, and T66ASATb, and their homologs in Aurantiochytrium limacinum SR21, AlASATa and AlASATb, were characterized. In A. limacinum SR21, genomic knockout of AlASATb reduced the amount of the steryl esters of palmitic acid, SE (16:0), and docosahexaenoic acid, SE (22:6). The double mutant of AlASATa and AlASATb produced even less of these steryl esters. The expression and overexpression of T66ASATb and AlASATb, respectively, enhanced SE (16:0) and SE (22:6) production more significantly than those of T66ASATa and AlASATa. In contrast, these mutations did not significantly change the level of triacylglycerols or other lipid classes. The results suggest that the four genes encoded proteins possessing acyl-CoA:sterol acyltransferase (ASAT) activity synthesizing both SE (16:0) and SE (22:6), but with the contribution from AlASATb and T66ASATb being more important than that of AlASATa and T66ASATa. Furthermore, the expression and overexpression of T66ASATb and AlASATb enhanced squalene accumulation in SR21 by up to 88%. The discovery highlights the functional diversity of DGAT2-like proteins and provides valuable information on steryl ester and squalene synthesis in thraustochytrids, paving the way to enhance squalene production through metabolic engineering.

Serum-free cultures of C2C12 cells show different muscle phenotypes which can be estimated by metabolic profiling.

In vitro skeletal muscle cell production is emerging in the field of artificial lab-grown meat as alternative future food. Currently, there is an urgent paradigm shift towards a serum replacement culture system. Surprisingly, little is known about the impact of serum-free culture on skeletal muscle cells to date. Therefore, we performed metabolic profiling of the C2C12 myoblasts and myotubes in serum-free mediums (B27, AIM-V) and compared it with conventional serum supplementation culture. Furthermore, cell morphology, viability, and myogenic differentiation were observed for 7 days of cultivation. Intriguingly, the metabolic difference is more dominant between the cell status than medium effects. In addition, proliferative myoblast showed more distinct metabolic differences than differentiated myotubes in different culture conditions. The intracellular levels of GL3P and UDP-GlcNAc were significantly increased in myotubes versus myoblast. Non-essential amino acids and pyruvate reduction and transamination showed significant differences among serum, B27, and AIM-V cultures. Intracellular metabolite profiles indicated that C2C12 myotubes cultured in serum and B27 had predominant glycolytic and oxidative metabolism, respectively, indicating fast and slow types of muscle confirmed by MHC immunostaining. This work might be helpful to understand the altered metabolism of skeletal muscle cells in serum-free culture and contribute to future artificial meat research work.

Experimental determination of Escherichia coli biomass composition for constraint-based metabolic modeling.

Genome-scale metabolic models (GEMs) are mathematical representations of metabolism that allow for in silico simulation of metabolic phenotypes and capabilities. A prerequisite for these predictions is an accurate representation of the biomolecular composition of the cell necessary for replication and growth, implemented in GEMs as the so-called biomass objective function (BOF). The BOF contains the metabolic precursors required for synthesis of the cellular macro- and micromolecular constituents (e.g. protein, RNA, DNA), and its composition is highly dependent on the particular organism, strain, and growth condition. Despite its critical role, the BOF is rarely constructed using specific measurements of the modeled organism, drawing the validity of this approach into question. Thus, there is a need to establish robust and reliable protocols for experimental condition-specific biomass determination. Here, we address this challenge by presenting a general pipeline for biomass quantification, evaluating its performance on Escherichia coli K-12 MG1655 sampled during balanced exponential growth under controlled conditions in a batch-fermentor set-up. We significantly improve both the coverage and molecular resolution compared to previously published workflows, quantifying 91.6% of the biomass. Our measurements display great correspondence with previously reported measurements, and we were also able to detect subtle characteristics specific to the particular E. coli strain. Using the modified E. coli GEM iML1515a, we compare the feasible flux ranges of our experimentally determined BOF with the original BOF, finding that the changes in BOF coefficients considerably affect the attainable fluxes at the genome-scale.

A comparative study at bioprocess and metabolite levels of superhost strain Streptomyces coelicolor M1152 and its derivative M1581 heterologously expressing chloramphenicol biosynthetic gene cluster.

Microbial superhost strains should provide an ideal platform for the efficient homologous or heterologous phenotypic expression of biosynthetic gene clusters (BGCs) of new and novel bioactive molecules. Our aim in the current study was to perform a comparative study at the bioprocess and metabolite levels of the previously designed superhost strain Streptomyces coelicolor M1152 and its derivative strain S. coelicolor M1581 heterologously expressing chloramphenicol BGC. Parent strain M1152 was characterized by a higher specific growth rate, specific CO2 evolution rate, and a higher specific l-glutamate consumption rate as compared with M1581. Intracellular primary central metabolites (nucleoside/sugar phosphates, amino acids, organic acids, and CoAs) were quantified using four targeted LC-MS/MS-based methods. The metabolite pathways in the nonantibiotic producing S. coelicolor host strain were flooded with carbon from both carbon sources, whereas in antibiotic-producing strain, the carbon of l-glutamate seems to be draining out through excreting synthesized antibiotic. The 13 C-isotope-labeling experiments revealed the bidirectionality in the glycolytic pathway and reversibility in the non-oxidative part of PPP even with continuous uptake of d-glucose. The change in the primary metabolites due to the insertion of BGC disclosed a clear linkage between the primary and secondary metabolites.