Characterization and validation of a preventative therapy for hypertrophic cardiomyopathy in a murine model of the disease.

Currently there is an unmet need for treatments that can prevent hypertrophic cardiomyopathy (HCM). Using a murine model we previously identified that HCM causing cardiac troponin I mutation Gly203Ser (cTnI-G203S) is associated with increased mitochondrial metabolic activity, consistent with the human condition. These alterations precede development of the cardiomyopathy. Here we examine the efficacy of in vivo treatment of cTnI-G203S mice with a peptide derived against the α-interaction domain of the cardiac L-type calcium channel (AID-TAT) on restoring mitochondrial metabolic activity, and preventing HCM. cTnI-G203S or age-matched wt mice were treated with active or inactive AID-TAT. Following treatment, targeted metabolomics was utilized to evaluate myocardial substrate metabolism. Cardiac myocyte mitochondrial metabolic activity was assessed as alterations in mitochondrial membrane potential and flavoprotein oxidation. Cardiac morphology and function were examined using echocardiography. Cardiac uptake was assessed using an in vivo multispectral imaging system. We identified alterations in six biochemical intermediates in cTnI-G203S hearts consistent with increased anaplerosis. We also reveal that AID-TAT treatment of precardiomyopathic cTnI-G203S mice, but not mice with established cardiomyopathy, restored cardiac myocyte mitochondrial membrane potential and flavoprotein oxidation, and prevented myocardial hypertrophy. Importantly, AID-TAT was rapidly targeted to the heart, and not retained by the liver or kidneys. Overall, we identify biomarkers of HCM resulting from the cTnI mutation Gly203Ser, and present a safe, preventative therapy for associated cardiomyopathy. Utilizing AID-TAT to modulate cardiac metabolic activity may be beneficial in preventing HCM in "at risk" patients with identified Gly203Ser gene mutations.

Simultaneous quantification of 26 NAD-related metabolites in plasma, blood, and liver tissue using UHPLC-MS/MS.

Nicotinamide adenine dinucleotide (NAD) is a key metabolic intermediate found in all cells and involved in numerous cellular functions. Perturbances in the NAD metabolome are linked to various diseases such as diabetes and schizophrenia, and to congenital malformations and recurrent miscarriage. Mouse models are central to the investigation of these and other NAD-related conditions because mice can be readily genetically modified and treated with diets with altered concentrations of NAD precursors. Simultaneous quantification of as many metabolites of the NAD metabolome as possible is required to understand which pathways are affected in these disease conditions and what are the functional consequences. Here, we report the development of a fit-for-purpose method to simultaneously quantify 26 NAD-related metabolites and creatinine in mouse plasma, whole blood, and liver tissue using ultra-high performance liquid chromatography - tandem mass spectrometry (UHPLC-MS/MS). The included metabolites represent dietary precursors, intermediates, enzymatic cofactors, and excretion products. Sample preparation was optimized for each matrix and included 21 isotope-labeled internal standards. The method reached adequate precision and accuracy for the intended context of use of exploratory pathway-related biomarker discovery in mouse models. The method was tested by determining metabolite concentrations in mice fed a special diet with defined precursor content.

Multi-omic profiling reveals associations between the gut mucosal microbiome, the metabolome, and host DNA methylation associated gene expression in patients with colorectal cancer.

BACKGROUND: The human gut microbiome plays a critical role in the carcinogenesis of colorectal cancer (CRC). However, a comprehensive analysis of the interaction between the host and microbiome is still lacking. RESULTS: We found correlations between the change in abundance of microbial taxa, butyrate-related colonic metabolites, and methylation-associated host gene expression in colonic tumour mucosa tissues compared with the adjacent normal mucosa tissues. The increase of genus Fusobacterium abundance was correlated with a decrease in the level of 4-hydroxybutyric acid (4-HB) and expression of immune-related peptidase inhibitor 16 (PI16), Fc Receptor Like A (FCRLA) and Lymphocyte Specific Protein 1 (LSP1). The decrease in the abundance of another potentially 4-HB-associated genus, Prevotella 2, was also found to be correlated with the down-regulated expression of metallothionein 1 M (MT1M). Additionally, the increase of glutamic acid-related family Halomonadaceae was correlated with the decreased expression of reelin (RELN). The decreased abundance of genus Paeniclostridium and genus Enterococcus were correlated with increased lactic acid level, and were also linked to the expression change of Phospholipase C Beta 1 (PLCB1) and Immunoglobulin Superfamily Member 9 (IGSF9) respectively. Interestingly, 4-HB, glutamic acid and lactic acid are all butyrate precursors, which may modify gene expression by epigenetic regulation such as DNA methylation. CONCLUSIONS: Our study identified associations between previously reported CRC-related microbial taxa, butyrate-related metabolites and DNA methylation-associated gene expression in tumour and normal colonic mucosa tissues from CRC patients, which uncovered a possible mechanism of the role of microbiome in the carcinogenesis of CRC. In addition, these findings offer insight into potential new biomarkers, therapeutic and/or prevention strategies for CRC.

Functional screening in human cardiac organoids reveals a metabolic mechanism for cardiomyocyte cell cycle arrest.

The mammalian heart undergoes maturation during postnatal life to meet the increased functional requirements of an adult. However, the key drivers of this process remain poorly defined. We are currently unable to recapitulate postnatal maturation in human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), limiting their potential as a model system to discover regenerative therapeutics. Here, we provide a summary of our studies, where we developed a 96-well device for functional screening in human pluripotent stem cell-derived cardiac organoids (hCOs). Through interrogation of >10,000 organoids, we systematically optimize parameters, including extracellular matrix (ECM), metabolic substrate, and growth factor conditions, that enhance cardiac tissue viability, function, and maturation. Under optimized maturation conditions, functional and molecular characterization revealed that a switch to fatty acid metabolism was a central driver of cardiac maturation. Under these conditions, hPSC-CMs were refractory to mitogenic stimuli, and we found that key proliferation pathways including ß-catenin and Yes-associated protein 1 (YAP1) were repressed. This proliferative barrier imposed by fatty acid metabolism in hCOs could be rescued by simultaneous activation of both ß-catenin and YAP1 using genetic approaches or a small molecule activating both pathways. These studies highlight that human organoids coupled with higher-throughput screening platforms have the potential to rapidly expand our knowledge of human biology and potentially unlock therapeutic strategies.

Barocycler-Based Concurrent Multiomics Method To Assess Molecular Changes Associated with Atherosclerosis Using Small Amounts of Arterial Tissue from a Single Mouse.

Atherosclerosis is a complex, multifactorial disease characterized by the buildup of plaque in the arterial wall. Apolipoprotein E gene deficient (Apoe-/-) mice serve as a commonly used tool to elucidate the pathophysiology of atherosclerosis because of their propensity to spontaneously develop arterial lesions. To date, however, an integrated omics assessment of atherosclerotic lesions in individual Apoe-/- mice has been challenging because of the small amount of diseased and nondiseased tissue available. To address this current limitation, we developed a multiomics method (Multi-ABLE) based on the proteomic method called accelerated Barocycler lysis and extraction (ABLE) to assess the depth of information that can be obtained from arterial tissue derived from a single mouse by splitting ABLE to allow for a combined proteomics-metabolomics-lipidomics analysis (Multi-ABLE). The new method includes tissue lysis via pressure cycling technology (PCT) in a Barocycler, followed by proteomic analysis of half the sample by nanoLC-MS and sequential extraction of lipids (organic extract) and metabolites (aqueous extract) combined with HILIC and reversed phase chromatography and time-of-flight mass spectrometry on the other half. Proteomic analysis identified 845 proteins, 93 of which were significantly altered in lesion-containing arteries. Lipidomic and metabolomic analyses detected 851 lipid and 362 metabolite features, which included 215 and 65 identified lipids and metabolites, respectively. The Multi-ABLE method is the first to apply a concurrent multiomics pipeline to cardiovascular disease using small (<5 mg) tissue samples, and it is applicable to other diseases where limited size samples are available at specific points during disease progression.

Systems-level engineering and characterisation of Clostridium autoethanogenum through heterologous production of poly-3-hydroxybutyrate (PHB).

Gas fermentation is emerging as an economically attractive option for the sustainable production of fuels and chemicals from gaseous waste feedstocks. Clostridium autoethanogenum can use CO and/or CO2 + H2 as its sole carbon and energy sources. Fermentation of C. autoethanogenum is currently being deployed on a commercial scale for ethanol production. Expanding the product spectrum of acetogens will enhance the economics of gas fermentation. To achieve efficient heterologous product synthesis, limitations in redox and energy metabolism must be overcome. Here, we engineered and characterised at a systems-level, a recombinant poly-3-hydroxybutyrate (PHB)-producing strain of C. autoethanogenum. Cells were grown in CO-limited steady-state chemostats on two gas mixtures, one resembling syngas (20% H2) and the other steel mill off-gas (2% H2). Results were characterised using metabolomics and transcriptomics, and then integrated using a genome-scale metabolic model reconstruction. PHB-producing cells had an increased expression of the Rnf complex, suggesting energy limitations for heterologous production. Subsequent optimisation of the bioprocess led to a 12-fold increase in the cellular PHB content. The data suggest that the cellular redox state, rather than the acetyl-CoA pool, was limiting PHB production. Integration of the data into the genome-scale metabolic model showed that ATP availability limits PHB production. Altogether, the data presented here advances the fundamental understanding of heterologous product synthesis in gas-fermenting acetogens.

Systems-based approaches enable identification of gene targets which improve the flavour profile of low-ethanol wine yeast strains.

Metabolic engineering has been vital to the development of industrial microbes such as the yeast Saccharomyces cerevisiae. However, sequential rounds of modification are often needed to achieve particular industrial design targets. Systems biology approaches can aid in identifying genetic targets for modification through providing an integrated view of cellular physiology. Recently, research into the generation of commercial yeasts that can produce reduced-ethanol wines has resulted in metabolically-engineered strains of S. cerevisiae that are less efficient at producing ethanol from sugar. However, these modifications led to the concomitant production of off-flavour by-products. A combination of transcriptomics, proteomics and metabolomics was therefore used to investigate the physiological changes occurring in an engineered low-ethanol yeast strain during alcoholic fermentation. Integration of 'omics data identified several metabolic reactions, including those related to the pyruvate node and redox homeostasis, as being significantly affected by the low-ethanol engineering methodology, and highlighted acetaldehyde and 2,4,5-trimethyl-1,3-dioxolane as the main off-flavour compounds. Gene remediation strategies were then successfully applied to decrease the formation of these by-products, while maintaining the 'low-alcohol' phenotype. The data generated from this comprehensive systems-based study will inform wine yeast strain development programmes, which, in turn, could potentially play an important role in assisting winemakers in their endeavour to produce low-alcohol wines with desirable flavour profiles.

Quantitative analysis of tetrahydrofolate metabolites from clostridium autoethanogenum.

INTRODUCTION: Quantification of tetrahydrofolates (THFs), important metabolites in the Wood-Ljungdahl pathway (WLP) of acetogens, is challenging given their sensitivity to oxygen. OBJECTIVE: To develop a simple anaerobic protocol to enable reliable THFs quantification from bioreactors. METHODS: Anaerobic cultures were mixed with anaerobic acetonitrile for extraction. Targeted LC-MS/MS was used for quantification. RESULTS: Tetrahydrofolates can only be quantified if sampled anaerobically. THF levels showed a strong correlation to acetyl-CoA, the end product of the WLP. CONCLUSION: Our method is useful for relative quantification of THFs across different growth conditions. Absolute quantification of THFs requires the use of labelled standards.

Improved performance of Pseudomonas putida in a bioelectrochemical system through overexpression of periplasmic glucose dehydrogenase.

It was recently demonstrated that a bioelectrochemical system (BES) with a redox mediator allowed Pseudomonas putida to perform anoxic metabolism, converting sugar to sugar acids with high yield. However, the low productivity currently limits the application of this technology. To improve productivity, the strain was optimized through improved expression of glucose dehydrogenase (GCD) and gluconate dehydrogenase (GAD). In addition, quantitative real-time RT-PCR analysis revealed the intrinsic self-regulation of GCD and GAD. Utilizing this self-regulation system, the single overexpression strain (GCD) gave an outstanding performance in the electron transfer rate and 2-ketogluconic acid (2KGA) productivity. The peak anodic current density, specific glucose uptake rate and 2KGA producing rate were 0.12 mA/cm2 , 0.27 ± 0.02 mmol/gCDW /hr and 0.25 ± 0.02 mmol/gCDW /hr, which were 327%, 477%, and 644% of the values of wild-type P. putida KT2440, respectively. This work demonstrates that expression of periplasmic dehydrogenases involved in electron transfer can significantly improve productivity in the BES.

Persistently Altered Metabolic Phenotype following Perinatal Excitotoxic Brain Injury.

Excitotoxicity plays a key role during insults to the developing brain such as neonatal encephalopathy, stroke, and encephalopathy of prematurity. Such insults affect many thousands of infants each year. Excitotoxicity causes frank lesions due to cell death and gliosis and disturbs normal developmental process, leading to deficits in learning, memory, and social integration that persist into adulthood. Understanding the underlying processes of the acute effects of excitotoxicity and its persistence during brain maturation provides an opportunity to identify mechanistic or diagnostic biomarkers, thus enabling and designing possible therapies. We applied mass spectrometry to provide metabolic profiles of brain tissue and plasma over time following an excitotoxic lesion (intracerebral ibotenate) to the neonatal (postnatal day 5) mouse brain. We found no differences between the plasma from the control (PBS-injected) and excitotoxic (ibotenate-injected) groups over time (on postnatal days 8, 9, 10, and 30). In the brain, we found that variations in amino acids (arginine, glutamine, phenylananine, and proline) and glycerophospholipids were sustaining acute and delayed (tertiary) responses to injury. In particular, the effect of the excitotoxic lesion on the normal profile of development was linked to alterations in a fingerprint of glycerophospolipids and amino acids. Specifically, we identified increases in the amino acids glutamine, proline, serine, threonine, tryptophan, valine, and the sphingolipid SM C26:1, and decreases in the glycerophospholipids, i.e., the arachidonic acid-containing phosphatidylcholine (PC aa) C30:2 and the PC aa C32:3. This study demonstrates that metabolic profiling is a useful approach to identify acute and tertiary effects in an excitotoxic lesion model, and generating a short list of targets with future potential in the hunt for identification, stratification, and possibly therapy.

A squalene synthase protein degradation method for improved sesquiterpene production in Saccharomyces cerevisiae.

Sesquiterpenes are C15 isoprenoids with utility as fragrances, flavours, pharmaceuticals, and potential biofuels. Microbial fermentation is being examined as a competitive approach for bulk production of these compounds. Competition for carbon allocation between synthesis of endogenous sterols and production of the introduced sesquiterpene limits yields. Achieving balance between endogenous sterols and heterologous sesquiterpenes is therefore required to achieve economical yields. In the current study, the yeast Saccharomyces cerevisiae was used to produce the acyclic sesquiterpene alcohol, trans-nerolidol. Nerolidol production was first improved by enhancing the upstream mevalonate pathway for the synthesis of the precursor farnesyl pyrophosphate (FPP). However, excess FPP was partially directed towards squalene by squalene synthase (Erg9p), resulting in squalene accumulation to 1% biomass; moreover, the specific growth rate declined. In order to re-direct carbon away from sterol production and towards the desired heterologous sesquiterpene, a novel protein destabilisation approach was developed for Erg9p. It was shown that Erg9p is located on endoplasmic reticulum and lipid droplets through a C-terminal ER-targeted transmembrane peptide. A PEST (rich in Pro, Glu/Asp, Ser, and Thr) sequence-dependent endoplasmic reticulum-associated protein degradation (ERAD) mechanism was established to decrease cellular levels of Erg9p without relying on inducers, repressors or specific repressing conditions. This improved nerolidol titre by 86% to ~100mgL-1. In this strain, squalene levels were similar to the wild-type control strain, and downstream ergosterol levels were slightly decreased relative to the control, indicating redirection of carbon away from sterols and towards sesquiterpene production. There was no negative effect on cell growth under these conditions. Protein degradation is an efficient mechanism to control carbon allocation at flux-competing nodes in metabolic engineering applications. This study demonstrates that an engineered ERAD mechanism can be used to balance flux competition between the endogenous sterol pathway and an introduced bio-product pathways at the FPP node. The approach of protein degradation in general might be more widely applied to improve metabolic engineering outcomes.

Arginine deiminase pathway provides ATP and boosts growth of the gas-fermenting acetogen Clostridium autoethanogenum.

Acetogens are attractive organisms for the production of chemicals and fuels from inexpensive and non-food feedstocks such as syngas (CO, CO2 and H2). Expanding their product spectrum beyond native compounds is dictated by energetics, particularly ATP availability. Acetogens have evolved sophisticated strategies to conserve energy from reduction potential differences between major redox couples, however, this coupling is sensitive to small changes in thermodynamic equilibria. To accelerate the development of strains for energy-intensive products from gases, we used a genome-scale metabolic model (GEM) to explore alternative ATP-generating pathways in the gas-fermenting acetogen Clostridium autoethanogenum. Shadow price analysis revealed a preference of C. autoethanogenum for nine amino acids. This prediction was experimentally confirmed under heterotrophic conditions. Subsequent in silico simulations identified arginine (ARG) as a key enhancer for growth. Predictions were experimentally validated, and faster growth was measured in media containing ARG (tD~4h) compared to growth on yeast extract (tD~9h). The growth-boosting effect of ARG was confirmed during autotrophic growth. Metabolic modelling and experiments showed that acetate production is nearly abolished and fast growth is realised by a three-fold increase in ATP production through the arginine deiminase (ADI) pathway. The involvement of the ADI pathway was confirmed by metabolomics and RNA-sequencing which revealed a ~500-fold up-regulation of the ADI pathway with an unexpected down-regulation of the Wood-Ljungdahl pathway. The data presented here offer a potential route for supplying cells with ATP, while demonstrating the usefulness of metabolic modelling for the discovery of native pathways for stimulating growth or enhancing energy availability.

Systems biology and metabolic modelling unveils limitations to polyhydroxybutyrate accumulation in sugarcane leaves; lessons for C4 engineering.

In planta production of the bioplastic polyhydroxybutyrate (PHB) is one important way in which plant biotechnology can address environmental problems and emerging issues related to peak oil. However, high biomass C4 plants such as maize, switch grass and sugarcane develop adverse phenotypes including stunting, chlorosis and reduced biomass as PHB levels in leaves increase. In this study, we explore limitations to PHB accumulation in sugarcane chloroplasts using a systems biology approach, coupled with a metabolic model of C4 photosynthesis. Decreased assimilation was evident in high PHB-producing sugarcane plants, which also showed a dramatic decrease in sucrose and starch content of leaves. A subtle decrease in the C/N ratio was found which was not associated with a decrease in total protein content. An increase in amino acids used for nitrogen recapture was also observed. Based on the accumulation of substrates of ATP-dependent reactions, we hypothesized ATP starvation in bundle sheath chloroplasts. This was supported by mRNA differential expression patterns. The disruption in ATP supply in bundle sheath cells appears to be linked to the physical presence of the PHB polymer which may disrupt photosynthesis by scattering photosynthetically active radiation and/or physically disrupting thylakoid membranes.

Tetanus toxin production is triggered by the transition from amino acid consumption to peptides.

Bacteria produce some of the most potent biomolecules known, of which many cause serious diseases such as tetanus. For prevention, billions of people and countless animals are immunised with the highly effective vaccine, industrially produced by large-scale fermentation. However, toxin production is often hampered by low yields and batch-to-batch variability. Improved productivity has been constrained by a lack of understanding of the molecular mechanisms controlling toxin production. Here we have developed a reproducible experimental framework for screening phenotypic determinants in Clostridium tetani under a process that mimics an industrial setting. We show that amino acid depletion induces production of the tetanus toxin. Using time-course transcriptomics and extracellular metabolomics to generate a 'fermentation atlas' that ascribe growth behaviour, nutrient consumption and gene expression to the fermentation phases, we found a subset of preferred amino acids. Exponential growth is characterised by the consumption of those amino acids followed by a slower exponential growth phase where peptides are consumed, and toxin is produced. The results aim at assisting in fermentation medium design towards the improvement of vaccine production yields and reproducibility. In conclusion, our work not only provides deep fermentation dynamics but represents the foundation for bioprocess design based on C. tetani physiological behaviour under industrial settings.

A novel anticonvulsant mechanism via inhibition of complement receptor C5ar1 in murine epilepsy models.

The role of complement system-mediated inflammation is of key interest in seizure and epilepsy pathophysiology, but its therapeutic potential has not yet been explored. We observed that the pro-inflammatory C5a receptor, C5ar1, is upregulated in two mouse models after status epilepticus; the pilocarpine model and the intrahippocampal kainate model. The C5ar1 antagonist, PMX53, was used to assess potential anticonvulsant actions of blocking this receptor pathway. PMX53 was found to be anticonvulsant in several acute models (6Hz and corneal kindling) and one chronic seizure model (intrahippocampal kainate model). The effects in the 6Hz model were not found in C5ar1-deficient mice, or with an inactive PMX53 analogue suggesting that the anticonvulsant effect of PMX53 is C5ar1-specific. In the pilocarpine model, inhibition or absence of C5ar1 during status epilepticus lessened seizure power and protected hippocampal neurons from degeneration as well as halved SE-associated mortality. C5ar1-deficiency during pilocarpine-induced status epilepticus also was accompanied by attenuation of TNFα upregulation by microglia, suggesting that C5ar1 activation results in TNFα release contributing to disease. Patch clamp studies showed that C5a-induced microglial K(+) outward currents were also inhibited with PMX53 providing a potential mechanism to explain acute anticonvulsant effects. In conclusion, our data indicate that C5ar1 activation plays a role in seizure initiation and severity, as well as neuronal degeneration following status epilepticus. The widespread anticonvulsant activity of PMX53 suggests that C5ar1 represents a novel target for improved anti-epileptic drug development which may be beneficial for pharmaco-resistant patients.

Increased sensitivity to tryptophan bioavailability is a positive adaptation by the human strains of Chlamydia pneumoniae.

One of the most significant activities induced by interferon-gamma against intracellular pathogens is the induction of IDO (indoleamine 2,3-dioxygenase) expression, which subsequently results in the depletion of tryptophan. We tested the hypothesis that human strains of Chlamydia pneumoniae are more sensitive to tryptophan limitation than animal C. pneumoniae strains. The human strains were significantly more sensitive to IFN-γ than the animal strains in a lung epithelia cell model (BEAS-2B), with exposure to 1 U ml(-1) IFN-γ resulting in complete loss of infectious yield of human strains, compared to the animal strains where reductions in infectious progeny were around 3.5-4.0 log. Strikingly, the IFN-γ induced loss of ability to form infectious progeny production was completely rescued by removal of the IFN-γ and addition of exogenous tryptophan for the human strains, but not the animal strains. In fact, a human heart strain was more capable of entering a non-infectious, viable persistent stage when exposed to IFN-γ and was also more effectively rescued, compared to a human respiratory strain. Exquisite susceptibility to IFN-γ, specifically due to tryptophan availability appears to be a core adaptation of the human C. pneumoniae strains, which may reflect the chronic nature of their infections in this host.

The use of an acetoacetyl-CoA synthase in place of a ß-ketothiolase enhances poly-3-hydroxybutyrate production in sugarcane mesophyll cells.

Engineering the production of polyhydroxyalkanoates (PHAs) into high biomass bioenergy crops has the potential to provide a sustainable supply of bioplastics and energy from a single plant feedstock. One of the major challenges in engineering C4 plants for the production of poly[(R)-3-hydroxybutyrate] (PHB) is the significantly lower level of polymer produced in the chloroplasts of mesophyll (M) cells compared to bundle sheath (BS) cells, thereby limiting the full PHB yield-potential of the plant. In this study, we provide evidence that the access to substrate for PHB synthesis may limit polymer production in M chloroplasts. Production of PHB in M cells of sugarcane is significantly increased by replacing ß-ketothiolase, the first enzyme in the bacterial PHA pathway, with acetoacetyl-CoA synthase. This novel pathway enabled the production of PHB reaching an average of 6.3% of the dry weight of total leaf biomass, with levels ranging from 3.6 to 11.8% of the dry weight (DW) of individual leaves. These yields are more than twice the level reported in PHB-producing sugarcane containing the ß-ketothiolase and illustrate the importance of producing polymer in mesophyll plastids to maximize yield. The molecular weight of the polymer produced was greater than 2 × 10(6)  Da. These results are a major step forward in engineering a high biomass C4 grass for the commercial production of PHB.

Systems analysis of methylerythritol-phosphate pathway flux in E. coli: insights into the role of oxidative stress and the validity of lycopene as an isoprenoid reporter metabolite.

BACKGROUND: High-throughput screening methods assume that the output measured is representative of changes in metabolic flux toward the desired product and is not affected by secondary phenotypes. However, metabolic engineering can result in unintended phenotypes that may go unnoticed in initial screening. The red pigment lycopene, a carotenoid with antioxidant properties, has been used as a reporter of isoprenoid pathway flux in metabolic engineering for over a decade. Lycopene production is known to vary between wild-type Escherichia coli hosts, but the reasons behind this variation have never been fully elucidated. RESULTS: In an examination of six E. coli strains we observed that strains also differ in their capacity for increased lycopene production in response to metabolic engineering. A combination of genetic complementation, quantitative SWATH proteomics, and biochemical analysis in closely-related strains was used to examine the mechanistic reasons for variation in lycopene accumulation. This study revealed that rpoS, a gene previously identified in lycopene production association studies, exerts its effect on lycopene accumulation not through modulation of pathway flux, but through alteration of cellular oxidative status. Specifically, absence of rpoS results in increased accumulation of reactive oxygen species during late log and stationary phases. This change in cellular redox has no effect on isoprenoid pathway flux, despite the presence of oxygen-sensitive iron-sulphur cluster enzymes and the heavy redox requirements of the methylerythritol phosphate pathway. Instead, decreased cellular lycopene in the ΔrpoS strain is caused by degradation of lycopene in the presence of excess reactive oxygen species. CONCLUSIONS: Our results demonstrate that lycopene is not a reliable indicator of isoprenoid pathway flux in the presence of oxidative stress, and suggest that caution should be exercised when using lycopene as a screening tool in genome-wide metabolic engineering studies. More extensive use of systems biology for strain analysis will help elucidate such unpredictable side-effects in metabolic engineering projects.

LC-MS-based metabolomics study of marine bacterial secondary metabolite and antibiotic production in Salinispora arenicola.

An LC-MS-based metabolomics approach was used to characterise the variation in secondary metabolite production due to changes in the salt content of the growth media as well as across different growth periods (incubation times). We used metabolomics as a tool to investigate the production of rifamycins (antibiotics) and other secondary metabolites in the obligate marine actinobacterial species Salinispora arenicola, isolated from Great Barrier Reef (GBR) sponges, at two defined salt concentrations and over three different incubation periods. The results indicated that a 14 day incubation period is optimal for the maximum production of rifamycin B, whereas rifamycin S and W achieve their maximum concentration at 29 days. A "chemical profile" link between the days of incubation and the salt concentration of the growth medium was shown to exist and reliably represents a critical point for selection of growth medium and harvest time.

Bacterial production of the fungus-derived cholesterol-lowering agent mevinolin.

Forty-five strains from two different species (Salinispora arenicola and Salinispora pacifica) were isolated from three different marine sponge species in the Great Barrier Reef region of Australia. We found that two of the strains of Salinispora arenicola (MV0335 and MV0029) produced mevinolin, a fungus-derived cholesterol-lowering agent. Compound structure was determined using an integrated approach: (a) high performance liquid chromatography-quadrupole time-of-flight-mass spectrometric analysis with multimode ionization (electrospray ionization and atmospheric pressure chemical ionization) and fast polarity switching; and (b) database searching and matching of monoisotopic masses, retention times and mass spectra of the precursor and product ions of the compounds of interest and the authentic reference standards thereof.