Metabolite secretion in microorganisms: the theory of metabolic overflow put to the test.

INTRODUCTION: Microbial cells secrete many metabolites during growth, including important intermediates of the central carbon metabolism. This has not been taken into account by researchers when modeling microbial metabolism for metabolic engineering and systems biology studies. MATERIALS AND METHODS: The uptake of metabolites by microorganisms is well studied, but our knowledge of how and why they secrete different intracellular compounds is poor. The secretion of metabolites by microbial cells has traditionally been regarded as a consequence of intracellular metabolic overflow. CONCLUSIONS: Here, we provide evidence based on time-series metabolomics data that microbial cells eliminate some metabolites in response to environmental cues, independent of metabolic overflow. Moreover, we review the different mechanisms of metabolite secretion and explore how this knowledge can benefit metabolic modeling and engineering.

Identification of New Natural Sources of Flavour and Aroma Metabolites from Solid-State Fermentation of Agro-Industrial By-Products.

Increasing consumer demand for natural flavours and fragrances has driven up prices and increased pressure on natural resources. A shift in consumer preference towards more sustainable and economical sources of these natural additives and away from synthetic production has encouraged research into alternative supplies of these valuable compounds. Solid-state fermentation processes support the natural production of secondary metabolites, which represents most flavour and aroma compounds, while agro-industrial by-products are a low-value waste stream with a high potential for adding value. Accordingly, four filamentous fungi species with a history of use in the production of fermented foods and food additives were tested to ferment nine different agro-industrial by-products. Hundreds of volatile compounds were produced and identified using headspace (HS) solid-phase microextraction (SPME) coupled to gas chromatography-mass spectrometry (GC-MS). Four compounds of interest, phenylacetaldehyde, methyl benzoate, 1-octen-3-ol, and phenylethyl alcohol, were extracted and quantified. Preliminary yields were encouraging compared to traditional sources. This, combined with the low-cost substrates and the high-value natural flavours and aromas produced, presents a compelling case for further optimisation of the process.

Can we predict the intracellular metabolic state of a cell based on extracellular metabolite data?

The analysis of extracellular metabolites presents many technical advantages over the analysis of intracellular compounds, which made this approach very popular in recent years as a high-throughput tool to assess the metabolic state of microbial cells. However, very little effort has been made to determine the actual relationship between intracellular and extracellular metabolite levels. The secretion of intracellular metabolites has been traditionally interpreted as a consequence of an intracellular metabolic overflow, which is based on the premise that for a metabolite to be secreted, it must be over-produced inside the cell. Therefore, we expect to find a secreted metabolite at increased levels inside the cells. Here we present a time-series metabolomics study of Saccharomyces cerevisiae growing on a glucose-limited chemostat with parallel measurements of intra- and extracellular metabolites. Although most of the extracellular metabolites were also detected in the intracellular samples and showed a typical metabolic overflow behaviour, we demonstrate that the secretion of many metabolites could not be explained by the metabolic overflow theory.

Biochemical biomarkers and hydrocarbons concentrations in the mangrove oyster Crassostrea brasiliana following exposure to diesel fuel water-accommodated fraction.

Understanding the toxic mechanisms by which organisms cope to environmental stressful conditions is a fundamental question for ecotoxicology. In this study, we evaluated biochemical responses and hydrocarbons bioaccumulation of the mangrove oyster Crassostrea brasiliana exposed for 96 h to four sublethal concentrations of diesel fuel water-accommodated fraction (WAF). For that purpose, enzymatic activities (SOD, CAT, GPx, GR, G6PDH, GST and GGT), HSP60 and HSP90 immunocontent and lipid peroxidation (LPO) levels were determined in the gill and digestive gland of oysters and related to the hydrocarbons accumulated in the whole soft tissues. The results of this study revealed clear biochemical responses to diesel fuel WAF exposure in both tissues of the oyster. The capacity of C. brasiliana to bioaccumulate aliphatic and aromatic hydrocarbons in a dose-dependent manner is a strong indication of its suitability as a model in biomonitoring programs along the Brazilian coast, which was also validated by the response of the antioxidant defenses, phase II biotransformation and chaperones. HSP60 levels and GGT activity were the most promising biomarkers in the gill, while GST and GR activities stood out as suitable biomarkers for the detection of diesel toxicity in the digestive gland. The decrease of SOD activity and HSP90 levels may also reflect a negative effect of diesel exposure regardless the tissue. The present results provide a sound preliminary report on the biochemical responses of C. brasiliana challenged with a petroleum by-product and should be carefully considered for use in the monitoring of oil and gas activities in Brazil.

Differential gene expression in Poecilia vivipara exposed to diesel oil water accommodated fraction.

Diesel fuel is a potential contaminant of estuarine and mangrove areas, particularly because it is the main fuel used in small boats and larger vessels. The aim of this work was to identify genes differentially expressed in the liver of Poecilia vivipara (Guppy) exposed to 10% diesel fuel water accommodated fraction (WAF), employing the subtractive suppressive hybridization (SSH) method. The results showed 27 differentially expressed gene fragments, 12 up-regulated and 15 down-regulated. Among the up-regulated genes were CYP1A, UDPGT1a, ABCC4, Methyltransferase and Apolipoprotein A1. Down-regulated genes included Vitellogenins, C1 Inhibitor and Complement Component 3c. The identified genes are associated with different metabolic functions like biotransformation, membrane transport and immune system, indicating the susceptibility and/or molecular responses of this organism to the toxic effects elicited by diesel fuel WSF.