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.The authors are thankful to Mia Jullig for assistance with Fig. 2. Callaghan Innovation and Bioresource Processing Alliance provided PhD stipends for James Daniell and Ninna Granucci respectively.info:eu-repo/semantics/publishedVersio

Grape and Wine Metabolomics to Develop New Insights Using Untargeted and Targeted Approaches

Chemical analysis of grape juice and wine has been performed for over 50 years in a targeted manner to determine a limited number of compounds using Gas Chromatography, Mass-Spectrometry (GC-MS) and High Pressure Liquid Chromatography (HPLC). Therefore, it only allowed the determination of metabolites that are present in high concentration, including major sugars, amino acids and some important carboxylic acids. Thus, the roles of many significant but less concentrated metabolites during wine making process are still not known. This is where metabolomics shows its enormous potential, mainly because of its capability in analyzing over 1000 metabolites in a single run due to the recent advancements of high resolution and sensitive analytical instruments. Metabolomics has predominantly been adopted by many wine scientists as a hypothesis-generating tool in an unbiased and non-targeted way to address various issues, including characterization of geographical origin (terroir) and wine yeast metabolic traits, determination of biomarkers for aroma compounds, and the monitoring of growth developments of grape vines and grapes. The aim of this review is to explore the published literature that made use of both targeted and untargeted metabolomics to study grapes and wines and also the fermentation process. In addition, insights are also provided into many other possible avenues where metabolomics shows tremendous potential as a question-driven approach in grape and wine research

Rapid Quantification of Major Volatile Metabolites in Fermented Food and Beverages Using Gas Chromatography-Mass Spectrometry

Here we present a method for the accurate quantification of major volatile metabolites found in different food and beverages, including ethanol, acetic acid and other aroma compounds, using gas chromatography coupled to mass spectrometry (GC-MS). The method is combined with a simple sample preparation procedure using sodium chloride and anhydrous ethyl acetate. The GC-MS analysis was accomplished within 4.75 min, and over 80 features were detected, of which 40 were positively identified using an in-house and a commercialmass spectrometry (MS) library. We determined different analytical parameters of these metabolites including the limit of detection (LOD), limit of quantitation (LOQ) and range of quantification. In order to validate the method, we also determined detailed analytical characteristics of five major fermentation end products including ethanol, acetic acid, isoamyl alcohol, ethyl-L-lactate and, acetoin. The method showed very low technical variability for the measurements of these metabolites in different matrices (<3%) with an excellent accuracy (100% ± 5%), recovery (100% ± 10%), reproducibility and repeatability [Coefficient of variation (CV) 1–10%)]. To demonstrate the applicability of the method, we analysed different fermented products including balsamic vinegars, sourdough, distilled (whisky) and non-distilled beverages (wine and beer)

Extracellular Microbial Metabolomics: The State of the Art

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

Analysis of Intracellular Metabolites from Microorganisms: Quenching and Extraction Protocols

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

Application of untargeted volatile profiling and data driven approaches in wine flavoromics research

Traditional flavor chemistry research usually makes use of targeted approaches by focusing on the detection and quantification of key flavor active metabolites that are present in food and beverages. In the last decade, flavoromics has emerged as an alternative to targeted methods where non-targeted and data driven approaches have been used to determine as many metabolites as possible with the aim to establish relationships among the chemical composition of foods and their sensory properties. Flavoromics has been successfully applied in wine research to gain more insights into the impact of a wide range of flavor active metabolites on wine quality. In this review, we aim to provide an overview of the applications of flavoromics approaches in wine research based on existing literature mainly by focusing on untargeted volatile profiling of wines and how this can be used as a powerful tool to generate novel insights. We highlight the fact that untargeted volatile profiling used in flavoromics approaches ultimately can assist the wine industry to produce different wine styles and to market existing wines appropriately based on consumer preference. In addition to summarizing the main steps involved in untargeted volatile profiling, we also provide an outlook about future perspectives and challenges of wine flavoromics research.Authors thank the support of the Spanish MINECO through the AGL201678936-R and PID2019-111734RB-100 (AEI/FEDER, UE) projects and for the pre-doctoral mobility grant to M.P.J. F.P. and E.S. would like to acknowledge the funding contribution from Strategic Science Investment Fund (project code P/471778/20) by Ministry of Business, Investment and Employment (MBIE).Peer reviewe

Correction: Pinu et al. The Effect of Yeast Inoculation Methods on the Metabolite Composition of Sauvignon Blanc Wines. <i>Fermentation</i> 2023, <i>9</i>, 759

There was a technical error in the calculation of yeast cell concentrations from cell numbers in the original publication [...

Application of untargeted volatile profiling to investigate the fate of aroma compounds during wine oral processing

We employed an untargeted volatile profiling approach in combination with spit off-odorant measurement procedure to investigate the fate of aroma compounds in mouth by determining how oral processing and intrinsic biological variables influence the overall volatile composition. A red wine before and after oral processing (expectorated wine), and control samples (expectorated water) were analyzed using GC-TOF-MS to determine as many volatile compounds as possible. We identified compounds in expectorated wines that originated in mouth from either an endogenous or exogenous source, while confirming that compounds might have metabolized by the action of salivary enzymes. Our data also show the changes in volatiles via hydrolysis from the corresponding molecules and may provide evidence of de novo formation of volatiles via transesterification reaction in mouth. While investigating the impact of intrinsic biological variables, we found age and gender specific differences in wine volatile composition due to oral processing and identified the key volatiles.This research was supported by the Spanish MINECO through the AGL201678936-R and PID 2019-11734RB-100 projects. M.P.-J. and C.M.-G. thank MINECO and CAM for their respective predoctoral and postdoctoral (Atracción de Talento CAM, 2019 T1/BIO13748) contracts. F.P and E.S would like to acknowledge financial contribution from The New Zealand Institute for Plant and Food Research Limited’s Strategic Science Investment Fund (SSIF) provided by the Ministry of Business, Innovation and Employment (MBIE).Peer reviewe

Vinegar Metabolomics: An Explorative Study of Commercial Balsamic Vinegars Using Gas Chromatography-Mass Spectrometry

Balsamic vinegar is a popular food condiment produced from cooked grape must by two successive fermentation (anaerobic and aerobic) processes. Although many studies have been performed to determine the composition of major metabolites, including sugars and aroma compounds, no study has been undertaken yet to characterize the comprehensive metabolite composition of balsamic vinegars. Here, we present the first metabolomics study of commercial balsamic vinegars by gas chromatography coupled to mass spectrometry (GC-MS). The combination of three GC-MS methods allowed us to detect &gt;1500 features in vinegar samples, of which 123 metabolites were accurately identified, including 25 amino acids, 26 carboxylic acids, 13 sugars and sugar alcohols, four fatty acids, one vitamin, one tripeptide and over 47 aroma compounds. Moreover, we identified for the first time in vinegar five volatile metabolites: acetin, 2-methylpyrazine, 2-acetyl-1-pyroline, 4-anisidine and 1,3-diacetoxypropane. Therefore, we demonstrated the capability of metabolomics for detecting and identifying large number of metabolites and some of them could be used to distinguish vinegar samples based on their origin and potentially quality