Targeted Metabolomics of Xylose-Fermenting Yeasts Based on Mass Spectrometry.

Mass spectrometry is a sensitive and selective analytical technique that enables detection and quantitation of low abundance compounds in a complex sample matrix. Targeted metabolomics allows for quantitative analysis of metabolites, providing kinetic information of production and consumption rates, an essential step to investigate microbial metabolism. Here, we describe a targeted metabolomics protocol for yeast samples, from sample preparation to mass spectrometry analysis, which enables the identification of metabolic fluxes after xylose consumption. Sample preparation methods were optimized for quenching of yeast metabolism followed by intracellular metabolite extraction, using cold methanol and boiling ethanol protocols. Ultrahigh performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) methods using ion pair chromatography (IPC) and hydrophilic interaction liquid chromatography (HILIC) allowed for the quantitation of 18 metabolites involved in central carbon metabolism (glycolysis, pentose phosphate pathway, and tricarboxylic acid cycle). The protocol here described was successfully applied to quantify metabolites in Scheffersomyces stipitis, Spathaspora passalidarum, Spathaspora arborariae, and Candida tenuis samples after xylose consumption.

Rhamnolipids production from sucrose by engineered Saccharomyces cerevisiae.

Biosurfactants are biological tensioactive agents that can be used in the cosmetic and food industries. Rhamnolipids are glycolipid biosurfactants naturally produced by Pseudomonas aeruginosa and are composed of one or two rhamnose molecules linked to beta-hydroxy fatty acid chains. These compounds are green alternatives to petrochemical surfactants, but their large-scale production is still in its infancy, hindered due to pathogenicity of natural producer, high substrate and purification costs and low yields and productivities. This study, for the first time, aimed at producing mono-rhamnolipids from sucrose by recombinant GRAS Saccharomyces cerevisiae strains. Six enzymes from P. aeruginosa involved in mono-rhamnolipid biosynthesis were functionally expressed in the yeast. Furthermore, its SUC2 invertase gene was disrupted and a sucrose phosphorylase gene from Pelomonas saccharophila was also expressed to reduce the pathway's overall energy requirement. Two strains were constructed aiming to produce mono-rhamnolipids and the pathway's intermediate dTDP-L-rhamnose. Production of both molecules was analyzed by confocal microscopy and mass spectrometry, respectively. These strains displayed, for the first time as a proof of concept, the potential of production of these molecules by a GRAS eukaryotic microorganism from an inexpensive substrate. These constructs show the potential to further improve rhamnolipids production in a yeast-based industrial bioprocess.

New Protocol Based on UHPLC-MS/MS for Quantitation of Metabolites in Xylose-Fermenting Yeasts.

Xylose fermentation is a bottleneck in second-generation ethanol production. As such, a comprehensive understanding of xylose metabolism in naturally xylose-fermenting yeasts is essential for prospection and construction of recombinant yeast strains. The objective of the current study was to establish a reliable metabolomics protocol for quantification of key metabolites of xylose catabolism pathways in yeast, and to apply this protocol to Spathaspora arborariae. Ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) was used to quantify metabolites, and afterwards, sample preparation was optimized to examine yeast intracellular metabolites. S. arborariae was cultivated using xylose as a carbon source under aerobic and oxygen-limited conditions. Ion pair chromatography (IPC) and hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) were shown to efficiently quantify 14 and 5 metabolites, respectively, in a more rapid chromatographic protocol than previously described. Thirteen and eleven metabolites were quantified in S. arborariae under aerobic and oxygen-limited conditions, respectively. This targeted metabolomics protocol is shown here to quantify a total of 19 metabolites, including sugars, phosphates, coenzymes, monosaccharides, and alcohols, from xylose catabolism pathways (glycolysis, pentose phosphate pathway, and tricarboxylic acid cycle) in yeast. Furthermore, to our knowledge, this is the first time that intracellular metabolites have been quantified in S. arborariae after xylose consumption. The results indicated that fine control of oxygen levels during fermentation is necessary to optimize ethanol production by S. arborariae. The protocol presented here may be applied to other yeast species and could support yeast genetic engineering to improve second generation ethanol production. Graphical Abstract ᅟ.