Brazilian Agricultural Research Corporation, Embrapa Agroenergy, W3 Norte, PqEB, Brasília, DF, 70770-901, Brazil.
Chemistry Institute, Federal University of Goiás, Campus Samambaia, Goiânia, GO, 74690-900, Brazil.
J Am Soc Mass Spectrom. 2017 Dec;28(12):2646-2657. doi: 10.1007/s13361-017-1786-9. Epub 2017 Sep 6.
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.
木糖发酵是第二代乙醇生产的瓶颈。因此，全面了解天然木糖发酵酵母中的木糖代谢对于展望和构建重组酵母菌株至关重要。本研究的目的是建立一种可靠的代谢组学方法，用于定量酵母中木糖分解代谢途径的关键代谢物，并将该方法应用于 Spathaspora arborariae。超高效液相色谱-串联质谱（UHPLC-MS/MS）用于定量代谢物，然后优化样品制备以检查酵母细胞内代谢物。S. arborariae 以木糖为碳源在有氧和缺氧条件下进行培养。离子对色谱（IPC）和亲水相互作用液相色谱-串联质谱（HILIC-MS/MS）分别被证明能够在比以前描述的更快速的色谱方案中有效地定量 14 和 5 种代谢物。在有氧和缺氧条件下，S. arborariae 分别定量了 13 和 11 种代谢物。该靶向代谢组学方法显示可定量总共 19 种代谢物，包括糖、磷酸盐、辅酶、单糖和醇，来自酵母的木糖分解代谢途径（糖酵解、戊糖磷酸途径和三羧酸循环）。此外，据我们所知，这是首次在 S. arborariae 消耗木糖后定量细胞内代谢物。结果表明，发酵过程中精细控制氧气水平对于优化 S. arborariae 的乙醇生产是必要的。这里提出的方案可应用于其他酵母物种，并可支持酵母遗传工程以提高第二代乙醇生产。
