Climate change directs the focus in biotechnology increasingly on one-carbon metabolism for fixation of CO₂ and CO₂-derived chemicals (e.g. methanol, formate) to reduce our reliance on both fossil and food-competing carbon sources. The tetrahydrofolate pathway is involved in several one-carbon fixation pathways. To study such pathways, stable isotope-labelled tracer analysis performed with mass spectrometry is state of the art. However, no such method is currently available for tetrahydrofolate vitamers. In the present work, we established a fit-for-purpose extraction method for the methylotrophic yeast Komagataella phaffii that allows access to intracellular methyl- and methenyl-tetrahydrofolate (THF) with demonstrated stability over several hours. To determine isotopologue distributions of methyl-THF, LC-QTOFMS provides a selective fragment ion with suitable intensity of at least two isotopologues in all samples, but not for methenyl-THF. However, the addition of ion mobility separation provided a critical selectivity improvement allowing accurate isotopologue distribution analysis of methenyl-THF with LC-IM-TOFMS. Application of these new methods for ¹³C-tracer experiments revealed a decrease from 83 ± 4 to 64 ± 5% in the M + 0 carbon isotopologue fraction in methyl-THF after 1 h of labelling with formate, and to 54 ± 5% with methanol. The M + 0 carbon isotopologue fraction of methenyl-THF was reduced from 83 ± 2 to 78 ± 1% over the same time when using ¹³C-methanol labelling. The labelling results of multiple strains evidenced the involvement of the THF pathway in the oxygen-tolerant reductive glycine pathway, the presence of the in vivo reduction of formate to formaldehyde, and the activity of the spontaneous condensation reaction of formaldehyde with THF in K. phaffii.

气候变化使生物技术的焦点越来越集中在固定CO ₂和CO ₂衍生化学品（例如甲醇、甲酸盐）的一碳代谢上，以减少我们对化石和与食物竞争的碳源的依赖。四氢叶酸途径参与多种一碳固定途径。为了研究这些途径，用质谱法进行稳定同位素标记的示踪剂分析是最先进的。然而，目前还没有这样的方法可用于四氢叶酸维生素体。在目前的工作中，我们为甲基营养酵母Komagataella phaffii建立了一种适合目的的提取方法，该方法可以获取细胞内的甲基四氢叶酸和次甲基四氢叶酸 (THF)，并在几个小时内表现出稳定性。为了确定甲基-THF 的同位素体分布，LC-QTOFMS 在所有样品中提供具有合适强度的至少两个同位素体的选择性碎片离子，但不适用于次甲基-THF。然而，离子淌度分离的增加提供了关键的选择性改进，允许使用 LC-IM-TOFMS 对次甲基-THF 进行准确的同位素分布分析。应用这些新方法进行¹³ C 示踪剂实验表明，用甲酸盐标记 1 小时后，甲基-THF 中的 M + 0 碳同位素体分数从 83 ± 4 减少到 64 ± 5%，而用甲酸盐标记后，则减少到 54 ± 5%。甲醇。当使用¹³ C-甲醇标记时，次甲基-THF 的 M + 0 碳同位素异构体分数在同一时间内从 83 ± 2 减少到 78 ± 1%。 多个菌株的标记结果证明了THF途径参与了耐氧还原甘氨酸途径，在体内存在甲酸还原为甲醛的现象，以及K. phaffii中甲醛与THF自发缩合反应的活性。 。