University of Natural Resources and Life Sciences Vienna, Department of Chemistry, Insitute of Analytical Chemistry, Muthgasse 18, 1190, Vienna, Austria.
University of Natural Resources and Life Sciences, Department of Biotechnology, Institute of Microbiology and Microbial Biotechnology, Vienna, Muthgasse 18, 1190 Vienna, Austria.
Anal Bioanal Chem. 2023 Sep;415(21):5151-5163. doi: 10.1007/s00216-023-04786-5. Epub 2023 Jun 22.
Climate change directs the focus in biotechnology increasingly on one-carbon metabolism for fixation of CO and CO-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 C-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 C-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.
气候变化使得生物技术越来越关注一碳代谢，以固定 CO 和 CO 衍生的化学品（例如甲醇、甲酸盐），从而减少对化石和与食物竞争的碳源的依赖。四氢叶酸途径参与了几种一碳固定途径。为了研究这些途径，使用质谱进行的稳定同位素标记示踪剂分析是最先进的方法。然而，目前没有针对四氢叶酸维生素的这种方法。在本工作中，我们建立了一种适用于甲基营养酵母 Komagataella phaffii 的提取方法，该方法可以在数小时内获得细胞内的甲基和亚甲基四氢叶酸（THF），并且具有稳定性。为了确定甲基-THF 的同位素分布，LC-QTOFMS 提供了一个选择性的片段离子，在所有样品中，至少有两个同位素的强度合适，但对于亚甲基-THF 则不然。然而，加入离子淌度分离提供了关键的选择性改进，允许使用 LC-IM-TOFMS 对亚甲基-THF 进行准确的同位素分布分析。这些新方法在 C 示踪实验中的应用表明，在用甲酸盐标记 1 小时后，甲基-THF 中 M+0 碳同位素分馏从 83±4%降低到 64±5%，而用甲醇标记则降低到 54±5%。在用 C-甲醇标记时，亚甲基-THF 的 M+0 碳同位素分馏在相同时间内从 83±2%降低到 78±1%。对多个菌株的标记结果表明，THF 途径参与了耐氧还原甘氨酸途径，体内存在将甲酸盐还原为甲醛的过程，以及甲醛与 THF 在 K. phaffii 中的自发缩合反应的活性。
