Tailored extraction and ion mobility-mass spectrometry enables isotopologue analysis of tetrahydrofolate vitamers

Abstract

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.

Single carbon metabolism – A new paradigm for microbial bioprocesses?

“…by M. extorquens [ 40 ] and other proteobacteria [ 41 ] while the reverse glycine cleavage system has been observed in nature only rarely, e.g. in Desulfovibrio desulfuricans [ 42 ] or Clostridium drakei [ 43 ] and recently also in the methylotrophic yeast K. phaffii [ 24 , 44 ]. Formatotrophic bacteria use formate not only as a carbon source, but gain their energy from oxidation of formate to CO 2 by NAD + dependent formate dehydrogenases, providing NADH as an electron donor for metabolic reactions and for the respiratory chain to produce ATP.…”

A native phosphoglycolate salvage pathway of the synthetic autotrophic yeastKomagataella phaffii

“…These data allow to conclude that methylene-THF, which forms serine from glycine, probably originates from non-labeled precursors. One possible precursor is methanol, which also partly enters the tetrahydrofolate pathway (Mitic et al, 2023(Mitic et al, , 2022. Later in the cultivation the fraction of M2 of the BB serine fragment increases indicating that labeled glycine is used for methylene-THF synthesis.…”