Production and Analysis of Perdeuterated Lipids from Pichia pastoris Cells

Abstract

Probing molecules using perdeuteration (i.e deuteration in which all hydrogen atoms are replaced by deuterium) is extremely useful in a wide range of biophysical techniques. In the case of lipids, the synthesis of the biologically relevant unsaturated perdeuterated lipids is challenging and not usually pursued. In this work, perdeuterated phospholipids and sterols from the yeast Pichia pastoris grown in deuterated medium are extracted and analyzed as derivatives by gas chromatography and mass spectrometry respectively. When yeast cells are grown in a deuterated environment, the phospholipid homeostasis is maintained but the fatty acid unsaturation level is modified while the ergosterol synthesis is not affected by the deuterated culture medium. Our results confirm that the production of well defined natural unsaturated perdeuterated lipids is possible and gives also new insights about the process of desaturase enzymes.

Multi-lamellar organization of fully deuterated lipid extracts of yeast membranes

“…Since for a given headgroup type the combination of fatty-acid chains can vary, and since the individual fatty-acid species were extracted after detaching them from the headgroup, the determination of the constituents as an entire molecule in terms of headgroup and chain pairs and triplets remained unknown. It is nevertheless clear that there were differences in fatty-acid composition between hydrogenous and deuterated extracts (de Ghellinck, 2013;de Ghellinck et al, 2014). These differences have been attributed to metabolic effects related to the use of perdeuterated growth media, and give rise to different bilayer structures for the hydrogenous and deuterated extracts (de Ghellinck et al, 2014) and to a different organization of the multi-lamellar stacks, as described in the present work.…”

“…It is nevertheless clear that there were differences in fatty-acid composition between hydrogenous and deuterated extracts (de Ghellinck, 2013;de Ghellinck et al, 2014). These differences have been attributed to metabolic effects related to the use of perdeuterated growth media, and give rise to different bilayer structures for the hydrogenous and deuterated extracts (de Ghellinck et al, 2014) and to a different organization of the multi-lamellar stacks, as described in the present work.…”

“…It was recently shown that the growth mechanism of P. pastoris cells is affected by the use of deuterated media instead of hydrogenous media (de Ghellinck et al, 2014). It was hypothesized that the action of fattyacid desaturase was partially inhibited if deuterated media are used.…”

Mobility of a Mononucleotide within a Lipid Matrix: A Neutron Scattering Study

“…X-ray diffraction results [12] confirmed that the AMP molecules are highly entangled, with the phosphate and ribose groups in close proximity to each other, a structure that may facilitate polymerization of the nucleotides into RNA-like polymers. We extended and compared polymerization of mononucleotides within a multilamellar lipid matrix using neutron scattering so as to investigate 5′-adenosine monophosphate (AMP) molecules captured in a multilamellar phospholipid matrix composed of a mixture of phospholipids extracted from Pichia pastoris cells [13]; this could help to understand the formation of RNA-like polymers.…”

“…We used neutron scattering to investigate AMP molecules captured in a multilamellar phospholipid matrix composed of a mixture of phospholipids extracted from Pichia pastoris cells [13] to study the formation of RNA-like polymers. Elastic incoherent neutron scattering experiments (EINS) were employed to investigate the changes of dynamical properties of AMP induced by embedding the molecules within the lipid matrix.…”