Isoprenoid glycerol dialkyl glycerol tetraethers (GDGTs), characteristic membrane lipids of archaea, are widely used in ecological and geochemical studies, especially for paleoenvironmental reconstruction. Glycerol monoalkyl glycerol tetraethers (GMGTs, also known as H-GDGTs), a unique variant of GDGTs, have covalent bonds linking the two alkyl chains. Despite some studies suggesting a link between GMGTs and high temperatures, the reliability and mechanisms remain unclear. Using molecular dynamics simulations, we elucidated the mechanism connecting GMGTs to high temperatures. Our findings show that H-bridging linkages reduce the distance between alkyl chains, leading to thicker and denser membranes with lower fluidity and permeability. The diffusion coefficient of GMGTs decreased by approximately 35 % compared to GDGTs, indicating their role as a archaeal high-temperature adaptation. This study provides a mechanistic basis for using archaeal GMGTs in geochemical studies and enhances confidence in their use for paleotemperature reconstruction.

Archaea produce unique membrane-spanning lipids (MSLs), termed glycerol dialkyl glycerol tetraethers (GDGTs), which aid in adaptive responses to various environmental challenges. GDGTs can be modified through cyclization, cross-linking, methylation, hydroxylation, and desaturation, resulting in structurally distinct GDGT lipids. Here, we report the identification of radical SAM proteins responsible for two of these modifications-a glycerol monoalkyl glycerol tetraether (GMGT) synthase (Gms), responsible for covalently cross-linking the two hydrocarbon tails of a GDGT to produce GMGTs, and a GMGT methylase (Gmm), capable of methylating the core hydrocarbon tail. Heterologous expression of Gms proteins from various archaea in Thermococcus kodakarensis results in the production of GMGTs in two isomeric forms. Further, coexpression of Gms and Gmm produces mono- and dimethylated GMGTs and minor amounts of trimethylated GMGTs with only trace GDGT methylation. Phylogenetic analyses reveal the presence of Gms homologs in diverse archaeal genomes spanning all four archaeal superphyla and in multiple bacterial phyla with the genetic potential to synthesize fatty acid-based MSLs, demonstrating that GMGT production may be more widespread than previously appreciated. We demonstrate GMGT production in three Gms-encoding archaea, identifying an increase in GMGTs in response to elevated temperature in two Archaeoglobus species and the production of GMGTs with up to six rings in Vulcanisaeta distributa. The occurrence of such highly cyclized GMGTs has been limited to environmental samples and their detection in culture demonstrates the utility of combining genetic, bioinformatic, and lipid analyses to identify producers of distinct archaeal membrane lipids.

The Black Sea is a permanently anoxic, marine basin serving as model system for the deposition of organic-rich sediments in a highly stratified ocean. In such systems, archaeal lipids are widely used as paleoceanographic and biogeochemical proxies; however, the diverse planktonic and benthic sources as well as their potentially distinct diagenetic fate may complicate their application. To track the flux of archaeal lipids and to constrain their sources and turnover, we quantitatively examined the distributions and stable carbon isotopic compositions (δ13 C) of intact polar lipids (IPLs) and core lipids (CLs) from the upper oxic water column into the underlying sediments, reaching deposits from the last glacial. The distribution of IPLs responded more sensitively to the geochemical zonation than the CLs, with the latter being governed by the deposition from the chemocline. The isotopic composition of archaeal lipids indicates CLs and IPLs in the deep anoxic water column have negligible influence on the sedimentary pool. Archaeol substitutes tetraether lipids as the most abundant IPL in the deep anoxic water column and the lacustrine methanic zone. Its elevated IPL/CL ratios and negative δ13 C values indicate active methane metabolism. Sedimentary CL- and IPL-crenarchaeol were exclusively derived from the water column, as indicated by non-variable δ13 C values that are identical to those in the chemocline and by the low BIT (branched isoprenoid tetraether index). By contrast, in situ production accounts on average for 22% of the sedimentary IPL-GDGT-0 (glycerol dibiphytanyl glycerol tetraether) based on isotopic mass balance using the fermentation product lactate as an endmember for the dissolved substrate pool. Despite the structural similarity, glycosidic crenarchaeol appears to be more recalcitrant in comparison to its non-cycloalkylated counterpart GDGT-0, as indicated by its consistently higher IPL/CL ratio in sediments. The higher TEX86 , CCaT, and GDGT-2/-3 values in glacial sediments could plausibly result from selective turnover of archaeal lipids and/or an archaeal ecology shift during the transition from the glacial lacustrine to the Holocene marine setting. Our in-depth molecular-isotopic examination of archaeal core and intact polar lipids provided new constraints on the sources and fate of archaeal lipids and their applicability in paleoceanographic and biogeochemical studies.

The sole unifying feature of the incredibly diverse Archaea is their isoprenoid-based ether-linked lipid membranes. Unique lipid membrane composition, including an abundance of membrane-spanning tetraether lipids, impart resistance to extreme conditions. Many questions remain, however, regarding the synthesis and modification of tetraether lipids and how dynamic changes to archaeal lipid membrane composition support hyperthermophily. Tetraether membranes, termed glycerol dibiphytanyl glycerol tetraethers (GDGTs), are generated by tetraether synthase (Tes) by joining the tails of two bilayer lipids known as archaeol. GDGTs are often further specialized through the addition of cyclopentane rings by GDGT ring synthase (Grs). A positive correlation between relative GDGT abundance and entry into stationary phase growth has been observed, but the physiological impact of inhibiting GDGT synthesis has not previously been reported. Here, we demonstrate that the model hyperthermophile Thermococcus kodakarensis remains viable when Tes (TK2145) or Grs (TK0167) are deleted, permitting phenotypic and lipid analyses at different temperatures. The absence of cyclopentane rings in GDGTs does not impact growth in T. kodakarensis, but an overabundance of rings due to ectopic Grs expression is highly fitness negative at supra-optimal temperatures. In contrast, deletion of Tes resulted in the loss of all GDGTs, cyclization of archaeol, and loss of viability upon transition to the stationary phase in this model archaea. These results demonstrate the critical roles of highly specialized, dynamic, isoprenoid-based lipid membranes for archaeal survival at high temperatures.

The asymmetric synthesis of polyunsaturated triene C18:3 n-3 and C18:3 n-6 methoxylated ether lipids (MEL) of the 1-O-alkyl-sn-glycerol type is described as possible structural candidates for a triene C18:3 MEL of an unknown identity found in a mixture of shark and dogfish liver oil. Their C18:3 hydrocarbon chains constitute an all-cis methylene skipped n-3 or n-6 triene framework, along with a methoxyl group at the 2\'-position and R-configuration of the resulting stereogenic center. The methoxylated polyenes are attached by an ether linkage to the pro-S hydroxymethyl group of the glycerol backbone. The syntheses were based on the polyacetylene approach that involves a semi-hydrogenation of the resulting triynes. Both syntheses were started from our previously described enantio- and diastereomerically pure isopropylidene-protected glyceryl glycidyl ether, a double-C3 building block that was designed as a head group synthon for the synthesis of various types of MELs.

Highly pure 2,3-dioleoyl-1-O-alkyl glyceryl ether (DOGE), whose 1-position is a lipase-tolerant ether bond, was chemically synthesized and its detailed regioselectivity and acyl transfer were confirmed. During ethanolysis using immobilized Candida antarctica lipase B (CAL-B) with DOGE as the substrate, monooleoyl-1-O-alkyl glyceryl ethers (MOGEs) and a few 1-alkyl glyceryl ethers were formed upon consumption of the substrate. The structure of MOGE was confirmed using nuclear magnetic resonance spectroscopy and only the isomer of 2-MOGE was formed, indicating that CAL-B has complete α- regiospecificity. During ethanolysis, 3-MOGE was formed via acyl migration. These results indicate that the formation of 1-alkyl glyceryl ethers is not due to the imperfect regiospecificity of CAL-B, but rather due to ethanolysis of the formed 3-MOGE. The ethanolysis rate at the 3-α-position of DOGE was faster and the rate of acyl transfer was slightly slower for chain lengths greater than 14. These results show for the first time that both deacylation at the 3-position and acyl migration from the 2- to 3-position are affected by the structure of 1-position.

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A new monoalkyl glycerol ether, 3-(n-henicosyloxy)propane-1,2-diol (1), was isolated from the CH2 Cl2 /MeOH crude extract of the Red Sea soft coral Nephthea mollis. Additionally, three known related analogs were identified: chimyl alcohol (2), batyl alcohol (3), and 3-(icosyloxy)propane-1,2-diol (4). The chemical structure of 3-(n-henicosyloxy)propane-1,2-diol was determined using advanced spectroscopic analyses, including 1D, 2D Nuclear Magnetic Resonance (NMR), Electron Ionization mass spectra (EI-MS), and High-Resolution Electron Spray Ionization mass spectra (HR-ESI-MS) analyses. Furthermore, the identification of chimyl alcohol, batyl alcohol and 3-(icosyloxy)propane-1,2-diol was achieved by studying their EI mass fragmentation analyses and comparing their mass data with those previously reported in the literature. The cytotoxic activity of the Nephthea mollis crude extract and 3-(n-henicosyloxy)propane-1,2-diol was evaluated against five human cancer cell lines: HepG2 (hepatocellular carcinoma), MCF-7 (breast carcinoma), NCI-1299 (lung carcinoma), HeLa (cervical cancer cell), and HT-29 (colon adenocarcinoma). Moreover, 3-(n-henicosyloxy)propane-1,2-diol revealed moderate cytotoxicity against the HeLa cell lines with an IC50 value of 24.1 μM, while showing inactivity against the remaining cell lines (IC50 >100 μM).

In this paper we discuss the effect of alkyl glycerol ethers (AGs) from the squid Berryteuthis magister on a chronic stress model in rats. The study was performed on 32 male Wistar rats. Animals received AGs at a dose of 200 mg/kg through a gavage for six weeks (1.5 months), and were divided into four groups: group 1 (control), group 2 (animals received AGs), group 3 (stress control), group 4 (animals received AGs and were subjected to stress). Chronic immobilization stress was induced by placing each rat into an individual plexiglass cages for 2 h daily for 15 days. The serum lipid spectrum was evaluated by the content of total cholesterol, triglycerides, high-density lipoprotein cholesterol, low lipoprotein cholesterol and very low-density lipoprotein cholesterol. The atherogenic coefficient was calculated. The hematological parameters of peripheral blood were evaluated. The neutrophil-lymphocyte ratio was counted. The levels of cortisol and testosterone in blood plasma were determined. AGs at the selected dose did not have a significant effect on the body weight of rats in the preliminary period of the experiment. Under stress, the body weight gain, the concentrations of very low-density lipoprotein cholesterol and blood triglycerides decreased significantly. The neutrophil-lymphocyte ratio in animals treated with AGs shifted towards lymphocytes. A favorable increase in the percentage of lymphocytes was found in the stressed group of animals treated with AGs. So, for the first time, it was found that AGs prevent stress-induced suppression of the immune system. This confirms the benefit of AGs for the immune system under chronic stress. Our results prove the efficiency of the use of AGs for treating chronic stress, a serious social problem in modern society.

Many Archaea produce membrane-spanning lipids that enable life in extreme environments. These isoprenoid glycerol dibiphytanyl glycerol tetraethers (GDGTs) may contain up to eight cyclopentyl and one cyclohexyl ring, where higher degrees of cyclization are associated with more acidic, hotter or energy-limited conditions. Recently, the genes encoding GDGT ring synthases, grsAB, were identified in two Sulfolobaceae; however, the distribution and abundance of grs homologs across environments inhabited by these and related organisms remain a mystery. To address this, we examined the distribution of grs homologs in relation to environmental temperature and pH, from thermal springs across Earth, where sequences derive from metagenomes, metatranscriptomes, single-cell and cultivar genomes. The abundance of grs homologs shows a strong negative correlation to pH, but a weak positive correlation to temperature. Archaeal genomes and metagenome-assembled genomes (MAGs) that carry two or more grs copies are more abundant in low pH springs. We also find grs in 12 archaeal classes, with the most representatives in Thermoproteia, followed by MAGs of the uncultured Korarchaeia, Bathyarchaeia and Hadarchaeia, while several Nitrososphaeria encodes >3 copies. Our findings highlight the key role of grs-catalysed lipid cyclization in archaeal diversification across hot and acidic environments.