Construction of an artificial biosynthetic pathway for hyperextended archaeal membrane lipids in the bacterium Escherichia coli

Archaea produce unique membrane lipids, which possess two fully saturated isoprenoid chains linked to the glycerol moiety via ether bonds. The isoprenoid chain length of archaeal membrane lipids is believed to be important for some archaea to thrive in extreme environments because the hyperthermophilic archaeon Aeropyrum pernix and some halophilic archaea synthesize extended C25,C25-archaeal diether-type membrane lipids, which have isoprenoid chains that are longer than those of typical C20,C20-diether lipids. Natural archaeal diether lipids possessing longer C30 or C35 isoprenoid chains, however, have yet to be isolated. In the present study, we attempted to synthesize such hyperextended archaeal membrane lipids. We investigated the substrate preference of the enzyme sn-2,3-(digeranylfarnesyl)glycerol-1-phosphate synthase from A. pernix, which catalyzes the transfer of the second C25 isoprenoid chain to the glycerol moiety in the biosynthetic pathway of C25,C25-archaeal membrane lipids. The enzyme was shown to accept sn-3-hexaprenylglycerol-1-phosphate, which has a C30 isoprenoid chain, as a prenyl acceptor substrate to synthesize sn-2-geranylfarnesyl-3-hexaprenylglycerol-1-phosphate, a supposed precursor for hyperextended C25,C30-archaeal membrane lipids. Furthermore, we constructed an artificial biosynthetic pathway by introducing 4 archaeal genes and 1 gene from Bacillus subtilis in the cells of Escherichia coli, which enabled the E. coli strain to produce hyperextended C25,C30-archaeal membrane lipids, which have never been reported so far.

Metabolism of Glycerol, Glucose, and Lactate in the Citric Acid Cycle Prior to Incorporation into Hepatic Acylglycerols

During hepatic lipogenesis, the glycerol backbone of acylglycerols originates from one of three sources: glucose, glycerol, or substrates passing through the citric acid cycle via glyceroneogenesis. The relative contribution of each substrate source to glycerol in rat liver acylglycerols was determined using 13C-enriched substrates and NMR. Animals received a fixed mixture of glucose, glycerol, and lactate; one group received [U-13C6]glucose, another received [U-13C3]glycerol, and the third received [U-13C3]lactate. After 3 h, the livers were harvested to extract fats, and the glycerol moiety from hydrolyzed acylglycerols was analyzed by 13C NMR. In either fed or fasted animals, glucose and glycerol provided the majority of the glycerol backbone carbons, whereas the contribution of lactate was small. In fed animals, glucose contributed >50% of the total newly synthesized glycerol backbone, and 35% of this contribution occurred after glucose had passed through the citric acid cycle. By comparison, the glycerol contribution was ∼40%, and of this, 17% of the exogenous glycerol passed first through the cycle. In fasted animals, exogenous glycerol became the major contributor to acylglycerols. The contribution from exogenous lactate did increase in fasted animals, but its overall contribution remained small. The contributions of glucose and glycerol that had passed through the citric acid cycle first increased in fasted animals from 35 to 71% for glucose and from 17 to 24% for glycerol. Thus, a substantial fraction from both substrate sources passed through the cycle prior to incorporation into the glycerol moiety of acylglycerols in the liver.

An easy α-glycosylation methodology for the synthesis and stereochemistry of mycoplasma α-glycolipid antigens

Mycoplasma fermentans possesses unique α-glycolipid antigens (GGPL-I and GGPL-III) at the cytoplasm membrane, which carry a phosphocholine group at the sugar primary (6-OH) position. This paper describes a practical synthetic pathway to a GGPL-I homologue (C16:0) and its diastereomer, in which our one-pot α-glycosylation method was effectively applied. The synthetic GGPL-I isomers were characterized with 1H NMR spectroscopy to determine the equilibrium among the three conformers (gg, gt, tg) at the acyclic glycerol moiety. The natural GGPL-I isomer was found to prefer gt (54%) and gg (39%) conformers around the lipid tail, while adopting all of the three conformers with equal probability around the sugar position. This property was very close to what we have observed with respect to the conformation of phosphatidylcholine (DPPC), suggesting that the Mycoplasma glycolipids GGPLs may constitute the cytoplasm fluid membrane together with ubiquitous phospholipids, without inducing stereochemical stress.

Measurement of TG synthesis and turnover in vivo by 2H2O incorporation into the glycerol moiety and application of MIDA

A method is presented for measurement of triglyceride (TG) synthesis that can be applied to slow-turnover lipids. The glycerol moiety of TG is labeled from 2H2O, and mass isotopomer distribution analysis (MIDA) is applied. Mice and rats were given 4-8% 2H2O in drinking water; TG-glycerol was isolated from adipose and liver during ≤12-wk of 2H2O labeling. Mass isotopomer abundances in the glycerol moiety of TG were measured by GC-MS. The combinatorial pattern of isotopomers revealed the number of H atoms in glycerol incorporating label from 2H2O (n) to be 3.8–4.0 of a possible 5 for adipose tissue and 4.6–4.8 for liver TG. Hepatic TG-glycerol in fact reached 97% predicted maximal value of label incorporation (4.4–4.6 × body 2H2O enrichment), indicating near-complete replacement of the liver TG pool. Label incorporation into adipose tissue revealed turnover of mesenteric TG to be faster ( k = 0.21 day–1) than other depots ( k = 0.04–0.06 day–1) in mice. TG isolated from subcutaneous depots of growing adult rats plateaued at 85–90% of calculated maximal values at 12 wk ( k = 0.05 day–1), excluding significant dilution by unlabeled α-glycerol phosphate. Turnover of plasma TG, modeled from 2H incorporation over 60 min, was 0.06 min–1 (half-life 11.5 min). In summary, use of 2H2O labeling with MIDA of TG-glycerol allows measurement of new α-glycerol phosphate-derived TG synthesis and turnover. The hypothesis that mesenteric TG is more lipolytically active than other depots, previously difficult to prove by isotope dilution techniques, was confirmed by this label incorporation approach.