Abstract
A part of the biosynthetic pathway of archaeal membrane lipids, comprised of 4 archaeal enzymes, was reconstructed in the cells of Escherichia coli. The genes of the enzymes were cloned from a mesophilic methanogen, Methanosarcina acetivorans, and the activity of each enzyme was confirmed using recombinant proteins. In vitro radioassay showed that the 4 enzymes are sufficient to synthesize an intermediate of archaeal membrane lipid biosynthesis, that is, 2,3-di-O-geranylgeranyl-sn-glycerol-1-phosphate, from precursors that can be produced endogenously in E. coli. Introduction of the 4 genes into E. coli resulted in the production of archaeal-type lipids. Detailed liquid chromatography/electron spray ionization-mass spectrometry analyses showed that they are metabolites from the expected intermediate, that is, 2,3-di-O-geranylgeranyl-sn-glycerol and 2,3-di-O-geranylgeranyl-sn-glycerol-1-phosphoglycerol. The metabolic processes, that is, dephosphorylation and glycerol modification, are likely catalyzed by endogenous enzymes of E. coli.
Highlights
Archaeal membrane lipids are very specific to the organisms in the domain Archaea and have structures that are distinct from those of bacterial/eukaryotic lipids [1,2,3]
Analogues of glycerolipids from bacteria or eukaryotes, they have specific structural features as follows: (1) hydrocarbon chains of archaeal lipids are multiply-branched isoprenoids typically derived from geranylgeranyl diphosphate (GGPP), while linear acyl groups are general in bacterial/eukaryotic lipids; (2) the isoprenoid chains are linked with the glycerol moiety with ether bonds, while ester bonds are general in bacterial/eukaryotic lipids; (3) the glycerol moiety of archaeal lipids is derived from sn-glycerol-1-phosphate (G-1P), which is the enantiomer of sn-glycerol-3-phosphate, the precursor for bacterial/eukaryotic glycerolipids; (4) dimerization of membrane lipids by the formation of carboncarbon bonds between the ω-terminals of hydrocarbon chains, which generates macrocyclic structures such as caldarchaeol-type lipids with a typically 72-membered ring, is often observed in thermophilic and methanogenic archaea
We evaluated the total amount and intact structures of the archaeal-type lipids extracted from the cells by liquid chromatography/electron spray ionization-mass spectrometry (LC/ESI-MS) analysis and showed that DGGGP was metabolized by enzymes endogenous to E. coli
Summary
Archaeal membrane lipids are very specific to the organisms in the domain Archaea and have structures that are distinct from those of bacterial/eukaryotic lipids [1,2,3]. The authors demonstrated the synthesis of precursors for archaeal membrane lipids, that is, 3-O-geranylgeranyl-snglycerol-1-phosphate (GGGP) and 2,3-di-O-geranylgeranylsn-glycerol-1-phosphate (DGGGP), in the recombinant E. coli, based on the detection of corresponding alcohols from the lipid extract from the cells after phosphatase treatment.
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