Archaeal Phospholipid Biosynthetic Pathway Reconstructed inEscherichia coli

Abstract

<jats:p>A part of the biosynthetic pathway of archaeal membrane lipids, comprised of 4 archaeal enzymes, was reconstructed in the cells of<jats:italic>Escherichia coli</jats:italic>. The genes of the enzymes were cloned from a mesophilic methanogen,<jats:italic>Methanosarcina acetivorans</jats:italic>, and the activity of each enzyme was confirmed using recombinant proteins.<jats:italic>In vitro</jats:italic>radioassay showed that the 4 enzymes are sufficient to synthesize an intermediate of archaeal membrane lipid biosynthesis, that is, 2,3-di-<jats:italic>O</jats:italic>-geranylgeranyl-<jats:italic>sn</jats:italic>-glycerol-1-phosphate, from precursors that can be produced endogenously in<jats:italic>E. coli</jats:italic>. Introduction of the 4 genes into<jats:italic>E. coli</jats:italic>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-<jats:italic>O</jats:italic>-geranylgeranyl-<jats:italic>sn</jats:italic>-glycerol and 2,3-di-<jats:italic>O</jats:italic>-geranylgeranyl-<jats:italic>sn</jats:italic>-glycerol-1-phosphoglycerol. The metabolic processes, that is, dephosphorylation and glycerol modification, are likely catalyzed by endogenous enzymes of<jats:italic>E. coli.</jats:italic></jats:p>

Geranylgeranyl Reductase and Ferredoxin from Methanosarcina acetivorans Are Required for the Synthesis of Fully Reduced Archaeal Membrane Lipid in Escherichia coli Cells

“…The chromosomal DNA of M. acetivorans was prepared as described in a previous study (4). The MA1485-MA1484 tandem genes encoding a ferredoxin-like protein and a GGR homologue, respectively, were amplified either simultaneously or separately with the primers shown in Table 1 and KOD DNA polymerase (Toyobo, Japan), using the genome of M. acetivorans as a template.…”

A novel geranylgeranyl reductase from the methanogenic archaeon Methanosarcina acetivorans displays unique regiospecificity

“…In our previous study, we reconstructed a portion of the biosynthetic pathway for archaeal membrane lipid in E. coli cells by developing the plasmid pBAD-ALB4 [26]. This plasmid contains four genes from M. acetivorans that are required for the early stage of membrane lipid biosynthesis, and E. coli transformed with the plasmid produced archaeal-type lipids such as digeranylgeranylglycerol (DGGGOH) and digeranylgeranylglyceryl phosphoglycerol (DGGGP-glycerol) as main products.…”

A phytoene desaturase homolog gene from the methanogenic archaeon Methanosarcina acetivorans is responsible for hydroxyarchaeol biosynthesis

“…An E. coli Top10 strain harboring pBAD-ALB4-ma0127 [13] or pBAD-ALB4 [14] was cultivated statically at 37 C for 48 h, in 1.5 L of LuriaeBertani (LB) medium containing 100 mg/ml ampicillin and 0.02%(w/v) L-arabinose. Lipid samples were extracted from the E. coli cells following a method described elsewhere [14].…”

“…LC-ESI-tandem-MS analyses of the lipid samples were performed with an Esquire 3000 ion trap system (Bruker Daltonics, USA) connected to an Agilent 1100 Series HPLC (Agilent Technologies, USA) as described elsewhere [14], with slight modifications as previously reported [13]: the mobile phase was replaced with methanol/2-propanol/100 mg L À1 sodium acetate (7:2:1), and the scanning range was changed to 50e2000 m/z.…”