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

Abstract

Archaea produce membrane lipids that typically possess fully saturated isoprenoid hydrocarbon chains attached to the glycerol moiety via ether bonds. They are functionally similar to, but structurally and biosynthetically distinct from, the fatty acid-based membrane lipids of bacteria and eukaryotes. It is believed that the characteristic lipid structure helps archaea survive under severe conditions such as extremely low or high pH, high salt concentrations, and/or high temperatures. We detail here the first successful production of an intact archaeal membrane lipid, which has fully saturated isoprenoid chains, in bacterial cells. The introduction of six phospholipid biosynthetic genes from a methanogenic archaeon, Methanosarcina acetivorans, in Escherichia coli enabled the host bacterium to synthesize the archaeal lipid, i.e., diphytanylglyceryl phosphoglycerol, while a glycerol modification of the phosphate group was probably catalyzed by endogenous E. coli enzymes. Reduction of the isoprenoid chains occurred only when archaeal ferredoxin was expressed with geranylgeranyl reductase, suggesting the role of ferredoxin as a specific electron donor for the reductase. This report is the first identification of a physiological reducer for archaeal geranylgeranyl reductase. On the other hand, geranylgeranyl reductase from the thermoacidophilic archaeon Sulfolobus acidocaldarius could, by itself, replace both its orthologue and ferredoxin from M. acetivorans, which indicated that an endogenous redox system of E. coli reduced the enzyme.

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

“…It was intriguing that the reductase was highly specific to the x-double bond of a geranylgeranyl group. This site-specific reduction was unprecedented and different from the reduction patterns of known archaeal GGRs that typically reduce a geranylgeranyl group to a phytanyl group [14][15][16][17] and of plant-type GGRs that convert it into a phytyl group [18]. The mechanisms responsible for these distinct patterns will be discussed later.…”

“…The almost complete conversion of DGGGP-glycerol in the cells bearing pBAD-ALB4-MA1492 suggested that sufficient reducing power was supplied for MA1492 from some endogenous redox system in E. coli. Although recombinant MA1492 can accept membrane lipid precursors, it should be emphasized that the enzyme probably plays a physiological role other than membrane lipid biosynthesis in the host cell because there exists another GGR (MA1484) that catalyzes the full saturation of prenyl chains in membrane lipids [17]. The most likely function of MA1492 is in the biosynthesis of glycosyl carrier lipids containing a saturated bond at the x-end, like the ones discovered in some archaeal species [10][11][12].…”

“…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. More recently, we succeeded in converting the parental DGGGOH lipids into diphytanylglycerol ones by adding a GGR gene (MA1484) together with its neighboring ferredoxin gene (MA1485) to pBAD-ALB4 [17]. Likewise, by introducing archaeal genes of unknown function into the cells harboring pBAD-ALB4 and examining the structural changes in the archaeal-type lipids that were produced, we initially attempted to search for genes involved in steps of archaeal membrane lipid biosynthesis that have not yet been identified.…”

“…geranylgeranyl reductase (GGR). Archaeal GGRs reduce all double bonds in the geranylgeranyl groups of substrate lipids to yield lipids with fully saturated phytanyl groups [14][15][16][17]. They are homologous with the plant GGRs that are responsible for the biosynthesis of chlorophyll, phylloquinone and tocopherol, which catalyze the partial reduction of a geranylgeranyl group to give a phytyl group [18].…”

Thermococcus kodakarensis : the key to affordable biohydrogen production

“…IPP synthase, GGPP synthase, GGGP synthase, etc. ), experimentally characterized genes, that have shown to be responsible for the reactions leading to isoprenoid and CDP-archaeol synthesis in various archaea, were BLASTed against the genome of T. kodakarensis [570][571][572][573][574][575][576][577]. T. kodakarensis genes that showed a high sequence similarity to these characterized genes were excluded from the list of 32 genes (Figure S5.1).…”