| The membrane lipids of archaea are believed to have evolved to increase membrane stability for survival at extreme conditions. Archaeal membrane lipids are characterized by ether-linked isoprenoid biochemistry, which typically is based on two core lipid structures, sn-2,3-diphytanylglycerol diether and sn-2,3-dibiphytanyldiglycerol tetraether. The biosynthetic pathway for tetraether lipid biosynthesis entails novel biochemistry involving unprecedented head-to-head coupling of isoprenoid intermediates. This study aimed to investigate isoprenoid ether lipid biosynthesis in the hyperthermophilic archaeon, Archaeoglobus fulgidus, with the overall goal of elucidating the tetraether lipid biosynthesis pathway and identifying the diether coupling enzyme.;To examine membrane lipid structure in A. fulgidus, lipid characterization primarily was achieved using thin-layer chromatography and mass spectrometry techniques. Core lipid analysis confirmed the presence of diether and tetraether structures and revealed that the degree of pentacyclization in tetraether lipid as well as the amount of tetraether lipid, relative to diether lipid, increased with growth temperature, thereby suggesting the existence and regulation of genes for tetraether lipid biosynthesis.;To demonstrate efficient isoprenoid ether lipid biosynthesis in E. coli, the lipid synthesis machinery of A. fulgidus was reconstructed in a metabolically engineered E. coli strain for the production of the intermediate, digeranylgeranylglyceryl phosphate (DGGGP) in vivo. The biosynthesis of DGGGP established roles for four A. fulgidus genes in the isoprenoid ether lipid pathway and was verified using a LC/MS/MS technique. This investigation provided a platform useful for identification of subsequent steps in tetraether lipid biosynthesis proceeding from DGGGP, which is the presumed substrate for the head-to-head diether coupling reaction yielding unsaturated caldarchaeol.;The search for the diether coupling enzyme and other genes for tetraether lipid biosynthesis in the A. fulgidus genome began with a combined bioinformatics and experimental approach. From analysis of gene homology and other bioinformatics information, approximately fifteen gene candidates for tetraether lipid biosynthesis were selected. These genes were screened experimentally by cloning into the DGGGP-producing E. coli strain and assaying for tetraether lipid biosynthetic product. However, alter acquiring preliminary data, the gene for the diether coupling enzyme has not yet been positively identified. |
