In archaea, the membrane phospholipids consist of isoprenoid hydrocarbon chains that are ether-linked to a sn-glycerol1-phosphate backbone. This unique structure is believed to be vital for the adaptation of these micro-organisms to extreme environments, but it also reflects an evolutionary marker that distinguishes archaea from bacteria and eukaryotes. CDP–archaeol is the central precursor for polar head group attachment. We examined various bacterial enzymes involved in the attachment of L-serine and glycerol as polar head groups for their promiscuity in recognizing CDP–archaeol as a substrate. Using a combination of mutated bacterial and archaeal enzymes, archaetidylethanolamine (AE) and archaetidylglycerol (AG) could be produced in vitro using nine purified enzymes while starting from simple building blocks. The ether lipid pathway constituted by a set of archaeal and bacterial enzymes was introduced into Escherichia coli, which resulted in the biosynthesis of AE and AG. This is a further step in the reprogramming of E. coli for ether lipid biosynthesis.