Fig. 2: Biochemical verification of Ca. Cloacimonetes GGGP and DGGGP synthase activities.

a Enzyme assay of purified GGGP synthase with G1P (a1–a4) or G3P (a5 and a6). Extracted ion chromatogram, within 3 ppm mass accuracy, of [GGGP + NH4]+ (m/z 462.298) of complete enzymatic assay (a1 and a5) or control assays lacking enzyme (a2 and a6), glycerol phosphate (a3) or geranylgeranyl-diphosphate, GGPP (a4). b Production of the lipid PG-archaeol with eight double bonds or unsaturations (PG-unsat(8)-archaeol) in E. coli. Extracted ion chromatogram (±0.5, mass units, mu) of [PG-unsat(8)-archaeol+H]+ (m/z 808); (blue filled area, left axis) or base peak chromatogram (dotted line, right axis) of optimized E. coli containing empty-vector (b1) or vector encoding GGGP synthase (b2), DGGGP synthase (b3), or both GGGP and DGGGP synthases (b4). Orange boxes spans the retention time of PG-unsat(8)-archaeol.