Extended Data Fig. 7: Identification of the five additional genes necessary for biosynthesis and addition of the arabinofuranosylated C18 acyl chain of QS-21 (2).

a–c, Investigation of the contributions of the five candidate enzymes to addition of the acyl chain. Mass spectrometry traces (EICs) are shown. Full details can be found in Supplementary Figs. 22–28. a,Co-expression of the acyl transferases ACT2 and ACT3 separately and together in combination with the enzymes needed for the biosynthesis of QA-TriX-FRXX (8) and the C9 acyl unit (6) and the keto-reductases KR1 and KR2. A putative QA-TriX-FRXX-C9 product (9) was synthesised in the presence of ACT2 but not ACT3, while co-expression of ACT2 and ACT3 together yielded a putative QA-TriX-FRXX-C18 product (10). Thus ACT2 is likely to ligate the first C9 acyl unit to the triterpene scaffold, and ACT3 the second one. b, Co-expression of the keto-reductases KR1 and KR2 separately and together in combination with the enzymes needed for the biosynthesis of QA-TriX-FRXX (8) and the C9 acyl unit (6) and the acyl transferases ACT2 and ACT3. KR1 and KR2 individually both yield detectable but low levels of the putative QA-TriX-FRXX-C18 (10), while co-expression of the two reductases together yields substantially more of the presumed (10). c, Co-expression of UGT73CZ2 with KR1, KR2, ACT2 and ACT3 yields a product with a mass and fragmentation pattern consistent with QS-21 (2). LC-MS extracted chromatograms (EIC) in negative mode are shown. The screened masses of the [M-H]^- pseudomolecular ions are: QA-TriX-FRXX-C9 (9), m/z = 1683.7563–1683.7731; QA-TriX-FRXX-C18 (10), m/z = 1855.8653–1855.8839; QS-21 (d-xyl) (2), m/z = 1987.9070–1987.9268.