Fig. 3.

A unified pathway featuring dedicated ent-kaurene and ent-atiserene synthases that channel the common precursor ent-copalyl diphosphate to PTM and PTN biosynthesis in S. platensis MA7327 and MA7339. (A) The common ADHBA moiety from ASA and DHAP. (B) The most advanced common intermediate ent-copalyl diphosphate (ent-CPP) for the terpenoid moieties via the MEP pathway. (C) The ent-kaurene synthase PtmT3 and ent-atiserene synthases PtmT1 and PtnT1 catalyzed divergence of ent-CPP en route to PTM and PTN with coupling between the terpenoid and benzoate moieties as the last step. (D) Inactivation of selected genes in S. platensis MA7327 supporting the proposed pathway. HPLC chromatograms of I, MA7327 wild type; II, SB12006 (i.e., ΔptmB2 mutant); III, SB12006 fermented with supplementation of AHBA; IV, SB12007 (i.e., ΔptmT1 mutant); V, SB12008 (i.e., ΔptmT3 mutant). (E) Production of PTN by expressing the ptn cluster from MA7339 in S. lividans K4-114 and PTM by expressing the PTM cassette from MA7327 in S. platensis MA7339. Extracted ion (m/z at 442 for the [PTM + H]⁺ ion in blue and m/z at 426 for the [PTN + H]⁺ ion in red) chromatograms from liquid chromatography–mass spectrometry (LC-MS) analyses of I, PTN standard; II PTM standard; III, MA7339 wild type [the two peaks shown in blue are platencin A1 and A3, which have been characterized previously from MA7339 and have the same molecular weight as PTM (26)]; IV, SB12606 (i.e., S. lividans K4-114/pBS12619); V, SB12604 (i.e., MA7339/pBS12603). HPLC chromatograms of the same analyses with UV detection at 240 nm are provided in SI Appendix. PTM (♦); PTN (●); platensic acid (◊); platencinic acid (○).