Dedicated ent-kaurene and ent-atiserene synthases for platensimycin and platencin biosynthesis

Abstract

Platensimycin (PTM) and platencin (PTN) are potent and selective inhibitors of bacterial and mammalian fatty acid synthases and have emerged as promising drug leads for both antibacterial and antidiabetic therapies. Comparative analysis of the PTM and PTN biosynthetic machineries in Streptomyces platensis MA7327 and MA7339 revealed that the divergence of PTM and PTN biosynthesis is controlled by dedicated ent-kaurene and ent-atiserene synthases, the latter of which represents a new pathway for diterpenoid biosynthesis. The PTM and PTN biosynthetic machineries provide a rare glimpse at how secondary metabolic pathway evolution increases natural product structural diversity and support the wisdom of applying combinatorial biosynthesis methods for the generation of novel PTM and/or PTN analogues, thereby facilitating drug development efforts based on these privileged natural product scaffolds.

Fig. 1.

PTM and PTN and their proposed biosynthetic relationship to ent-kaurene and ent-atiserene. (A) Structures of PTM and PTN with the 3-amino-2,4-dihydroxybenzoic acid moiety common to PTM and PTN shown in red and with the ent-kaurene and ent-atiserene-derived diterpenoid moieties of PTM and PTN shown in blue. Wavy lines indicate bonds broken during biosynthesis en route to PTM and PTN. (B) Commonly accepted proposal for ent-kaurene synthase-catalyzed formation of ent-kaurene (path a) and ent-atiserene (paths b and c) from ent-copalyl diphosphate.

Fig. 2.

Genetic organization of the PTM and PTN biosynthetic gene clusters. (A) The ptm cluster encoding PTM and PTN dual production from S. platensis MA7327. (B) The ptn cluster encoding PTN production from S. platensis MA7339. Genes are color-coded to highlight their predicted functions in PTM and PTN biosynthesis, resistance, and regulation. The PTM cassette that endows the ptm cluster the capacity for dual PTM and PTN production is shaded in gray and is absent from the ptn cluster. The asterisks denote the location of the 1-kb repeats in the ptm gene cluster. Functional annotations of the genes within the ptm and ptn clusters are summarized in Table 1.

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 (○).

Fig. 4.

Primary sequence alignments of PtmT1 and PtmT3 with characterized terpene synthases and homologues of unknown functions. (A) PtmT3 contains the two conserved metal-binding active-site motifs, DDxxD/E (motif i) and NSE/DTE (motif ii), present in characterized type-I terpene synthases. KS, ent-kaurene synthase (Q39548); AS, aristolochene synthase (AAF13264); TS, trichodiene synthase (AAN05035); IS, epi-isozizaene synthase (Q9K499); PtmT3, ent-kaurene synthase (ACO31279). Given in parentheses are GenBank accession numbers. The numbers between the active site motifs indicate the number of amino acids. (B) PtmT1 contains two atypical putative active site motifs, deviated from the two canonical DDxxD (motif iii) motifs present in characterized prenyltransferases. FPPS, farnesyl pyrophosphate synthase (P08836); OPT, octoprenyltransverase (1V4E_B); HOPT, HBA oligoprenyltransferase (AEE59373); PtmT1, ent-atiserene synthase (ACO31274). (C) PtmT1 shows high sequence homology, including the two deviated putative active site motifs DxxxD (motif iii), to proteins of unknown function uncovered in genome sequencing projects. Sros_3708 (ACZ86631), annotated as a hypothetic protein in Streptosporangium roseum DSM43021. Svir_08340 (ACU95894), annotated a 4-hydroxybenzoate prenyltransferase-like prenyltransferase in Saccharomonospora viridis DSM 43017.