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.antibiotic | metabolic pathway engineering | biosynthetic gene cluster | ent-copalyl diphosphate | terpene synthase I nfectious disease is the second leading cause of death worldwide, and the growing number of antibiotic-resistant microbes threatens to worsen this problem; only two previously undescribed classes of antibiotics have been introduced into the clinic since the 1960s (1, 2). Diabetes affects nearly 24 million people in the United States, and current therapies suffer from serious limitations (3). Platensimycin (PTM) and platencin (PTN) are recently discovered natural products (4) that are potent and selective inhibitors of bacterial (5, 6) and mammalian (7) fatty acid synthases. Remarkably, they have emerged as promising drug leads for both antibacterial (5,6,8,9) and antidiabetic (7) therapies. The efficacy of PTM and PTN in treating bacterial infections (5, 6), including those that are resistant to commercially available drugs, and the efficacy of PTM in treating diabetes and related metabolic disorders (7) have been demonstrated in mouse models.Structurally, PTM and PTN are composed of two distinct moieties-a substituted benzoic acid and an aliphatic cage moiety joined together by a flexible propionamide chain (Fig. 1A) (Fig. 1A) and diterpenoid natural products of both ent-kaurene and ent-atiserene origin are well known (SI Appendix, Fig. S1 C and D). Although numerous terpene synthase genes have been cloned from eukaryotes, only a few have been cloned from prokaryotes (17-19). The only ent-kaurene synthase of bacterial origin was reported in 2009 (20), and no gene or enzyme of eukaryotic or prokaryotic origin for ent-atiserene biosynthesis has ever been reported. Interestingly, ent-kaurene synthase-catalyzed biosynthesis of ent-kaurene from ent-copalyl diphosphate (ent-CPP) can produce ent-atiserene as a minor metabolite (21). Minor mutations to terpene synthases in general (22) and CPPutilizing terpene synthases in particular (21,23,24) are also known to alter product specificity. These observations, together with the fact that no ent-atiserene synthase is known, has become the basis of the current proposal th...

A genetically amenable platensimycin- and platencin-overproducer as a platform for biosynthetic explorations: a showcase of PtmO4, a long-chain acyl-CoA dehydrogenase

“…The idea that an acyl-CoA dehydrogenase may be necessary for both PTM and PTN biosynthesis was also confounding, as the PTN-only producer S. platensis MA7339 does not contain a ptmO4 homologue within its gene cluster. 7 Furthermore, heterologous expression of the ptn cluster in S. lividans successfully produced PTN, 15 suggesting all the necessary genes for PTN production are included within the ptn cluster.…”

“…The PTN-only producer S. platensis MA7339 and the heterologous PTN-production strains S. lividans SB12606 and SB12608 do not contain the PTM cassette, and therefore lack ptmO4 . 7, 15 In large-scale fermentation of SB12030, the overproducing Δ ptmO4 mutant, small amounts of PTM and PTN (<1 mg L −1 ) were isolated. In comparison, the Δ ptmR1 dual PTM-PTN overproducing strain SB12029 produces >300 and 150 mg L −1 , respectively.…”

“…8 Comparison of the ptm and ptn gene clusters revealed high conservation in both sequence and organization; however, a striking difference between them was the absence of a 5.4 kb cassette containing five open reading frames (ORFs) in the PTN gene cluster. 7 The five ORFs include ptmO3 , ptmO4 , ptmT3 , ptmO5 , and ptmR3 . These genes encode an α-ketoglutarate-dependent dioxygenase, a long-chain acyl-CoA dehydrogenase, an ent -kaurene synthase, a P450 monooxygenase, and a hypothetical regulatory kinase, respectively.…”

“…Furthermore, introduction of this “PTM” cassette into the PTN producer S. platensis MA7339 conferred the ability to produce both PTM and PTN and thus revealed that the PTM cassette encodes the genes necessary for PTM production in a sole PTN producer. 7 …”

Functional Conservation of the Capacity for ent-Kaurene Biosynthesis and an Associated Operon in Certain Rhizobia

“…In the organization of the rhizobial diterpenoid biosynthesis operons, it is notable that the genes predicted to be involved in oxidation are in the 5= region, with all those predicted to be involved in the formation of the cyclized olefin ent-kaurene falling in the 3= region. This includes the putative GGPP synthase (GGPS), as bacteria do not necessarily produce GGPP, leading to the presence of a GGPS in all of the identified bacterial diterpenoid biosynthetic gene clusters (21,(32)(33)(34)(35)(36)(37). The observed organization of the rhizobial diterpenoid biosynthetic operon suggests that the 3= and 5= regions might form nominally independent subclusters, although no such subclusters appear in the currently available sequence information.…”