Available online at www.sciencedirect.com Bacterial diterpene synthases: new opportunities for mechanistic enzymology and engineered biosynthesis 1 2,3 3 Michael J Smanski , Ryan M Peterson , Sheng-Xiong Huang and 1,2,3,4,5 Ben Shen Diterpenoid biosynthesis has been extensively studied in plants potential for terpenoids in bacteria, particularly in the and fungi, yet cloning and engineering diterpenoid pathways in actinomycetes, may be significantly underestimated [1,2]. these organisms remain challenging. Bacteria are emerging as prolific producers of diterpenoid natural products, and bacterial Diterpenoid biosynthesis has been extensively studied diterpene synthases are poised to make significant in plants and fungi [3–6], yet cloning the respective contributions to our understanding of terpenoid biosynthesis. genes and characterizing and engineering diterpenoid Here we will first survey diterpenoid natural products of pathways in these higher organisms remain challenging bacterial origin and briefly review their biosynthesis with [7,8]. Scattering of the biosynthetic genes on the geno- emphasis on diterpene synthases (DTSs) that channel mic DNA of these higher organisms substantially geranylgeranyl diphosphate to various diterpenoid scaffolds. increases the effort to clone all the genes encoding We will then highlight differences of DTSs of bacterial and the complete biosynthetic machinery for a given diter- higher organism origins and discuss the challenges in penoid natural product. By contrast, genes encoding discovering novel bacterial DTSs. We will conclude by secondary metabolite biosynthesis in actinomycetes are discussing new opportunities for DTS mechanistic enzymology nearly always arranged on the bacterial chromosome as and applications of bacterial DTS in biocatalysis and metabolic a cluster. Recent characterization of terpene synthases pathway engineering. (TSs) from several actinomycete species demonstrated Addresses that these enzymes are not membrane-bound and can 1 Microbiology Doctoral Training Program, University of Wisconsin- be overproduced with relative ease as soluble, func- Madison, Madison, WI 53705, USA tional recombinant proteins in heterologous hosts such 2 Division of Pharmaceutical Sciences, University of Wisconsin-Madison, as Escherichia coli [9]. Diterpenoid biosynthesis in bac- Madison, WI 53705, USA 3 teria therefore may provide new opportunities to Department of Chemistry, The Scripps Research Institute, Jupiter, FL 33458, USA characterize these enzymes and to engineer their bio- 4 Department of Molecular Therapeutics, The Scripps Research Institute, synthetic machinery for diterpenoid natural product Jupiter, FL 33458, USA 5 structural diversity. Natural Products Library Initiative at The Scripps Research Institute, The Scripps Research Institute, Jupiter, FL 33458, USA Diterpenoids are all derived from (E,E,E)-geranylgeranyl Corresponding author: Shen, Ben ([email protected]) diphosphate (GGDP). Diterpene synthases (DTSs), also known as diterpene cyclases, catalyze the critical step in diterpenoid biosynthesis by morphing GGDP into one of Current Opinion in Chemical Biology 2012, 16:132–141 the many diterpenoid scaffolds, further transformations of This review comes from a themed issue on which by the downstream enzymes afford the enormous Biocatalysis and Biotransformation structural diversity known for diterpenoid natural pro- Edited by Jon S Thorson and Ben Shen ducts. TSs in general, DTSs included, can display incred- ible fidelity, catalyzing multi-step cyclization reactions Available online 22nd March 2012 with exquisite regiochemical and stereochemical control 1367-5931/$ – see front matter [10] or display marked product promiscuity, with a single # 2012 Elsevier Ltd. All rights reserved. enzyme generating over 50 unique products from a single substrate [11 ]. It is the fidelity and promiscuity in this DOI 10.1016/j.cbpa.2012.03.002 chemistry that has inspired a great interest in exploiting TSs for engineered biosynthesis of novel terpenoid natural products [7,8,12 ]. Introduction Terpenoids comprise the largest, structurally most Here we will first survey diterpenoid natural products of diverse family of natural products and play important bacterial origin and briefly review their biosynthesis with roles in all living organisms. Among the 60 000 mem- emphasis on DTSs that channel GGDP to various diter- bers known to date, 12 000 are diterpenoids, most of penoid scaffolds. We will then highlight differences of which are produced in plants and fungi. Diterpenoids of DTSs of bacterial and higher organism origins and discuss bacterial origin are known but rare, however recent the challenges in discovering novel bacterial DTSs. We advances in genomics have revealed that the biosynthetic will conclude by discussing new opportunities for DTS Current Opinion in Chemical Biology 2012, 16:132–141 www.sciencedirect.com Bacterial diterpene synthases Smanski et al. 133 mechanistic enzymology and applications of bacterial isotuberculosinols from Mycobacterium tuberculosis H37Rv DTS in biocatalysis and metabolic pathway engineering. [25,26 ,27–29], platensimycin from Streptomyces platensis MA7327 [30 ,31,32], platencin from Streptomyces platensis MA7339 [33–35], the neoverrucosanes from Saprospira Bacterial diterpenoids grandis [36,37], cyslabdan from Streptomyces sp. K04-0144 The discovery of gibberellins (GAs) from Rhizobium [38], the gifhornenolones from Verrucosispora gifhornesis phaseoli in 1988, originally only known to fungi and plants, YM28-088 [39], and JBIR-65 from Actinomadura sp. may represent the first report of bacterial diterpenoids SpB081030SC-15 [40]. The actinomycetes have emerged [13,14]. It was followed by the discovery of verrucosan- as prolific producers of bacterial diterpenoids [1,2]. Bac- 2b-ol from Chloroflexus aurantiacus in 1993 [15] and iso- terial producers of paclitaxel have also been reported, agathenediol from Rhodospirillum rubrum in 1995 [16]. many of which were actinomycetes, however definitive Since then, the list of bacterial diterpenoids has grown evidence supporting their paclitaxel production remains steadily, and Figure 1 summarizes the bacterial diterpe- elusive [41]. noids known to date. These include terpentecin from Streptomyces griseolosporeus MF730-N6 [17,18 ,19], the phenalinolactones from Streptomyces sp. Tu6071 [20], Bacterial DTSs the brasilicardins from Nocardia brasiliensis IFM 0406 DTS classification follows other TSs. Type I TSs initiate [21], viguiepinol and the oxaloterpins from Streptomyces a cyclization reaction via a heterolytic cleavage of the sp. KO-3988 [22,23], cyclooctatin from Streptomyces mel- polyprenyl diphosphate, while type II TSs initiate the anosporofaciens MI614-43F2 [24], tuberculosinol and the cyclization reaction via protonation of a double bond or an Figure 1 O (a) OH OH O O O O O H OH O H OH OCH3 O O O OH O HO O O CO H H H HO O 2 HO HO O H H CO H NH NH 2 O O HO 2 N O HO H OH H Gibberellin A3 O O OCH3 O Brasilicardin A Terpentecin Phenalinolactone A OH OH 13 H OH OH O HO H H H N H O H HO H H O O HO Viguiepinol Oxaloterpin A Cyclooctatin Tuberculosinol Isotuberculosinol (13R or 13S) OH OH O O O O HO C N HO2C N 2 H OH H OH O Platensimycin Platencin (b) O OH NH OH OH O O H OH H CO2H OH H H H H H S HO O H H H HO OH OH HO H HO Verrucosan-2β-ol Isoagathenediol Neoverrucosane Cyslabdan Gifhornenolone A JBIR-65 Current Opinion in Chemical Biology Bacterial diterpenoid natural products with their diterpenoid carbon scaffolds highlighted in red: (a) the biosynthetic gene clusters for these natural products have been cloned and partially characterized and (b) biosynthesis for these natural products has not been studied. www.sciencedirect.com Current Opinion in Chemical Biology 2012, 16:132–141 134 Biocatalysis and Biotransformation epoxide ring. In both cases, the resulting carbocation Bacterial type I DTSs undergoes a cascade of cyclization, the fate of which is The staggering sequence diversity present in bacterial type determined by a combination of steric and electrostatic I DTSs hinders sequence-gazing efforts but heightens our forces within the active site cavity. The cyclization understanding of the minimal requirements for catalysis. cascade is ultimately terminated by abstraction of a Terpentetriene synthase (Cyc2) from S. griseolosporeus proton or electrophilic attack by water (Figure 2a) MF730-N6, the first bacterial type I DTS reported, was [4,5]. Because type II TSs leave the diphosphate group readily identified on the basis of its sequence homology to intact, their products can serve as substrates for further known bacterial type I TSs, and presence of the charac- cyclization by type I TSs. The high frequency with which teristic DDXXD and NSE/DTE motifs [18 ,19]. The such two-step cyclizations are employed differentiates other bacterial type I DTSs characterized since – including diterpenoid biosynthesis from that of smaller terpenoids, the tuberculosinol/isotuberculosinol synthase (Rv3378c) which rarely implement a type II mechanism. from M. tuberculosis [26 ,27,29], the cyclooctatenol synthase (CotB2) from S. melanosporofaciens [24], the pimaradiene Terpentedienyl diphosphate synthase and terpentetriene synthase (ORF3) from S. sp. KO-3988 [22], and two ent- synthase were the first two characterized bacterial DTSs, kaurene synthases (BjKS and PtmT3) from B. japonicum reported in 2001 for terpentecin biosynthesis from S. [42] and S. platensis MA7327 [43 ], respectively – however, griseolosporeus MF730-N6 [18 ,19].