Onocerin Biosynthesis Requires Two Highly Dedicated Triterpene Cyclases in a Fern Lycopodium clavatum

Onocerin is known for its unusual structure among triterpenoids, with a symmetrical structure that is formed by cyclizations at the both termini of dioxidosqualene. The nature of the enzyme catalyzing these unusual cyclizations has remained elusive for decades. Here, we report the cloning of genes responsible for these reactions; they exhibited unprecedented substrate specificities among oxidosqualene cyclase family members. Two genes, LCC and LCD, were identified from the fern Lycopodium clavatum. Expression in yeast revealed that both were required to produce α‐onocerin. LCC, the first dioxidosqualene cyclase, catalyzed the production of a novel intermediate pre‐α‐onocerin from only dioxidosqualene as a substrate; LCD catalyzed the second half of the cyclization, exclusively from pre‐α‐onocerin. These results demonstrated that these two most unusual oxidosqualene cyclases were involved in onocerin biosynthesis.     

Structural and Mechanistic Insight into Terpene Synthases that Catalyze the Irregular Non-Head-to-Tail Coupling of Prenyl Substrates

Terpenoids have diverse structures and thus represent an important class of biologically active natural products. The structural diversity of terpenoids originates from the coupling of prenyl diphosphate substrates, such as isopentenyl diphosphate and dimethylallyl diphosphate. These isoprenyl diphosphates undergo canonical and sequential “head-to-tail” coupling catalyzed by terpene synthases, followed by modifications such as cyclization, hydroxylation, and glycosylation. In recent years, several terpene synthases that catalyze irregular “non-head-to-tail” couplings to afford branched terpenoids have been identified. This minireview describes structural and mechanistic insights into these unusual coupling reactions that provide a new strategy for the structural diversification of natural products.     

Discovery of the Tiancilactone Antibiotics by Genome Mining of Atypical Bacterial Type II Diterpene Synthases

Although genome mining has advanced the identification, discovery, and study of microbial natural products, the discovery of bacterial diterpenoids continues to lag behind. Herein, we report the identification of 66 putative producers of novel bacterial diterpenoids, and the discovery of the tiancilactone (TNL) family of antibiotics, by genome mining of type II diterpene synthases that do not possess the canonical DXDD motif. The TNLs, which are broad‐spectrum antibiotics with moderate activities, are produced by both Streptomyces sp. CB03234 and Streptomyces sp. CB03238 and feature a highly functionalized diterpenoid skeleton that is further decorated with chloroanthranilate and γ‐butyrolactone moieties. Genetic manipulation of the tnl gene cluster resulted in TNL congeners, which provided insights into their biosynthesis and structure–activity relationships. This work highlights the biosynthetic potential that bacteria possess to produce diterpenoids and should inspire continued efforts to discover terpenoid natural products from bacteria.     

Molecular Evolution of Fern Squalene Cyclases

Triterpenes, a diverse group of natural products comprising six isoprene units, are distributed across various organisms from bacteria to higher plants. Ferns are sporophytes that produce triterpenes and are lower on the evolutionary scale than higher plants. Among ferns that produce triterpenes analogous to bacterial hopanoids, Polypodiodes niponica produces migrated dammaranes and oleananes, which are also widely found in higher plants. Because the study of terpene‐producing ferns could help us to understand the molecular basis of triterpene biosynthesis, cDNA cloning of squalene cyclases (SCs) from P. niponica was carried out. Two SCs (PNT and PNG) were obtained. The heterologously expressed PNT produces tirucalla‐7,21‐diene (67 % major), and PNG produces germanicene (69 %). Phylogenetic analysis revealed that PNT and PNG, which produce higher‐plant‐type migrated dammaranes and oleananes, are closely related to bacterial‐type SCs. Furthermore, analysis of the minor products indicated that fern SCs gained the ability to directly form dammarenyl cations, which are key intermediates in oleanane formation during molecular evolution.     

Ribosylhopane,a Novel Bacterial Hopanoid,as Precursor of C35 Bacteriohopanepolyols in Streptomyces coelicolor A3(2)

Wild‐type Streptomyces coelicolor A3(2) produces aminobacteriohopanetriol as the only elongated C₃₅ hopanoid. The hopanoid phenotype of two mutants bearing a deletion of genes from a previously identified hopanoid biosynthesis gene cluster provides clues to the formation of C₃₅ bacteriohopanepolyols. orf14 encodes a putative nucleosidase; its deletion induces the accumulation of adenosylhopane as it cannot be converted into ribosylhopane. orf18 encodes a putative transaminase; its deletion results in the accumulation of adenosylhopane, ribosylhopane, and bacteriohopanetetrol. Ribosylhopane was postulated twenty years ago as a precursor for bacterial hopanoids but was never identified in a bacterium. Absence of the transaminase encoded by orf18 prevents the reductive amination of ribosylhopane into aminobacteriohopanetriol and induces its accumulation. Its reduction by an aldose‐reductase‐like enzyme produces bacteriohopanetetrol, which is normally not present in S. coelicolor.     

A Case of Convergent Evolution: The Bacterial Sesquiterpene Synthase for 1-epi-Cubenol from Nonomuraea coxensis

A terpene synthase from Nonomuraea coxensis was identified as (+)-1-epi-cubenol synthase. The enzyme is phylogenetically unrelated to the known enzyme of the same function that is widespread in streptomycetes. Isotopic labelling experiments were performed to unambiguously assign the NMR data and to investigate hydrogen migrations during terpene cyclisations. Epoxidations of (+)-1-epi-cubenol and of the plant derived compounds (−)-cubenol and (−)-1-epi-cubenol confirmed the structure of a natural product isolated from the brown alga Dictyopteris divaricata and allowed to conclude on its absolute configuration. The crystal structures of the epoxides from (+)- and (−)-1-epi-cubenol and the acid catalysed conversion into an isomeric ketone are reported.     

Biosynthetic Machinery of Diterpene Pleuromutilin Isolated from Basidiomycete Fungi

The diterpene pleuromutilin is a ribosome‐targeting antibiotic isolated from basidiomycete fungi, such as Clitopilus pseudo‐pinsitus. The functional characterization of all biosynthetic enzymes involved in pleuromutilin biosynthesis is reported and a biosynthetic pathway proposed. In vitro enzymatic reactions and mutational analysis revealed that a labdane‐related diterpene synthase, Ple3, catalyzed two rounds of cyclization from geranylgeranyl diphosphate to premutilin possessing a characteristic 5–6–8‐tricyclic carbon skeleton. Biotransformation experiments utilizing Aspergillus oryzae transformants possessing modification enzyme genes allowed the biosynthetic pathway from premutilin to pleuromutilin to be proposed. The present study sets the stage for the enzymatic synthesis of natural products isolated from basidiomycete fungi, which are a prolific source of structurally diverse and biologically active terpenoids.     

Characterization of the Rv3377c Gene Product,a Type‐B Diterpene Cyclase,from the Mycobacterium tuberculosis H37 Genome

The Rv3377c gene from the Mycobacterium tuberculosis H37 genome is specifically limited to those Mycobacterium species that cause tuberculosis. We have demonstrated that the gene product of Rv3377c is a diterpene cyclase that catalyzes the formation of tuberculosinol from geranylgeranyl diphosphate (GGPP). However, the characteristics of this enzyme had not previously been studied in detail with homogeneously purified enzyme. The purified enzyme catalyzed the synthesis of tuberculosinyl diphosphate from GGPP, but it did not bring about the synthesis of tuberculosinol. Optimal conditions for the highest activity were found to be as follows: pH 7.5, 30 °C, Mg^II (0.1 mM ), and Triton X‐100 (0.1 %). Under these conditions, the kinetic values of K_M and k_cat were determined to be 11.7±1.9 μM for GGPP and 12.7±0.7 min^?1, respectively, whereas the specific activity was 186 nmol min^?1 mg^?1. The enzyme activity was inhibited at substrate concentrations higher than 50 μM . The catalytic activity was strongly inhibited by 15‐aza‐dihydrogeranylgeraniol and 5‐isopropyl‐N,N,N,2‐tetramethyl‐4‐(piperidine‐1‐carbonyloxy)benzenaminium chloride (Amo‐1618). The DXDTT_293–297 motif, corresponding to the DXDDTA motif conserved among terpene cyclases, was mutated in order to investigate its function. The middle D295 was found to be the most crucial entity for the catalysis. D293 and two threonine residues function synergistically to enhance the acidity of D295, possibly through hydrogen‐bonding networks. The Rv3377c enzyme could also react with (14R/S)‐14,15‐oxidoGGPP to generate 3α‐ and 3β‐hydroxytuberculosinyl diphosphate. Conformational analyses were carried out with deuterium‐labeled GGPP and oxidoGGPP. We found that GGPP and (14R)‐oxidoGGPP adopted a chair/chair conformation, but (14S)‐oxidoGGPP adopted a boat/chair conformation. Interestingly, the conformations of oxidoGGPP for the A‐ring formation are the opposite of those of oxidosqualene when it is used as a substrate by squalene cyclases for the biosynthesis of hopene and tetrahymanol. (3R)‐Oxidosqualene is folded in a boat conformation, whereas (3S)‐2,3‐oxidosqualene folds into a chair conformation, for the formation of the A‐rings of the hopene and tetrahymanol skeletons, respectively.     

Analysis of the Catalytic Mechanism of Bifunctional Triterpene/Sesquarterpene Cyclase: Tyr167 Functions To Terminate Cyclization of Squalene at the Bicyclic Step

Onoceroids are a group of triterpenes biosynthesized from squalene or dioxidosqualene by cyclization from both termini. We previously identified a bifunctional triterpene/sesquarterpene cyclase (TC) that constructs a tetracyclic scaffold from tetraprenyl‐β‐curcumene (C₃₅) but a bicyclic scaffold from squalene (C₃₀) in the first reaction. TC also accepts the bicyclic intermediate as a substrate and generates tetracyclic and pentacyclic onoceroids in the second reaction. In this study, we analyzed the catalytic mechanism of an onoceroid synthase by using mutated enzymes. TC^Y167A produced an unnatural tricyclic triterpenol, but TC^Y167L, TC^Y167F, and TC^Y167W formed small quantities of tricyclic compounds, which suggested that the bulk size at Y167 contributed to termination of the cyclization of squalene at the bicyclic step. Our findings provide insight into the unique catalytic mechanism of TC, which triggers different cyclization modes depending on the substrate. These findings may facilitate the large‐scale production of an onoceroid for which natural sources are limited.     

Identification of a New Diterpene Biosynthetic Gene Cluster that Produces O‐Methylkolavelool in Herpetosiphon aurantiacus

Diterpenoids are usually found in plants and fungi, but are rare in bacteria. We have previously reported new diterpenes, named tuberculosinol and isotuberculosinol, which are generated from the Mycobacterium tuberculosis gene products Rv3377c and Rv3378c. No homologous gene was found at that time, but we recently found highly homologous proteins in the Herpetosiphon aurantiacus ATCC 23779 genome. Haur_2145 was a class II diterpene cyclase responsible for the conversion of geranylgeranyl diphosphate into kolavenyl diphosphate. Haur_2146, homologous to Rv3378c, synthesized (+)‐kolavelool through the nucleophilic addition of a water molecule to the incipient cation formed after the diphosphate moiety was released. Haur_2147 afforded (+)‐O‐methylkolavelool from (+)‐kolavelool, so this enzyme was an O‐methyltransferase. This new diterpene was indeed detected in H. aurantiacus cells. This is the first report of the identification of a (+)‐O‐methylkolavelool biosynthetic gene cluster.     

An Improved Technique for the Rapid Chemical Characterisation of Bacterial Terpene Cyclases

A derivative of the pET28c(+) expression vector was constructed. It contains a yeast replication system (2μ origin of replication) and a yeast selectable marker (URA3), and can be used for gene cloning in yeast by efficient homologous recombination, and for heterologous expression in E. coli. The vector was used for the expression and chemical characterisation of three bacterial terpene cyclases.     

Revision of the Cyclisation Mechanism for the Diterpene Spiroviolene and Investigations of Its Mass Spectrometric Fragmentation

The diterpene spiroviolene, its diterpene synthase from Streptomyces violens and the experimentally determined terpene cyclisation mechanism were reported in 2017. Recently, the structure of spiroviolene was revised based on a total synthesis, with consequences for the cyclisation mechanism. Herein, a reinvestigation of the terpene cyclisation to spiroviolene and the mass spectrometric fragmentation mechanism investigated by ¹³C-labelling experiments are presented.     

Bifunctionality of ActIV as a Cyclase‐Thioesterase Revealed by in Vitro Reconstitution of Actinorhodin Biosynthesis in Streptomyces coelicolor A3(2)

Type II polyketide synthases iteratively generate a nascent polyketide thioester of the acyl carrier protein (ACP); this is structurally modified to produce an ACP‐free intermediate towards the final metabolite. However, the timing of ACP off‐loading is not well defined because of the lack of an apparent thioesterase (TE) among relevant biosynthetic enzymes. Here, ActIV, which had been assigned as a second ring cyclase (CYC) in actinorhodin (ACT) biosynthesis, was shown to possess TE activity in vitro with a model substrate, anthraquinone‐2‐carboxylic acid‐N‐acetylcysteamine. In order to investigate its function further, the ACT biosynthetic pathway in Streptomyces coelicolor A3(2) was reconstituted in vitro in a stepwise fashion up to (S)‐DNPA, and the product of ActIV reaction was characterized as an ACP‐free bicyclic intermediate. These findings indicate that ActIV is a bifunctional CYC‐TE and provide clear evidence for the release timing of the intermediate from the ACP anchor.     

Identification and Characterization of the Carbapenem MM 4550 and its Gene Cluster in Streptomyces argenteolus ATCC 11009

Nearly 50 naturally occurring carbapenem β‐lactam antibiotics, most produced by Streptomyces, have been identified. The structural diversity of these compounds is limited to variance of the C‐2 and C‐6 side chains as well as the stereochemistry at C‐5/C‐6. These structural motifs are of interest both for their antibiotic effects and their biosynthesis. Although the thienamycin gene cluster is the only active gene cluster publically available in this group, more comparative information is needed to understand the genetic basis of these structural differences. We report here the identification of MM 4550, a member of the olivanic acids, as the major carbapenem produced by Streptomyces argenteolus ATCC 11009. Its gene cluster was also identified by degenerate PCR and targeted gene inactivation. Sequence analysis revealed that the genes encoding the biosynthesis of the bicyclic core and the C‐6 and C‐2 side chains are well conserved in the MM 4550 and thienamycin gene clusters. Three new genes, cmmSu, cmm17 and cmmPah were found in the new cluster, and their putative functions in the sulfonation and epimerization of MM 4550 are proposed. Gene inactivation showed that, in addition to cmmI, two new genes, cmm22 and ‐23, encode a two‐component response system thought to regulate the production of MM 4550. Overexpression of cmmI, cmm22 and cmm23 promoted MM 4550 production in an engineered strain. Finally, the involvement and putative roles of all genes in the MM 4550 cluster are proposed based on the results of bioinformatics analysis, gene inactivation, and analysis of disruption mutants. Overall, the differences between the thienamycin and MM 4550 gene clusters are reflected in characteristic structural elements and provide new insights into the biosynthesis of the complex carbapenems.     

Identification and Characterization of the Streptazone E Biosynthetic Gene Cluster in Streptomyces sp. MSC090213JE08

Streptazone derivatives isolated from Streptomyces species are piperidine alkaloids with a cyclopenta[b]pyridine scaffold. Previous studies indicated that these compounds are polyketides, but the biosynthetic enzymes responsible for their synthesis are unknown. Here, we have identified the streptazone E biosynthetic gene cluster in Streptomyces sp. MSC090213JE08, which encodes a modular type I PKS and tailoring enzymes that include an aminotransferase, three oxidoreductases, and two putative cyclases. The functions of the six tailoring enzymes were analyzed by gene disruption, and two putative biosynthetic intermediates that accumulated in particular mutants were structurally elucidated. On the basis of these results, we propose a pathway for the biosynthesis of streptazone E in which the two putative cyclases of the nuclear transport factor 2–like superfamily are responsible for C?C bond formation coupled with epoxide ring opening to give the five‐membered ring of streptazone E.