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1. Mechanistic Insights into the Formation of the 6,10‐Bicyclic Eunicellane Skeleton by the Bacterial Diterpene Synthase Bnd4

   https://doi.org/10.1002/ange.202109641  

   Xu, Baofu ; Tantillo, Dean J. ; Rudolf, Jeffrey D. ( September 2021 , Angewandte Chemie)

   The eunicellane diterpenoids are a unique family of natural products seen in marine organisms, plants, and bacteria. We used a series of biochemical, bioinformatics, and theoretical experiments to investigate the mechanism of the first diterpene synthase known to form the eunicellane skeleton. Deuterium labeling studies and quantum chemical calculations support that Bnd4, fromStreptomycessp. (CL12‐4), forms the 6,10‐bicyclic skeleton through a 1,10‐cyclization, 1,3‐hydride shift, and 1,14‐cyclization cascade. Bnd4 also demonstrated sesquiterpene cyclase activity and the ability to prenylate small molecules. Bnd4 possesses a unique D⁹⁴NxxxD motif and mutation experiments confirmed an absolute requirement for D94 as well as E169.

    

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2. Mechanistic Insights into the Formation of the 6,10‐Bicyclic Eunicellane Skeleton by the Bacterial Diterpene Synthase Bnd4

   https://doi.org/10.1002/anie.202109641  

   Xu, Baofu ; Tantillo, Dean J. ; Rudolf, Jeffrey D. ( September 2021 , Angewandte Chemie International Edition)

   The eunicellane diterpenoids are a unique family of natural products seen in marine organisms, plants, and bacteria. We used a series of biochemical, bioinformatics, and theoretical experiments to investigate the mechanism of the first diterpene synthase known to form the eunicellane skeleton. Deuterium labeling studies and quantum chemical calculations support that Bnd4, fromStreptomycessp. (CL12‐4), forms the 6,10‐bicyclic skeleton through a 1,10‐cyclization, 1,3‐hydride shift, and 1,14‐cyclization cascade. Bnd4 also demonstrated sesquiterpene cyclase activity and the ability to prenylate small molecules. Bnd4 possesses a unique D⁹⁴NxxxD motif and mutation experiments confirmed an absolute requirement for D94 as well as E169.

    

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3. Ecology shapes the genomic and biosynthetic diversification of Streptomyces bacteria from insectivorous bats

   https://doi.org/10.1099/mgen.0.001238  

   Montoya-Giraldo, Manuela ; Piper, Kathryn R ; Ikhimiukor, Odion O ; Park, Cooper J ; Caimi, Nicole A ; Buecher, Debbie C ; Valdez, Ernest W ; Northup, Diana E ; Andam, Cheryl P ( April 2024 , Microbial Genomics)

   Streptomycesare prolific producers of secondary metabolites from which many clinically useful compounds have been derived. They inhabit diverse habitats but have rarely been reported in vertebrates. Here, we aim to determine to what extent the ecological source (bat host species and cave sites) influence the genomic and biosynthetic diversity ofStreptomycesbacteria. We analysed draft genomes of 132Streptomycesisolates sampled from 11 species of insectivorous bats from six cave sites in Arizona and New Mexico, USA. We delineated 55 species based on the genome-wide average nucleotide identity and core genome phylogenetic tree.Streptomycesisolates that colonize the same bat species or inhabit the same site exhibit greater overall genomic similarity than they do withStreptomycesfrom other bat species or sites. However, when considering biosynthetic gene clusters (BGCs) alone, BGC distribution is not structured by the ecological or geographical source of theStreptomycesthat carry them. Each genome carried between 19–65 BGCs (median=42.5) and varied even among members of the sameStreptomycesspecies. Nine major classes of BGCs were detected in ten of the 11 bat species and in all sites: terpene, non-ribosomal peptide synthetase, polyketide synthase, siderophore, RiPP-like, butyrolactone, lanthipeptide, ectoine, melanin. Finally,Streptomycesgenomes carry multiple hybrid BGCs consisting of signature domains from two to seven distinct BGC classes. Taken together, our results bring critical insights to understandingStreptomyces-bat ecology and BGC diversity that may contribute to bat health and in augmenting current efforts in natural product discovery, especially from underexplored or overlooked environments.

    

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4. Promiscuous terpene synthases from Prunella vulgaris highlight the importance of substrate and compartment switching in terpene synthase evolution

   https://doi.org/10.1111/nph.15778  

   Johnson, Sean R. ; Bhat, Wajid Waheed ; Sadre, Radin ; Miller, Garret P. ; Garcia, Alekzander Sky ; Hamberger, Björn ( April 2019 , New Phytologist)

   The mint family (Lamiaceae) is well documented as a rich source of terpene natural products. More than 200 diterpene skeletons have been reported from mints, but biosynthetic pathways are known for just a few of these.

   We crossreferenced chemotaxonomic data with publicly available transcriptomes to select common selfheal (Prunella vulgaris) and its highly unusual vulgarisin diterpenoids as a case study for exploring the origins of diterpene skeletal diversity in Lamiaceae. Four terpene synthases (TPS) from the TPS‐a subfamily, including two localised to the plastid, were cloned and functionally characterised. Previous examples of TPS‐a enzymes from Lamiaceae were cytosolic and reported to act on the 15‐carbon farnesyl diphosphate. Plastidial TPS‐a enzymes using the 20‐carbon geranylgeranyl diphosphate are known from other plant families, having apparently arisen independently in each family.

   All four new enzymes were found to be active on multiple prenyl‐diphosphate substrates with different chain lengths and stereochemistries. One of the new enzymes catalysed the cyclisation of geranylgeranyl diphosphate into 11‐hydroxy vulgarisane, the likely biosynthetic precursor of the vulgarisins.

   We uncovered the pathway to a rare diterpene skeleton. Our results support an emerging paradigm of substrate and compartment switching as important aspects of TPS evolution and diversification.

    

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5. Uncovering a miltiradiene biosynthetic gene cluster in the Lamiaceae reveals a dynamic evolutionary trajectory

   https://doi.org/10.1038/s41467-023-35845-1  

   Bryson, Abigail E. ; Lanier, Emily R. ; Lau, Kin H. ; Hamilton, John P. ; Vaillancourt, Brieanne ; Mathieu, Davis ; Yocca, Alan E. ; Miller, Garret P. ; Edger, Patrick P. ; Buell, C. Robin ; et al ( December 2023 , Nature Communications)

   Abstract The spatial organization of genes within plant genomes can drive evolution of specialized metabolic pathways. Terpenoids are important specialized metabolites in plants with diverse adaptive functions that enable environmental interactions. Here, we report the genome assemblies of Prunella vulgaris , Plectranthus barbatus , and Leonotis leonurus . We investigate the origin and subsequent evolution of a diterpenoid biosynthetic gene cluster (BGC) together with other seven species within the Lamiaceae (mint) family. Based on core genes found in the BGCs of all species examined across the Lamiaceae, we predict a simplified version of this cluster evolved in an early Lamiaceae ancestor. The current composition of the extant BGCs highlights the dynamic nature of its evolution. We elucidate the terpene backbones generated by the Callicarpa americana BGC enzymes, including miltiradiene and the terpene (+)-kaurene, and show oxidization activities of BGC cytochrome P450s. Our work reveals the fluid nature of BGC assembly and the importance of genome structure in contributing to the origin of metabolites. 

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