Baofu Xu

Ancestral marine sponges produce terpenoid‐based compounds, and recent studies have begun to reveal the genetic basis in some species, including the first report of type I terpene synthases (T1TSs) in a Bubarida sponge Obruta collector . Here, we present a further genomic and functional characterization of terpenoid biosynthesis in the isonitrile terpenoid‐rich sponge Acanthella cavernosa by integrating multi‐omics with comparative genomic, phylogenetic, and biochemical analyses. We found that A. cavernosa retains the full eukaryotic ancestral mevalonate pathway for terpene precursor biosynthesis. Their single α‐domain classical T1TSs form a basal, independently evolved lineage related to plant T1TS α‐domains, while UbiA‐type TSs drive lineage‐specific structural diversification. Notably, in contrast to the clustering of T1TSs with other biosynthetic genes observed in some corals, we did not observe such clustering in the sponge we sequenced, which is consistent with the observation in O. collector . Our findings provide additional evidence for the ancestral origin of metazoan terpenoid biosynthesis and further our understanding of early metazoan adaptive evolution, and facilitate biotechnological exploitation of sponge‐derived terpenoids.

Marine fungi are known to produce natural products possessing significant therapeutic potential. Here, chemical exploration of a marine‐derived Aspergillus strain, coupled with comprehensive spectroscopic analyses (NMR spectroscopy and x‐ray diffraction) and chemical calculations, resulted in the isolation and identification of six aryl‐type compounds, including three new metabolites: aspergiterpenamine ( 1 ), norbisaboid ( 3 ), and 3’‐carboxy‐4’,7‐dihydroxyisoflavone ( 4 ). Notably, compound 1 features an unprecedented benzylamine moiety fused to a bisabolane sesquiterpene scaffold, while compound 3 is distinguished by the elimination of three carbons from the isopropyl side chain of its terpene skeleton, which expands the structural diversity of aryl‐containing natural products.

Over the past decade, an increasing number of functional microbial-like terpene synthases (MTPSLs) have been reported in non-seed plants. However, whether the traditional Chinese medicinal plant H. serrata harbors such enzymes and their corresponding functions remains unexplored. In this study, we mined the transcriptome of H. serrata and identified a microbial-like terpene synthase, HsMTPSL1, which produces multiple diterpene products. Following isolation and structural elucidation, seven distinct compounds were obtained, representing three skeletal types: spatane, prenylkelsoene-type, and biflorane. Among these, compound 7 is a novel biflorane diterpene. Structural analysis and subsequent mutagenesis revealed critical residues governing the formation of distinct skeletons, uncovering the multifunctional nature of this enzyme. Notably, the S224A mutation significantly enhanced the production of spatane diterpene compound 1 by 11.6-fold, demonstrating the potential for protein engineering to improve the yield of this bioactive marine-specific diterpene. Transcriptomic profiling revealed that HsMTPSL1 is highly expressed in sporangia, and co-expression analysis with cytochrome P450s identified the CYP781 subfamily as candidates potentially involved in the downstream modification of these skeletons. Collectively, we report the first MTPSL from H. serrata and characterize it as a multifunctional diterpene synthase. Through structure-guided mutagenesis, we uncovered the molecular basis of its functional versatility, with the S224A mutation providing a powerful tool for enhancing the yields of all three diterpene skeletons, thereby laying a foundation for future protein engineering and synthetic biology applications.

Aspergillus fungi are renowned for producing various natural products, including meroterpenoids that show significant bioactivities. In this study, chemical investigation of a newly identified Aspergillus fungus led to the discovery of seven aromatic meroterpenoids, including two new ones designated as pantaromerotin (1) and pantanitrimerotin (2). Pantaromerotin is distinguished by containing a rare pyran ring formed from a prenyl unit, which are not observed in the structurally similar compound stocksiloate. In comparison, pantanitrimerotin harbors a rare cyanide group, which is not found in the previously reported asperterreusine C. In terms of bioactivity, compounds 2-7 exhibited significant biofilm eradication effects. Notably, compound 5 not only inhibited biofilm formation by 60.4% at sub-MIC concentrations but also demonstrated synergistic antimicrobial effects in combination with kanamycin. Furthermore, genome sequencing and in vitro assays enabled us to identify AtUbiA3 and Pan9 as two functional prenyltransferases, which catalyzes the conversion of the coisolated compound 8 to form 5, providing biochemical insights into the biosynthesis. Collectively, our work identifies aromatic meroterpenoids with promising antibiofilm potential and lays a solid foundation for the subsequent elucidation of their biosynthetic pathways.

Odyverdienes are a rare class of bacterial diterpenes possessing 6/8/4- or 6/7/5-tricyclic skeletons and the cyclization mechanism underlying their formation remains unknown. Based on their skeletons, we hypothesized that their tricyclic skeletons pass through a neutral or cationic 6/10-eunicellane intermediate. Here, we investigated the mechanism of odyverdiene B2 synthase, SalkS from Streptomyces alkaliterrae, using site-directed mutagenesis, isotopic labeling experiments, and quantum chemical calculations. Two key shunt products, the 10-membered monocyclic 13-deoxolobophytumin A and a trans-eunicellane named alkacellene, isolated from SalkS variants and quantum chemical calculations supported the proposed mechanism to odyverdiene B2 via initial 1,10-cyclization and a cationic eunicellanyl intermediate. Deuterium labeling studies also supported the occurrence of two sequential 1,2-hydride shifts that set distinctive stereocenters during eunicellanyl formation. Alkacellene was also shown to undergo acid-catalyzed cyclization to the 6/6/6/6-tetracyclic isohydropyrene, an isomer of the bacterial diterpene synthase product hydropyrene. This mechanistic study of SalkS highlights the frequent formation and use of eunicellanyl intermediates in diversifying bacterial diterpene biosynthesis and provides a model system to address fundamental questions involving 6/10-bicyclic ring systems.

Odyverdienes are a unique class of bacterial diterpenes possessing 6/8/4- or 6/7/5-tricyclic skeletons and the cyclization mechanism underlying their formation remains unknown. Based on their skeletons, we hypothesized that their tricyclic skel-etons pass through a neutral or cationic 6/10-eunicellane intermediate. Here, we investigated the mechanism of odyverdiene B2 synthase, SalkS from Streptomyces alkaliterrae, using site-directed mutagenesis, isotopic labeling experiments, and quantum chemical calculations. Two key shunt products, the 10-membered monocyclic 13-deoxolobophytumin A and a trans-eunicellane named alkacellene, isolated from SalkS variants and quantum chemical calculations supported the proposed mechanism to odyverdiene B2 via initial 1,10-cyclization and a cationic eunicellanyl intermediate. Deuterium labeling stud-ies also supported the occurrence of two sequential 1,2-hydride shifts that set distinctive stereocenters during eunicellanyl formation. Alkacellene was also shown to undergo acid-catalyzed cyclization to the 6/6/6/6-tetracyclic isohydropyrene, an isomer of the bacterial diterpene synthase product hydropyrene. This mechanistic study of SalkS highlights the frequent for-mation and use of eunicellanyl intermediates in diversifying bacterial diterpene biosynthesis and provides a model system to address fundamental questions involving 6/10-bicyclic ring systems.

Plants have evolved an extensive repertoire of specialized metabolites to adapt to complex environmental changes. Here, we identify two paralogous dirigent proteins (DPs) in cotton that serve as gatekeepers of extracellular terpenoid phytoalexin production in green organs, directing the transition of hemigossypol away from gossypol synthesis toward a hydroxylation pathway that leads to the biosynthesis of highly toxic hemigossypolone and heliocides. Under oxidative conditions, these proteins function synergistically with aldo-keto reductases to catalyze the hydroxylation of hemigossypol, followed by spontaneous oxidation that yields hemigossypolone, revealing a noncanonical role for aldo-keto reductases in extracellular terpenoid metabolism. Notably, mutants lacking these dirigent proteins produce gossypol but are devoid of hemigossypolone and heliocides in green organs exhibit heightened susceptibility to multiple biotic stresses, underscoring the enhanced protective role of these metabolites. This study describes a DPs-mediated mechanism of extracellular hydroxylation and highlights the potential ecological advantages of redirecting specialized metabolism extracellularly for enhanced defense against varying types of pathogens and herbivores.

Eunicellane diterpenoids, characterized by a 6,10-bicyclic core, are prone to undergo further cyclization into gersemiane diterpenoids via acid- or epoxidation-mediated protonation. However, it remains unclear whether classical type I terpene synthases (TSs), phylogenetically related to reported eunicellane synthases, can catalyze this protonation reaction, a process typically driven by type II TSs. To address this question, we employed a phylogeny-directed discovery approach starting from the eunicellane synthase MicA. This led to the discovery of SsFitTS, a tricyclic gersemiane synthase capable of catalyzing the protonation of a natural eunicellane intermediate within its classical type I TS active pocket. Through mutagenesis and molecular docking studies, we elucidated the mechanistic basis for this type I TS's ability to mediate a typically type II-like reaction while retaining its canonical type I fold. Our study not only demonstrated that the phylogeny-based genome mining method can unveil cryptic TSs but also facilitated the decoding of the structural basis underlying the determination of terpene scaffolds.