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Terpenoids are the largest family of natural products, but prokaryotes are vastly underrepresented in this chemical space. However, genomics supports vast untapped biosynthetic potential for terpenoids in bacteria. We discovered the first trans-eunicellane terpene synthase (TS), AlbS from Streptomyces albireticuli NRRL B-1670, in nature. Mutagenesis, deuterium labeling studies, and quantum chemical calculations provided extensive support for its cyclization mechanism. In addition, parallel stereospecific labeling studies with Bnd4, a cis-eunicellane TS, revealed a key mechanistic distinction between these two enzymes. AlbS highlights bacteria as a valuable source of novel terpenoids, expands our understanding of the eunicellane family of natural products and the enzymes that biosynthesize them, and provides a model system to address fundamental questions about the chemistry of 6,10-bicyclic ring systems.

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, from Streptomyces sp. (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 D94 NxxxD motif and mutation experiments confirmed an absolute requirement for D94 as well as E169.

Mangrove ecosystems are widely distributed in the intertidal zone of tropical and subtropical estuaries or coasts, containing abundant biological communities, for example, mangrove plants and diverse groups of microorganisms, featuring various bioactive secondary metabolites. We surveyed the literature from 2010 to 2022, resulting in a collection of 134 secondary metabolites, and classified them into two major families in terms of the biological sources and 15 subfamilies according to the chemical structures. To highlight the structural diversity and bioactivities of the mangrove ecosystem-associated secondary metabolites, we presented the chemical structures, bioactivities, biosynthesis, and chemical syntheses. [ABSTRACT FROM AUTHOR]

A new bicyclic diterpenoid, benditerpenoic acid, was isolated from soil‐dwelling Streptomyces sp. (CL12‐4). We sequenced the bacterial genome, identified the responsible biosynthetic gene cluster, verified the function of the terpene synthase, and heterologously produced the core diterpene. Comparative bioinformatics indicated this Streptomyces strain is phylogenetically unique and possesses nine terpene synthases. The absolute configurations of the new trans‐fused bicyclo[8.4.0]tetradecanes were achieved by extensive spectroscopic analyses, including Mosher's analysis, J‐based coupling analysis, and computations based on sparse NMR‐derived experimental restraints. Interestingly, benditerpenoic acid exists in two distinct ring‐flipped bicyclic conformations with a rotational barrier of ≈16 kcal mol−1 in solution. The diterpenes exhibit moderate antibacterial activity against Gram‐positive bacteria including methicillin and multi‐drug resistant Staphylococcus aureus. This is a rare example of an eunicellane‐type diterpenoid from bacteria and the first identification of a diterpene synthase and biosynthetic gene cluster responsible for the construction of the eunicellane scaffold. [ABSTRACT FROM AUTHOR]

Ein neues bicyclisches Diterpenoid, Benditerpensäure, wurde aus einem im Erdreich vorkommenden Streptomyceten‐Stamm (CL12‐4) isoliert. Wir sequenzierten das Bakteriengenom, identifizierten den verantwortlichen Biosynthesegencluster, verifizierten die Funktion der Terpensynthase und produzierten heterolog das Diterpenoid‐Grundgerüst. Vergleichende Bioinformatik zeigte, dass dieser Streptomyceten‐Stamm phylogenetisch einzigartig ist und neun Terpensynthasen besitzt. Die absoluten Konfigurationen der neuen trans‐fusionierten Bicyclo[8.4.0]tetradecane wurden durch umfangreiche spektroskopische Analysen erreicht, einschließlich Moshers Analyse, NMR‐basierter Kopplungsanalyse, sowie DFT Berechnungen auf Basis von experimentellen NMR Parametern. Interessanterweise liegt Benditerpensäure in Lösung in zwei Ringkonformationen vor. Die Rotationsbarriere beträgt ca. 16 kcal mol−1. Die Diterpenoide zeigen mäßige antibakterielle Aktivität gegen Gram‐positive Bakterien, einschließlich Methicillin‐resistenten und multiresistenten Staphylococcus aureus. Hier präsentieren wir die erste Isolierung eines bakteriellen Eunicellan‐Diterpenoids und die Identifizierung der Diterpensynthase und des Biosynthesegenclusters, der für den Aufbau des bakteriellen Eunicellan‐Gerüsts verantwortlich ist. [ABSTRACT FROM AUTHOR]

Covering: up to mid-2020 Terpenoids, also called isoprenoids, are the largest and most structurally diverse family of natural products. Found in all domains of life, there are over 80 000 known compounds. The majority of characterized terpenoids, which include some of the most well known, pharmaceutically relevant, and commercially valuable natural products, are produced by plants and fungi. Comparatively, terpenoids of bacterial origin are rare. This is counter-intuitive to the fact that recent microbial genomics revealed that almost all bacteria have the biosynthetic potential to create the C5 building blocks necessary for terpenoid biosynthesis. In this review, we catalogue terpenoids produced by bacteria. We collected 1062 natural products, consisting of both primary and secondary metabolites, and classified them into two major families and 55 distinct subfamilies. To highlight the structural and chemical space of bacterial terpenoids, we discuss their structures, biosynthesis, and biological activities. Although the bacterial terpenome is relatively small, it presents a fascinating dichotomy for future research. Similarities between bacterial and non-bacterial terpenoids and their biosynthetic pathways provides alternative model systems for detailed characterization while the abundance of novel skeletons, biosynthetic pathways, and bioactivies presents new opportunities for drug discovery, genome mining, and enzymology. [ABSTRACT FROM AUTHOR]

Natural products have been playing an indispensable role in drug discovery. However, it seems that the golden period of discovering new compounds has passed since the first antibiotic–penicillin. With the development of genome sequencing, it has been found that marine fungi contain various biosynthetic gene clusters (BGCs), which are silent under standard laboratory conditions. Therefore, it might be envisioned that once these BGCs are expressed, a large quantity of new secondary metabolites with biological activities could be generated. This paper reviewed several activation techniques implemented from 2020 to 2022, including epigenetics regulation, co-culture, precursor feeding, heterologous expression, and changing fermentation parameters to activate silent BGCs of marine fungi. We also described the diversity and bioactivities of these newly discovered uncommon marine fungi-derived compounds based on the classification of activation techniques, facilitating research groups focusing on natural products to enhance discovering efficiency. [Display omitted] [ABSTRACT FROM AUTHOR]

Penicillium strains are renowned for producing diverse secondary metabolites with unique structures and promising bioactivities. Our chemical investigations, accompanied by fermentation media optimization, of a newly isolated fungus, Penicillium shentong XL-F41, led to the isolation of twelve compounds. Among these are two novel indole terpene alkaloids, shentonins A and B ( 1 and 2 ), and a new fatty acid 3 . Shentonin A ( 1 ) is distinguished by an unusual methyl modification at the oxygen atom of the typical succinimide ring, a feature not seen in the structurally similar brocaeloid D. Additionally, shentonin A ( 1 ) exhibits a cis relationship between H-3 and H-4, as opposed to the trans configuration in brocaeloid D, suggesting a divergent enzymatic ring-expansion process in their respective fungi. Both shentonins A ( 1 ) and B ( 2 ) also feature a reduction of a carbonyl to a hydroxy group within the succinimide ring. All isolated compounds were subjected to antimicrobial evaluations, and compound 12 was found to have moderate inhibitory activity against Candia albicans . Moreover, genome sequencing of Penicillium shentong XL-F41 uncovered abundant silent biosynthetic gene clusters, indicating the need for future efforts to activate these clusters and unlock the full chemical potential of the fungus., (Copyright © 2024, Zou et al.)

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, from Streptomyces sp. (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 D 94 NxxxD motif and mutation experiments confirmed an absolute requirement for D94 as well as E169., (© 2021 Wiley-VCH GmbH.)

N-Methylpyrrolinium-derived alkaloids like tropane alkaloids, nicotine, and calystegines are valuable plant source specialized metabolites bearing pharmaceutical or biological activity. Microbial synthesis of the critical common intermediate N-methylpyrrolinium would allow for sustainable production of N-methylpyrrolinium-derived alkaloids. Here, we achieve the production of N-methylpyrrolinium both in Escherichia coli and in Saccharomyces cerevisiae by employing the biosynthetic genes derived from three different plants. Specifically, the diamine oxidases (DAOs) from Anisodus acutangulus were first characterized. Then, we produced N-methylpyrrolinium in vitro from l-ornithine via a combination of the three cascade enzymes, ornithine decarboxylase from Erythroxylum coca, putrescine N-methyltransferase from Anisodus tanguticus, and DAOs from A. acutangulus. Construction of the plant biosynthetic pathway in E. coli and S. cerevisiae resulted in de novo bioproduction of N-methylpyrrolinium with titers of 3.02 and 2.07 mg/L, respectively. Metabolic engineering of the yeast strain to produce N-methylpyrrolinium via decreasing the flux to the product catabolism pathway and improving the cofactor supply resulted in a final titer of 17.82 mg/L. This study not only presents the first microbial synthesis of N-methylpyrrolinium but also lays the foundation for heterologous biosynthesis of N-methylpyrrolinium-derived alkaloids. More importantly, the strains constructed herein can serve as important alternative tools for identifying undiscovered pathway enzymes with a synthetic biology strategy.

For the biosynthesis of Pip in Huperzia serrata, the mechanistic studies were evaluated. Through a series of biochemical analyses, Pip is biosynthesized through a two-step cascade reaction. Three intermediates possibly exist simultaneously as an equilibrium matter in the first-step reaction catalyzed by HsAld1, while HsSard4 performs as a ketimine reductase and chemoselectively and stereoselectively takes 1,2-dehydropipecolic acid as the preferred substrate in vitro.

Background: Huperzia serrata (H. serrata) is an economically important traditional Chinese herb with the notably medicinal value. As a representative member of the Lycopodiaceae family, the H. serrata produces various types of effectively bioactive lycopodium alkaloids, especially the huperzine A (HupA) which is a promising drug for Alzheimer's disease. Despite their medicinal importance, the public genomic and transcriptomic resources are very limited and the biosynthesis of HupA is largely unknown. Previous studies on comparison of 454-ESTs from H. serrata and Phlegmariurus carinatus predicted putative genes involved in lycopodium alkaloid biosynthesis, such as lysine decarboxylase like (LDC-like) protein and some CYP450s. However, these gene annotations were not carried out with further biochemical characterizations. To understand the biosynthesis of HupA and its regulation in H. serrata, a global transcriptome analysis on H. Serrata tissues was performed., Results: In this study, we used the Illumina Highseq4000 platform to generate a substantial RNA sequencing dataset of H. serrata. A total of 40.1 Gb clean data was generated from four different tissues: root, stem, leaf, and sporangia and assembled into 181,141 unigenes. The total length, average length, N50 and GC content of unigenes were 219,520,611 bp, 1,211 bp, 2,488 bp and 42.51%, respectively. Among them, 105,516 unigenes (58.25%) were annotated by seven public databases (NR, NT, Swiss-Prot, KEGG, COG, Interpro, GO), and 54 GO terms and 3,391 transcription factors (TFs) were functionally classified, respectively. KEGG pathway analysis revealed that 72,230 unigenes were classified into 21 functional pathways. Three types of candidate enzymes, LDC, CAO and PKS, responsible for the biosynthesis of precursors of HupA were all identified in the transcripts. Four hundred and fifty-seven CYP450 genes in H. serrata were also analyzed and compared with tissue-specific gene expression. Moreover, two key classes of CYP450 genes BBE and SLS, with 23 members in total, for modification of the lycopodium alkaloid scaffold in the late two stages of biosynthesis of HupA were further evaluated., Conclusion: This study is the first report of global transcriptome analysis on all tissues of H. serrata, and critical genes involved in the biosynthesis of precursors and scaffold modifications of HupA were discovered and predicted. The transcriptome data from this work not only could provide an important resource for further investigating on metabolic pathways in H. serrata, but also shed light on synthetic biology study of HupA.