Characterization and catalytic investigation of fungal single-module nonribosomal peptide synthetase in terpene-amino acid meroterpenoid biosynthesis.

Hybrid natural products are compounds that originate from diverse biosynthetic pathways and undergo a conjugation process, which enables them to expand their chemical diversity and biological functionality. Terpene-amino acid meroterpenoids have garnered increasing attention in recent years, driven by the discovery of noteworthy examples such as the anthelmintic CJ-12662, the insecticidal paeciloxazine, and aculene A (1). In the biosynthesis of terpene-amino acid natural products, single-module nonribosomal peptide synthetases (NRPSs) have been identified to be involved in the esterification step, catalyzing the fusion of modified terpene and amino acid components. Despite prior investigations into these NRPSs through gene deletion or in vivo experiments, the enzymatic basis and mechanistic insights underlying this family of single-module NRPSs remain unclear. In this study, we performed biochemical characterization of AneB by in vitro characterization, molecular docking, and site-directed mutagenesis. The enzyme reaction analyses, performed with L-proline and daucane/nordaucane sesquiterpene substrates, revealed that AneB specifically esterifies the C10-OH of aculenes with L-proline. Notably, in contrast to ThmA in CJ-12662 biosynthesis, which exclusively recognizes oxygenated amorpha-4,11-diene sesquiterpenes for L-tryptophan transfer, AneB demonstrates broad substrate selectivity, including oxygenated amorpha-4,11-diene and 2-phenylethanol, resulting in the production of diverse unnatural prolyl compounds. Furthermore, site-directed mutagenesis experiments indicated the involvement of H794 and D798 in the esterification catalyzed by AneB. Lastly, domain swapping between AneB and ThmA unveiled that the A‒T domains of ThmA can be effectively harnessed by the C domain of AneB for L-tryptophan transfer, thus highlighting the potential of the C domain of AneB for generating various terpene-amino acid meroterpenoid derivatives. ONE-SENTENCE SUMMARY: The enzymatic basis and mechanistic insights into AneB, a single-module NRPS, highlight its capacity to generate various terpene-amino acid meroterpenoid derivatives.

The Biosynthetic Gene Cluster of Mushroom-Derived Antrocin Encodes Two Dual-Functional Haloacid Dehalogenase-like Terpene Cyclases.

(-)-Antrocin (1), produced by the medicinal mushroom Antrodia cinnamomea, is a potent antiproliferative compound. The biosynthetic gene cluster of 1 was identified, and the pathway was characterized by heterologous expression. We characterized a haloacid dehalogenase-like terpene cyclase AncC that biosynthesizes the drimane-type sesquiterpene (+)-albicanol (2) from farnesyl pyrophosphate (FPP). Biochemical characterization of AncC, including kinetic studies and mutagenesis, demonstrated the functions of two domains: a terpene cyclase (TC) and a pyrophosphatase (PPase). The TC domain first cyclizes FPP to albicanyl pyrophosphate, and the PPase domain then removes the pyrophosphate to form 2. Intriguingly, AncA (94 % sequence identity to AncC), in the same gene cluster, converts FPP into (R)-trans-γ-monocyclofarnesol instead of 2. Notably, Y283/F375 in the TC domain of AncA serve as a gatekeeper in controlling the formation of a cyclofarnesoid rather than a drimane-type scaffold.

The Biosynthesis of Norsesquiterpene Aculenes Requires Three Cytochrome P450 Enzymes to Catalyze a Stepwise Demethylation Process.

Aculenes are a unique class of norsequiterpenes (C14 ) that are produced by Aspergillus aculeatus. The nordaucane skeleton in aculenes A-D may be derived from an ent-daucane precursor through demethylation, however, the enzymes involved remain unexplored. We identified the biosynthetic gene cluster and characterized the biosynthetic pathway based on gene inactivation, feeding experiments, and heterologous reconstitution in Saccharomyces cerevisiae and Aspergillus oryzae. We discovered that three cytochrome P450 monoxygenases are required to catalyze the stepwise demethylation process. AneF converts the 12-methyl group into a carboxylic acid and AneD installs the 10-hydroxy group for later tautomerization and stabilization. Finally, AneG installs an electron-withdrawing carbonyl group at the C-2 position, which triggers C-12 decarboxylation to yield the nordaucane skeleton. Additionally, a terpene cyclase (AneC) was found that forms a new product (dauca-4,7-diene).

Biosynthesis of Complex Indole Alkaloids: Elucidation of the Concise Pathway of Okaramines.

The okaramines are a class of complex indole alkaloids isolated from Penicillium and Aspergillus species. Their potent insecticidal activity arises from selectively activating glutamate-gated chloride channels (GluCls) in invertebrates, not affecting human ligand-gated anion channels. Okaramines B (1) and D (2) contain a polycyclic skeleton, including an azocine ring and an unprecedented 2-dimethyl-3-methyl-azetidine ring. Owing to their complex scaffold, okaramines have inspired many total synthesis efforts, but the enzymology of the okaramine biosynthetic pathway remains unexplored. Here, we identified and characterized the biosynthetic gene cluster (oka) of 1 and 2, then elucidated the pathway with target gene inactivation, heterologous reconstitution, and biochemical characterization. Notably, we characterized an α-ketoglutarate-dependent non-heme FeII dioxygenase that forged the azetidine ring on the okaramine skeleton.

Iron oxide nanopropellers prepared by a low-temperature solution approach.

The alpha-Fe(2)O(3) (hematite) nanopropellers were synthesized via a low-temperature solution-based method using FeCl(2) as a precursor in the presence of urea and glycine hydrochloride. The formation of alpha-Fe(2)O(3) nanopropellers is strongly depended on the addition of glycine hydrochloride, which serves as a pH modulator and affects the oxidation rate of Fe(2+). The structural evolution of the propeller-structured hematite was found to follow dissolution and recrystallization processes. For the structural conformation, each nanopropeller presents a hexagonal central column closed by six equivalent surfaces of {(-)1100} and the six arrays of the nanopropeller structure are a result of growth along +/- [(-)1100], +/- [(-)1010], and +/-[0(-)110]. Preliminary results show that the magnetic maghemite (gamma-Fe(2)O(3)) nanopropellers could also be prepared by a reduction and reoxidation process from the alpha-Fe(2)O(3) (hematite) nanopropeller precursors.