Genome mining for unknown-unknown natural products.

Genome mining of biosynthetic pathways with no identifiable core enzymes can lead to discovery of the so-called unknown (biosynthetic route)-unknown (molecular structure) natural products. Here we focused on a conserved fungal biosynthetic pathway that lacks a canonical core enzyme and used heterologous expression to identify the associated natural product, a highly modified cyclo-arginine-tyrosine dipeptide. Biochemical characterization of the pathway led to identification of a new arginine-containing cyclodipeptide synthase (RCDPS), which was previously annotated as a hypothetical protein and has no sequence homology to non-ribosomal peptide synthetase or bacterial cyclodipeptide synthase. RCDPS homologs are widely encoded in fungal genomes; other members of this family can synthesize diverse cyclo-arginine-Xaa dipeptides, and characterization of a cyclo-arginine-tryptophan RCDPS showed that the enzyme is aminoacyl-tRNA dependent. Further characterization of the biosynthetic pathway led to discovery of new compounds whose structures would not have been predicted without knowledge of RCDPS function.

Biosynthesis of Polycyclic Natural Products from Conjugated Polyenes via Tandem Isomerization and Pericyclic Reactions.

We report biosynthetic pathways that can synthesize and transform conjugated octaenes and nonaenes to complex natural products. The biosynthesis of (-)-PF1018 involves an enzyme PfB that can control the regio-, stereo-, and periselectivity of multiple reactions starting from a conjugated octaene. Using PfB as a lead, we discovered a homologous enzyme, BruB, that facilitates diene isomerization, tandem 8π-6π-electrocyclization, and a 1,2-divinylcyclobutane Cope rearrangement to generate a new-to-nature compound.

Genome Mining from Agriculturally Relevant Fungi Led to a d-Glucose Esterified Polyketide with a Terpene-like Core Structure.

Comparison of biosynthetic gene clusters (BGCs) found in devastating plant pathogens and biocontrol fungi revealed an uncharacterized and conserved polyketide BGC. Genome mining identified the associated metabolite to be treconorin, which has a terpene-like, trans-fused 5,7-bicyclic core that is proposed to derive from a (4 + 3) cycloaddition. The core is esterified with d-glucose, which derives from the glycosidic cleavage of a trehalose ester precursor. This glycomodification strategy is different from the commonly observed glycosylation of natural products.

Hypascyrins A-E, Prenylated Acylphloroglucinols from Hypericum ascyron.

Six new prenylated acylphloroglucinols with menthane moieties, hypascyrins A-E (1-5) and ent-hyphenrone J (6), together with four known analogues, were isolated from Hypericum ascyron roots. Detailed spectroscopic data analyses resulted in the assignment of their structures. The absolute configuration of 1 was deduced by experimental and calculated ECD data, while those of 2-6 were assigned by ECD data analyses as well as chemical conversions. Hypascyrins A (1), C (3), and E (5) and ent-hyphenrone J (6) exhibited antimicrobial activity against MRSA (MIC50 values of 4.0, 8.0, 2.0, and 4.0 µM, respectively) and Bacillus subtilis (MIC values of 4.0, 4.0, 2.0, and 4.0 µM, respectively).

High-Throughput Identification of Crystalline Natural Products from Crude Extracts Enabled by Microarray Technology and microED.

The structural determination of natural products (NPs) can be arduous because of sample heterogeneity. This often demands iterative purification processes and characterization of complex molecules that may be available only in miniscule quantities. Microcrystal electron diffraction (microED) has recently shown promise as a method to solve crystal structures of NPs from nanogram quantities of analyte. However, its implementation in NP discovery remains hampered by sample throughput and purity requirements, akin to traditional NP-discovery workflows. In the methods described herein, we leverage the resolving power of transmission electron microscopy (TEM) and the miniaturization capabilities of deoxyribonucleic acid (DNA) microarray technology to address these challenges through the establishment of an NP screening platform, array electron diffraction (ArrayED). In this workflow, an array of high-performance liquid chromatography (HPLC) fractions taken from crude extracts was deposited onto TEM grids in picoliter-sized droplets. This multiplexing of analytes on TEM grids enables 1200 or more unique samples to be simultaneously inserted into a TEM instrument equipped with an autoloader. Selected area electron diffraction analysis of these microarrayed grids allows for the rapid identification of crystalline metabolites. In this study, ArrayED enabled structural characterization of 14 natural products, including four novel crystal structures and two novel polymorphs, from 20 crude extracts. Moreover, we identify several chemical species that would not be detected by standard mass spectrometry (MS) or ultraviolet-visible (UV/vis) spectroscopy and crystal forms that would not be characterized using traditional methods.

Engineered Production of Strictosidine and Analogues in Yeast.

Monoterpene indole alkaloids (MIAs) are an expansive class of plant natural products, many of which have been named on the World Health Organization's List of Essential Medicines. Low production from native plant hosts necessitates a more reliable source of these drugs to meet global demand. Here, we report the development of a yeast-based platform for high-titer production of the universal MIA precursor, strictosidine. Our fed-batch platform produces ∼50 mg/L strictosidine, starting from the commodity chemicals geraniol and tryptamine. The microbially produced strictosidine was purified to homogeneity and characterized by NMR. Additionally, our approach enables the production of halogenated strictosidine analogues through the feeding of modified tryptamines. The MIA platform strain enables rapid access to strictosidine for reconstitution and production of downstream MIA natural products.

Hyperdioxanes, dibenzo-1,4-dioxane derivatives from the roots of Hypericum ascyron.

Six dibenzo-1,4-dioxane derivatives (1-6) were isolated from the roots of a Hypericaceous plant Hypericum ascyron. Spectroscopic analyses revealed 2 and 4-6 to be new compounds. The partial racemic natures of 1-3 were concluded by chiral HPLC analyses, while 5 was confirmed to be a racemate. The absolute configurations 1-4 were deduced on the basis of ECD calculations. Biological activity evaluation of the dibenzo-1,4-dioxane derivatives along with two related compounds: hyperdioxanes A (7) and B (8), previously isolated from the same plant material by our group demonstrated that 7 exhibit an anti-HIV activity (IC50 5.3 µM, TI 7.2) while 8 showed an inhibitory effect on IL-1ß production (inhibition rate: 72.3% at 6.3 µM) from LPS-stimulated microglial cells.

Prenylated benzophenone derivatives from Hypericum patulum.

A new prenylated benzophenone, hypatulin C (1), was isolated from the leaves of Hypericum patulum together with a biogenetically related analog, hypelodin B (2). Hypatulin C (1) had a tricyclic [4.3.1.03,7]-decane moiety substituted by four prenyl groups. The structure of 1 was elucidated by detailed spectroscopic analyses, while the absolute stereochemistries of 1 and 2 were assigned by comparison of their ECD spectra with TDDFT calculated spectra.

C28 Terpenoids from Lamiaceous Plant Perovskia scrophulariifolia: Their Structures and Anti-neuroinflammatory Activity.

Structurally unique C28 terpenoids, perovsfolins A (1) and B (2), were isolated from the aerial parts of an Uzbek medicinal plant, Perovskia scrophulariifolia (Lamiaceae). Their chemical structures including an unprecedented 6/8/6/6/6 pentacyclic carbon skeleton with a C6-C3 ester moiety were elucidated on the basis of spectroscopic analyses aided by density functional theory calculations as well as chemical evidence. Perovsfolin B (2) exhibited an anti-neuroinflammatory activity.

Linaburiosides A-D, acylated iridoid glucosides from Linaria buriatica.

Four previously undescribed acylated iridoid glucosides, linaburiosides A‒D, one undescribed iridoid, 7-deoxyiridolactonic acid, and one known acylated iridoid glucoside, iridolinarin C, were isolated from the aerial parts of a Mongolian traditional herbal medicine, Linaria buriatica. Linaburiosides A‒D had an acyl moiety corresponding to 7-deoxyiridolactonic acid. Detailed spectroscopic analyses of linaburiosides A‒D and 7-deoxyiridolactonic acid led to the assignment of their structures. The absolute configuration of 7-deoxyiridolactonic acid was elucidated by application of the PGME method; those of linaburiosides A‒D were assigned on the basis of chemical conversions, as well as application of the modified Mosher's method. The absolute configuration of iridolinarin C was also elucidated in this study. Anti-inflammatory and antiproliferative activities of isolated compounds and their derivatives were evaluated.

Hyperdioxane A, a Conjugate of Dibenzo-1,4-dioxane and Sesquiterpene from Hypericum ascyron.

Two new dibenzo-1,4-dioxane derivatives, hyperdioxanes A (1) and B (2), were isolated from the roots of a Hypericaceous plant, Hypericum ascyron. Hyperdioxane A (1) is a conjugate of dibenzo-1,4-dioxane and sesquiterpene with an unprecedented heptacyclic ring system. The structures of 1 and 2 were assigned by detailed spectroscopic analyses, including application of a modified Mosher's method. A possible biogenetic pathway of hyperdioxane A (1) from hyperdioxane B (2) and a sesquiterpene, eremophil-9,11(13)-dien-8ß,12-olide (3), is presented.