Unveiling the fungal diversity and associated secondary metabolism on black apples.

Black apples are the result of late-stage microbial decomposition after falling to the ground. This phenomenon is highly comparable from year to year, with the filamentous fungus Monilinia fructigena most commonly being the first invader, followed by Penicillium expansum. Motivated by the fact that only little chemistry has been reported from apple microbiomes, we set out to investigate the chemical diversity and potential ecological roles of secondary metabolites (SMs) in a total of 38 black apples. Metabolomics analyses were conducted on either whole apples or small excisions of fungal biomass derived from black apples. Annotation of fungal SMs in black apple extracts was aided by the cultivation of 15 recently isolated fungal strains on 9 different substrates in a One Strain Many Compounds (OSMAC) approach, leading to the identification of 3,319 unique chemical features. Only 6.4% were attributable to known compounds based on analysis of high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-HRMS/MS) data using spectral library matching tools. Of the 1,606 features detected in the black apple extracts, 32% could be assigned as fungal-derived, due to their presence in the OSMAC-based training data set. Notably, the detection of several antifungal compounds indicates the importance of such compounds for the invasion of and control of other microbial competitors on apples. In conclusion, the diversity and abundance of microbial SMs on black apples were found to be much higher than that typically observed for other environmental microbiomes. Detection of SMs known to be produced by the six fungal species tested also highlights a succession of fungal growth following the initial invader M. fructigena.IMPORTANCEMicrobial secondary metabolites constitute a significant reservoir of biologically potent and clinically valuable chemical scaffolds. However, their usefulness is hampered by rapidly developing resistance, resulting in reduced profitability of such research endeavors. Hence, the ecological role of such microbial secondary metabolites must be considered to understand how best to utilize such compounds as chemotherapeutics. Here, we explore an under-investigated environmental microbiome in the case of black apples; a veritable "low-hanging fruit," with relatively high abundances and diversity of microbially produced secondary metabolites. Using both a targeted and untargeted metabolomics approach, the interplay between metabolites, other microbes, and the apple host itself was investigated. This study highlights the surprisingly low incidence of known secondary metabolites in such a system, highlighting the need to study the functionality of secondary metabolites in microbial interactions and complex microbiomes.

Maramycin, a Cytotoxic Isoquinolinequinone Terpenoid Produced through Heterologous Expression of a Bifunctional Indole Prenyltransferase/Tryptophan Indole-Lyase in S. albidoflavus.

Isoquinolinequinones represent an important family of natural alkaloids with profound biological activities. Heterologous expression of a rare bifunctional indole prenyltransferase/tryptophan indole-lyase enzyme from Streptomyces mirabilis P8-A2 in S. albidoflavus J1074 led to the activation of a putative isoquinolinequinone biosynthetic gene cluster and production of a novel isoquinolinequinone alkaloid, named maramycin (1). The structure of maramycin was determined by analysis of spectroscopic (1D/2D NMR) and MS spectrometric data. The prevalence of this bifunctional biosynthetic enzyme was explored and found to be a recent evolutionary event with only a few representatives in nature. Maramycin exhibited moderate cytotoxicity against human prostate cancer cell lines, LNCaP and C4-2B. The discovery of maramycin (1) enriched the chemical diversity of natural isoquinolinequinones and also provided new insights into crosstalk between the host biosynthetic genes and the heterologous biosynthetic genes in generating new chemical scaffolds.

Fungal Duel between Penicillium brasilianum and Aspergillus nomius Results in Dual Induction of Miktospiromide A and Kitrinomycin A.

Cocultivation of the fungi Penicillium brasilianum MST-FP1927 and Aspergillus nomius MST-FP2004 resulted in the reciprocal induction of two new compounds, miktospiromide A (1) from A. nomius and kitrinomycin A (2) from P. brasilianum. A third new compound, kitrinomycin B (3), was also identified from an axenic culture of P. brasilianum, along with the previously reported compounds austalide K (4), 17S-dihydroaustalide K (5), verruculogen (6), and fumitremorgin B (7). The structures of 1-3 were elucidated by detailed spectroscopic analysis and DFT calculations, while 4-7 were identified by comparison to authentic standards. The genome of A. nomius MST-FP2004 was sequenced, and a putative biosynthetic gene cluster for 1 was identified. Compound 2 showed activity against murine melanoma NS-1 cells (LD99 7.8 µM) and the bovine parasite Tritrichomonas foetus (LD99 4.8 µM).

Stereodivergent Hydroxylation of Berkeleylactones by Penicillium turbatum.

Penicillium turbatum has previously been reported to produce A26771B, a 16-membered macrocyclic polyketide with activity against Gram-positive bacteria, mycoplasma, and fungi, as well as the structurally related compounds berkeleylactone E and berkeleylactones I-O. In this work, large-scale cultivation of P. turbatum NRRL 5630 on rice yielded seven new berkeleylactone analogues, berkeleylactone E methyl ester, 14-epi-berkeleylactone F, berkeleylactones P-R, 12-epi-berkeleylactone Q, and 13-epi-berkeleylactone R, and six previously reported analogues, A26771B and berkeleylactones E-G and J-K. The structures of the berkeleylactones were elucidated by detailed analysis of spectroscopic data, molecular modeling, and comparison with literature values. Interestingly, six of the berkeleylactone analogues were isolated as pairs of hydroxy epimers, highlighting how Nature can exploit stereodivergence in biosynthetic pathways to increase chemical diversity. The genome of P. turbatum was sequenced, and a putative gene cluster (bekl) responsible for the biosynthesis of the berkeleylactones was identified. The new berkeleylactone analogues exhibited no significant biological activity against a panel of bacteria, fungi, the parasite Giardia duodenalis, or NS-1 murine myeloma cells, suggesting a hitherto undiscovered biological role.

Yeppoonic acids A - D: 1,2,4-trisubstituted arene carboxylic acid co-metabolites of conglobatin from an Australian Streptomyces sp.

Streptomyces sp. MST-91080 was isolated from a soil sample collected in Queensland, Australia. From this strain, yeppoonic acids A - D were purified and spectroscopically characterised. The yeppoonic acids are a family of diene enecarboxylic acids on a 1,2,4-trisubstituted benzene scaffold, structurally related to other Streptomyces secondary metabolites MF-EA-705α/ß, NFAT-133 and the lorneic acids. Yeppoonic acids B and C show strong cytotoxicity against the NS-1 mouse myeloma cell line (IC50 2.3 µg ml-1 and 3.8 µg ml-1, respectively) and moderate activity against the DU 145 human prostate cancer cell line (IC50 32.8 µg ml-1 and 49.6 µg ml-1, respectively).

Rechoreographing Enterocin's Ballet of Isomers: Structure Revision of Enterocins C, D, and F.

The tricyclic scaffold of the bacterial polyketide enterocin was recently shown to undergo stereodiversification through a series of abiotic isomerizations, described metaphorically as a "ballet of isomers". However, some confusion remains regarding the exact nature of these interconversions, with two independent reports proposing different reaction mechanisms and intermediates. Herein, we have rechoreographed enterocin's ballet of isomers to provide a unified mechanism and revised the structures reported for enterocins C, D, and F.

The chemical gymnastics of enterocin: evidence for stereodivergence in Nature.

Stereodivergence in Nature encapsulates both enzymatic (biosynthetic) and non-enzymatic (chemical) diversification of natural product scaffolds arising from a single biosynthetic pathway. Herein, we report a fascinating example of stereodivergence for the bacterial polyketide enterocin, which we observed to undergo a series of facile skeletal rearrangements in solution, leading to four distinct isomeric structures. The final distribution of the four isomers was found to be highly sensitive to the conditions used, including solvent, temperature and pH. In this study, we have investigated the kinetics of these isomeric conversions, and using a combination of DFT and thermochemical calculations, were able to establish a mechanism detailing a concerted rearrangement and an unusual "gymnastic" sequence of pseudo-chair-boat conformational interconversions. In addition to these kinetic and mechanistic studies, we also performed a semisynthetic study aimed at stabilising the enterocin scaffold. In total, seven analogues of enterocin were synthesised and investigated for their stability and in vitro activity against a panel of bacteria, fungi, plants and mammalian cells.