Structure Elucidation of Antibiotics.

To date, there are hundreds of characterized natural products with antibacterial activity against pathogenic bacteria, and several have become bonafide antibiotic drugs. The development of antibacterial natural products into antibiotic drugs, both in the past and in the future, hinges upon an accurate description of the exact chemical structure of the compound. Bolstered by some form of mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy is the primary technique for elucidating the chemical structure of organic molecules including natural products. By combining various one-dimensional (1D) and two-dimensional (2D) experiments, the connectivity between atoms is established and a complete "picture" of the molecule is thereby revealed.

R-Hexitronic acid, a new tetronic acid derivative isolated from a soil fungus FG9RK.

A new tetronic acid derivative (1) together with terrestric acid (2), a known metabolite of Penicillium species, was isolated from the soil fungus, FG9RK following fermentation on solid rice medium. The structure of 1 was elucidated by one- and two-dimensional NMR and MS measurements. The absolute configuration of the oxygenated carbon in the side chain of 1 was identified as S by converting the compound into its Mosher ester whereas the absolute configuration of the lactone ring was deduced based on biogenetic considerations and comparison with 2.

Dithiodiketopiperazine derivatives from endophytic fungi Trichoderma harzianum and Epicoccum nigrum.

A new epidithiodiketopiperazine (ETP), pretrichodermamide G (1), along with three known (epi)dithiodiketopiparazines (2-4) were isolated from cultures of Trichoderma harzianum and Epicoccum nigrum, endophytic fungi associated with medicinal plants Zingiber officinale and Salix sp., respectively. The structure of the new compound (1) was established on the basis of spectroscopic data, including 1D/2D NMR and HRESIMS. The isolated compounds were investigated for their antifungal, antibacterial and cytotoxic potential against a panel of microorganisms and cell lines. Pretrichodermamide A (2) displayed antimicrobial activity towards the plant pathogenic fungus Ustilago maydis and the human pathogenic bacterium Mycobacterium tuberculosis with MIC values of 1 mg/mL (2 mM) and 25 µg/mL (50 µM), respectively. Meanwhile, epicorazine A (3) exhibited strong to moderate cytotoxicity against L5178Y, Ramos, and Jurkat J16 cell lines with IC50 values ranging from 1.3 to 28 µM. Further mechanistic studies indicated that 3 induces apoptotic cell death.

Azacoccones F-H, new flavipin-derived alkaloids from an endophytic fungus Epicoccum nigrum MK214079.

Three new flavipin-derived alkaloids, azacoccones F-H (1-3), along with six known compounds (4-9) were isolated from the endophytic fungus Epicoccum nigrum MK214079 associated with leaves of Salix sp. The structures of the new compounds were established by analysis of their 1D/2D nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass spectroscopy (HRESIMS) data. The absolute configuration of azacoccones F-H (1-3) was determined by comparison of experimental electronic circular dichroism (ECD) data with reported ones and biogenetic considerations. Epicocconigrone A (4), epipyrone A (5), and epicoccolide B (6) exhibited moderate antibacterial activity against Staphylococcus aureus ATCC 29213 with minimal inhibitory concentration (MIC) values ranging from 25 to 50 µM. Furthermore, epipyrone A (5) and epicoccamide A (7) displayed mild antifungal activity against Ustilago maydis AB33 with MIC values of 1.6 and 1.8 mM, respectively. Epicorazine A (8) showed pronounced cytotoxicity against the L5178Y mouse lymphoma cell line with an IC50 value of 1.3 µM.

Protein engineering strategies for microbial production of isoprenoids.

Isoprenoids comprise one of the most chemically diverse family of natural products with high commercial interest. The structural diversity of isoprenoids is mainly due to the modular activity of three distinct classes of enzymes, including prenyl diphosphate synthases, terpene synthases, and cytochrome P450s. The heterologous expression of these enzymes in microbial systems is suggested to be a promising sustainable way for the production of isoprenoids. Several limitations are associated with native enzymes, such as low stability, activity, and expression profiles. To address these challenges, protein engineering has been applied to improve the catalytic activity, selectivity, and substrate turnover of enzymes. In addition, the natural promiscuity and modular fashion of isoprenoid enzymes render them excellent targets for combinatorial studies and the production of new-to-nature metabolites. In this review, we discuss key individual and multienzyme level strategies for the successful implementation of enzyme engineering towards efficient microbial production of high-value isoprenoids. Challenges and future directions of protein engineering as a complementary strategy to metabolic engineering are likewise outlined.

Novel Strategies and Platforms for Industrial Isoprenoid Engineering.

Metabolic engineering has emerged as an important tool for reconstructing heterologous isoprenoid metabolic pathways in microbial hosts. Here, we provide an overview of promising engineering strategies that have proven to be successful for the high-yield production of isoprenoids. Besides 'conventional' approaches, such as the 'push-pull' and protein engineering to optimize the isoprenoid flux and limited catalytic activity of enzymes, we review emerging strategies in the field, including compartmentalization between synthetic consortia members, novel bypass pathways for isoprenoid synthesis, cell-free systems, and improvement of the lipid content to overcome storage isoprenoid limitations. Pitfalls, along with lessons learned from the application of these strategies, will be addressed with the hope of guiding future efforts toward cost-effective and sustainable production of isoprenoids.

Bioactive Secondary Metabolites from Endophytic Fungi.

BACKGROUND: Endophytes represent a complex community of microorganisms colonizing asymptomatically internal tissues of higher plants. Several reports have shown that endophytes enhance the fitness of their host plants by direct production of bioactive secondary metabolites, which are involved in protecting the host against herbivores and pathogenic microbes. In addition, it is increasingly apparent that endophytes are able to biosynthesize medicinally important "phytochemicals", originally believed to be produced only by their host plants. OBJECTIVE: The present review provides an overview of secondary metabolites from endophytic fungi with pronounced biological activities covering the literature between 2010 and 2017. Special focus is given on studies aiming at exploration of the mode of action of these metabolites towards the discovery of leads from endophytic fungi. Moreover, this review critically evaluates the potential of endophytic fungi as alternative sources of bioactive "plant metabolites". RESULTS: Over the past few years, several promising lead structures from endophytic fungi have been described in the literature. In this review, 65 metabolites are outlined with pronounced biological activities, primarily as antimicrobial and cytotoxic agents. Some of these metabolites have shown to be highly selective or to possess novel mechanisms of action, which hold great promises as potential drug candidates. CONCLUSION: Endophytes represent an inexhaustible reservoir of pharmacologically important compounds. Moreover, endophytic fungi could be exploited for the sustainable production of bioactive "plant metabolites" in the future. Towards this aim, further insights into the dynamic endophyte - host plant interactions and origin of endophytic fungal genes would be of utmost importance.

Xanthone, benzophenone and bianthrone derivatives from the hypersaline lake-derived fungus Aspergillus wentii.

Five new metabolites, including the xanthone derivative wentixanthone A (1), the benzophenone wentiphenone A (2), the diastereomeric mixtures of the bianthrones wentibianthrone A (3a, b) and wentibianthrone B (4a, b), as well as (10R,10'S)-wentibianthrone C (5a) and (10R,10'R)-wentibianthrone C (5b) were obtained from the fungus Aspergillus wentii, isolated from soil of the hypersaline lake El Hamra in Wadi El-Natrun, Egypt. The structures of the isolated compounds were established by one and two-dimensional NMR and MS spectroscopic analysis. The relative configuration of bianthrones (3-5) was elucidated by comparison of experimental and computed 1H NMR chemical shifts. Results of biological assays are reported.

Polyketides and a Dihydroquinolone Alkaloid from a Marine-Derived Strain of the Fungus Metarhizium marquandii.

Three new natural products (1-3), including two butenolide derivatives (1 and 2) and one dihydroquinolone derivative (3), together with nine known natural products were isolated from a marine-derived strain of the fungus Metarhizium marquandii. The structures of the new compounds were unambiguously deduced by spectroscopic means including HRESIMS and 1D/2D NMR spectroscopy, ECD, VCD, OR measurements, and calculations. The absolute configuration of marqualide (1) was determined by a combination of modified Mosher's method with TDDFT-ECD calculations at different levels, which revealed the importance of intramolecular hydrogen bonding in determining the ECD features. The (3R,4R) absolute configuration of aflaquinolone I (3), determined by OR, ECD, and VCD calculations, was found to be opposite of the (3S,4S) absolute configuration of the related aflaquinolones A-G, suggesting that the fungus M. marquandii produces aflaquinolone I with a different configuration (chiral switching). The absolute configuration of the known natural product terrestric acid hydrate (4) was likewise determined for the first time in this study. TDDFT-ECD calculations allowed determination of the absolute configuration of its chirality center remote from the stereogenic unsaturated γ-lactone chromophore. ECD calculations aided by solvent models revealed the importance of intramolecular hydrogen bond networks in stabilizing conformers and determining relationships between ECD transitions and absolute configurations.

Indole Diterpenoids from an Endophytic Penicillium sp.

A chemical investigation of the endophyte Penicillium sp. (strain ZO-R1-1), isolated from roots of the medicinal plant Zingiber officinale, yielded nine new indole diterpenoids (1-9), together with 13 known congeners (10-22). The structures of the new compounds were elucidated by 1D and 2D NMR analysis in combination with HRESIMS data. The absolute configuration of the new natural products 1, 3, and 7 was determined using the TDDFT-ECD approach and confirmed for 1 by single-crystal X-ray determination through anomalous dispersion. The isolated compounds were tested for cytotoxicity against L5178Y, A2780, J82, and HEK-293 cell lines. Compound 1 was the most active metabolite toward L5178Y cells, with an IC50 value of 3.6 µM, and an IC50 against A2780 cells of 8.7 µM. Interestingly, 1 features a new type of indole diterpenoid scaffold with a rare 6/5/6/6/6/6/5 heterocyclic system bearing an aromatic ring C, which is suggested to be important for the cytotoxic activity of this natural product against L5278Y and A2780 cells.

Induction of Secondary Metabolites from the Marine-Derived Fungus Aspergillus versicolor through Co-cultivation with Bacillus subtilis.

A new cyclic pentapeptide, cotteslosin C (1: ), a new aflaquinolone, 22-epi-aflaquinolone B (3: ), and two new anthraquinones (9: and 10: ), along with thirty known compounds (2, 4:  - 8, 11:  - 34: ) were isolated from a co-culture of the sponge-associated fungus Aspergillus versicolor with Bacillus subtilis. The new metabolites were only detected in the co-culture extract, but not when the fungus was grown under axenic conditions. Furthermore, the co-culture extract exhibited an enhanced accumulation of the known constituents versicolorin B (14: ), averufin (16: ), and sterigmatocyctin (19: ) by factors of 1.5, 2.0, and 4.7, respectively, compared to the axenic fungal culture. The structures of the isolated compounds were elucidated on the basis of 1D and 2D NMR spectra and mass spectrometry as well as by comparison with literature data. The absolute configuration of compounds 3, 9: , and 10: was determined by ECD (electronic circular dichroism) analysis aided by TDDFT-ECD (time-dependent density functional theory electronic circular dichroism) calculations. Compounds 15, 18:  - 21: , and 26: exhibited strong to moderate cytotoxic activity against the mouse lymphoma cell line L5178Y, with IC50 values ranging from 2.0 to 21.2 µM, while compounds 14, 16, 31, 32: , and 33: displayed moderate inhibitory activities against several gram-positive bacteria, with MIC values ranging from 12.5 to 50 µM.

Expanding the chemical diversity of an endophytic fungus Bulgaria inquinans, an ascomycete associated with mistletoe, through an OSMAC approach.

An endophytic fungus Bulgaria inquinans (isolate MSp3-1), isolated from mistletoe (Viscum album), was subjected to fermentation on solid Czapek medium. Chromatographic workup of the crude EtOAc extract yielded five new natural products (1-5). Subsequent application of the "One Strain, MAny Compounds" (OSMAC) strategy on this strain by the addition of a mixture of salts (MgSO4, NaNO3 and NaCl) to solid Czapek medium induced the accumulation of nine additional new secondary metabolites (6-13, 16), with most of them (8, 10-12) not detectable in cultures lacking the salt mixture. The structures of the new compounds were established on the basis of the 1D/2D NMR and HRESIMS data. The TDDFT-ECD method was applied to determine the absolute configurations of the new compounds 1, 4 and 6 as well as of the previously reported bulgarialactone B (14), for which the absolute configuration was unknown so far. The modified Mosher's method was performed to assign the absolute configurations of 12 and 13. TDDFT-ECD analysis also allowed determining the absolute configuration of (+)-epicocconone, which had an enantiomeric absolute configuration in the tricyclic moiety compared to that of bulgarialactone B (14). All the isolated metabolites were evaluated for their cytotoxic activity. Compound 2 was found to possess strong cytotoxic activity against the murine lymphoma cell line L5178Y with an IC50 value of 1.8 µM, while the remaining metabolites were shown to be inactive.

Chaetolines A and B, Pyrano[3,2- f]isoquinoline Alkaloids from Cultivation of Chaetomium sp. in the Presence of Autoclaved Pseudomonas aeruginosa.

The first members of a new alkaloid class, chaetolines A (1) and B (2), which feature a pyrano[3,2- f]isoquinoline core structure, were obtained from a crude extract of the fungal endophyte Chaetomium sp. after cultivation in the presence of autoclaved Pseudomonas aeruginosa. The structures of the new compounds, including the absolute configuration of the major stereoisomer, were determined through detailed analysis of HRESIMS, 1D/2D NMR, and calculation of ECD data. The possible biosynthetic origin of the unprecedented scaffold of 1 and 2 is proposed. The current study provides further evidence for mixed fermentation as a powerful tool to induce the accumulation of cryptic fungal natural products even in the absence of viable bacterial cells.

Lead Compounds from Mangrove-Associated Microorganisms.

The mangrove ecosystem is considered as an attractive biodiversity hotspot that is intensively studied in the hope of discovering new useful chemical scaffolds, including those with potential medicinal application. In the past two decades, mangrove-derived microorganisms, along with mangrove plants, proved to be rich sources of bioactive secondary metabolites as exemplified by the constant rise in the number of publications, which suggests the great potential of this important ecological niche. The present review summarizes selected examples of bioactive compounds either from mangrove endophytes or from soil-derived mangrove fungi and bacteria, covering the literature from 2014 to March 2018. Accordingly, 163 natural products are described in this review, possessing a wide range of potent bioactivities, such as cytotoxic, antibacterial, antifungal, α-glucosidase inhibitory, protein tyrosine phosphatase B inhibitory, and antiviral activities, among others.

Tanzawaic acid derivatives from freshwater sediment-derived fungus Penicillium sp.

Chemical investigation of a freshwater sediment-derived fungus, Penicillium sp. (S1a1), led to the isolation of three new tanzawaic acid derivatives, including penitanzchroman (1), tanzawaic acids Y (2) and Z (3), along with six known tanzawaic acid analogues (4-9), three known isochromans (10-12) and two known benzoquinones (13 and 14). The structures of the new compounds were established based on high-resolution mass spectrometry, and detailed analysis of one- and two-dimensional NMR spectroscopy. The relative configuration of the new compounds was assigned on the basis of NMR spectroscopic data including ROESY spectra. The absolute configuration was determined based on the specific optical rotation, in addition to biogenetic considerations in comparison with related co-isolated known metabolites. Penitanzchroman (1) constitutes a hitherto unprecedented skeleton, formed of tanzawaic acid A (5) and (3S)-6-hydroxy-8-methoxy-3,5-dimethylisochroman (10) linked by a CC bond. Moreover, all compounds were evaluated for their antibacterial and cytotoxic activities.

Chlorflavonin Targets Acetohydroxyacid Synthase Catalytic Subunit IlvB1 for Synergistic Killing of Mycobacterium tuberculosis.

The flavonoid natural compound chlorflavonin was isolated from the endophytic fungus Mucor irregularis, which was obtained from the Cameroonian medicinal plant Moringa stenopetala. Chlorflavonin exhibited strong growth inhibitory activity in vitro against Mycobacterium tuberculosis (MIC90 1.56 µM) while exhibiting no cytotoxicity toward the human cell lines MRC-5 and THP-1 up to concentrations of 100 µM. Mapping of resistance-mediating mutations employing whole-genome sequencing, chemical supplementation assays, and molecular docking studies as well as enzymatic characterization revealed that chlorflavonin specifically inhibits the acetohydroxyacid synthase catalytic subunit IlvB1, causing combined auxotrophies to branched-chain amino acids and to pantothenic acid. While exhibiting a bacteriostatic effect in monotreatment, chlorflavonin displayed synergistic effects with the first-line antibiotic isoniazid and particularly with delamanid, leading to a complete sterilization in liquid culture in combination treatment. Using a fluorescent reporter strain, intracellular activity of chlorflavonin against Mycobacterium tuberculosis inside infected macrophages was demonstrated and was superior to streptomycin treatment.

Natural Products from Deep-Sea-Derived Fungi ̶ A New Source of Novel Bioactive Compounds?

BACKGROUND: Over the last two decades, deep-sea-derived fungi are considered to be a new source of pharmacologically active secondary metabolites for drug discovery mainly based on the underlying assumption that the uniqueness of the deep sea will give rise to equally unprecedented natural products. Indeed, up to now over 200 new metabolites have been identified from deep-sea fungi, which is in support of the statement made above. RESULTS: This review summarizes the new and/or bioactive compounds reported from deepsea- derived fungi in the last six years (2010 - October 2016) and critically evaluates whether the data published so far really support the notion that these fungi are a promising source of new bioactive chemical entities.

Cyclic Cystine-Bridged Peptides from the Marine Sponge Clathria basilana Induce Apoptosis in Tumor Cells and Depolarize the Bacterial Cytoplasmic Membrane.

Investigation of the sponge Clathria basilana collected in Indonesia afforded five new peptides, including microcionamides C (1) and D (2), gombamides B (4), C (5), and D (6), and an unusual amide, (E)-2-amino-3-methyl-N-styrylbutanamide (7), along with 11 known compounds, among them microcionamide A (3). The structures of the new compounds were elucidated by one- and two-dimensional NMR spectroscopy as well as by high-resolution mass spectrometry. The absolute configurations of the constituent amino acid residues in 1-7 were determined by Marfey's analysis. Microcionamides A, C, and D (1-3) showed in vitro cytotoxicity against lymphoma (Ramos) and leukemia cell lines (HL-60, Nomo-1, Jurkat J16), as well as against a human ovarian carcinoma cell line (A2780) with IC50 values ranging from 0.45 to 28 µM. Mechanistic studies showed that compounds 1-3 rapidly induce apoptotic cell death in Jurkat J16 and Ramos cells and that 1 and 2 potently block autophagy upon starvation conditions, thereby impairing pro-survival signaling of cancer cells. In addition, microcionamides C and A (1 and 3) inhibited bacterial growth of Staphylococcus aureus and Enterococcus faecium with minimal inhibitory concentrations between 6.2 and 12 µM. Mechanistic studies indicate dissipation of the bacterial membrane potential.

New 2-Methoxy Acetylenic Acids and Pyrazole Alkaloids from the Marine Sponge Cinachyrella sp.

Three new 2-methoxy acetylenic acids (1-3) and a known derivative (4), in addition to three new natural pyrazole alkaloids (5-7) were isolated from an Indonesian marine sponge of the genus Cinachyrella. Compounds 5 and 6 have previously been reported as synthetic compounds. The structures of the new compounds were established on the basis of one- and two-dimensional NMR spectroscopy as well as by mass spectrometric data. The absolute configuration of the new acetylenic acid derivatives (1-3) was established by ECD spectroscopy. All isolated compounds were evaluated for their cytotoxicity against L5178Y mouse lymphoma cells. Compounds 1-4 exhibited strong activity with an IC50 value of 0.3 µM. A plausible biosynthetic pathway for the pyrazole metabolites 5-7 is proposed.

OSMAC approach leads to new fusarielin metabolites from Fusarium tricinctum.

Using the OSMAC (One Strain MAny Compounds) approach, the fungal endophyte Fusarium tricinctum was cultivated on fruit and vegetable juice-supplemented solid rice media. This led to an up to 80-fold increase in the accumulation of the new natural product fusarielin J (1), as well as to the induction of two new natural products fusarielin K (2) and fusarielin L (3) and the known derivatives fusarielins A (4) and B (5). Compounds 2-5 were not detected when the fungus was grown on rice media lacking either fruit or vegetable juice. The highest increase in the accumulation of compound 1 was observed in the presence of apple and carrot juice, whereas the stimulating effect was weaker for banana juice. Compound 1 exhibited cytotoxicity against the human ovarian cancer cell line A2780, with an IC50 value of 12.5 µM.