A novel 7-base pair deletion at a splice site in MS-2 impairs male fertility via premature tapetum degradation in common bean (Phaseolis vulgaris L.).

KEY MESSAGE: A novel splice-site mutation in the P. vulgarisgene for TETRAKETIDE α-PYRONE REDUCTASE 2 impairs male fertility, and parthenocarpic pod development can be improved by external application of IAA. Snap bean (Phaseolus vulgaris L.) is an important vegetable crop in many parts of the world, and the main edible part is the fresh pod. Here, we report the characterization of the genic male sterility (ms-2) mutant in common bean. Loss of function of MS-2 accelerates degradation of the tapetum, resulting in a complete male sterility. Through fine-mapping, co-segregation, and re-sequencing analysis, we identified Phvul.003G032100, which encodes the TETRAKETIDE α-PYRONE REDUCTASE 2 (PvTKPR2) protein in common bean, as the causal gene for MS-2. PvTKPR2 is predominantly expressed at the early stages of flower development. A novel 7-bp (+ 6028 bp to + 6034 bp) deletion mutation spans the splice site between the fourth intron and fifth exon, leading to a 9-bp deletion in transcribed mRNA and a 3-amino acid (G210M211V212) deletion in the protein coding sequence of the PvTKPR2ms-2 gene. The 3-D structural changes in the protein due to the mutation may impair the activities of NAD-dependent epimerase/dehydratase and the NAD(P)-binding domains of PvTKPR2ms-2 protein. The ms-2 mutant plants produce many small parthenocarpic pods, and the size of the pods can be doubled by external application of 2 mM indole-3-acetic acid (IAA). Our results demonstrate that a novel mutation in PvTKPR2 impairs male fertility through premature degradation of the tapetum.

Effective production of kojic acid in engineered Aspergillus niger.

BACKGROUND: Kojic acid (KA) is a widely used compound in the cosmetic, medical, and food industries, and is typically produced by Aspergillus oryzae. To meet increasing market demand, it is important to optimize KA production through seeking alternatives that are more economic than current A. oryzae-based methods. RESULTS: In this study, we achieved the first successful heterologous production of KA in Aspergillus niger, an industrially important fungus that does not naturally produce KA, through the expression of the kojA gene from A. oryzae. Using the resulting KA-producing A. niger strain as a platform, we identified four genes (nrkA, nrkB, nrkC, and nrkD) that negatively regulate KA production. Knocking down nrkA or deleting any of the other three genes resulted in a significant increase in KA production in shaking flask cultivation. The highest KA titer (25.71 g/L) was achieved in a pH controlled batch bioreactor using the kojA overexpression strain with a deletion of nrkC, which showed a 26.7% improvement compared to the KA titer (20.29 g/L) that was achieved in shaking flask cultivation. CONCLUSION: Our study demonstrates the potential of using A. niger as a platform for studying KA biosynthesis and regulation, and for the cost-effective production of KA in industrial strain development.

Domain Truncation in Hispidin Synthase Orthologs from Non-Bioluminescent Fungi Does Not Lead to Hispidin Biosynthesis.

Hispidin is a polyketide found in plants and fungi. In bioluminescent fungi, hispidin serves as a precursor of luciferin and is produced by hispidin synthases. Previous studies revealed that hispidin synthases differ in orthologous polyketide synthases from non-bioluminescent fungi by the absence of two domains with predicted ketoreductase and dehydratase activities. Here, we investigated the hypothesis that the loss of these domains in evolution led to the production of hispidin and the emergence of bioluminescence. We cloned three orthologous polyketide synthases from non-bioluminescent fungi, as well as their truncated variants, and assessed their ability to produce hispidin in a bioluminescence assay in yeast. Interestingly, expression of the full-length enzyme hsPKS resulted in dim luminescence, indicating that small amounts of hispidin are likely being produced as side products of the main reaction. Deletion of the ketoreductase and dehydratase domains resulted in no luminescence. Thus, domain truncation by itself does not appear to be a sufficient step for the emergence of efficient hispidin synthases from orthologous polyketide synthases. At the same time, the production of small amounts of hispidin or related compounds by full-length enzymes suggests that ancestral fungal species were well-positioned for the evolution of bioluminescence.

Lasiodiplodiapyrones A and B, Pyrone-Preussomerin Adducts with Highly Strained Polycyclic Ring Systems from Lasiodiplodia pseudotheobromae.

Lasiodiplodiapyrones A and B (1 and 2), two new preussomerin derivatives, possessing an unexpected 6-methyl-4H-furo[3,2-c]pyran-4-one moiety and a highly functionalized conjoint and complicated polycyclic ring system, along with two known congeners (3 and 4), were isolated from the fungus Lasiodiplodia pseudotheobromae. Their structures including absolute configurations were determined by spectroscopic analyses, Mosher's method, and ECD calculations. A biosynthetic pathway was proposed to explain the origin of lasiodiplodiapyrones A and B as well as their relationship with preussomerins. Compounds 1-4 showed suppressive effects on the production of NO with IC50 values of 4.8 ± 0.3, 8.5 ± 1.1, 5.9 ± 0.8, and 12.8 ± 1.3 µM, respectively.

Five new verrucosidin derivatives from Penicillium polonicum, a deep-sea cold-seep sediment isolated fungus.

Five new verrucosidin derivatives, poloncosidins G-K (1-5), were isolated from the deep sea cold-seep sediment-derived fungus Penicillium polonicum CS-252. Their planar structures were elucidated by discreet analysis of the NMR spectroscopic and HRESIMS spectrometric data. The absolute configurations of compounds 1-5 were deduced from the combination of the modified Mosher's method and quantum chemical calculations of their ECD and NMR (with DP4+ probability analysis) data. The antimicrobial activities against several human- and aquatic-pathogenic bacteria of all the isolated compounds were evaluated and the structure-bioactivity relationship was briefly discussed.

Neuropyrones A-E, five undescribed α-pyrone derivatives with tyrosinase inhibitory activity from the endophytic fungus Neurospora dictyophora WZ-497.

Five undescribed α-pyrone derivatives, named neuropyrones A-E, were isolated from the endophytic fungus Neurospora dictyophora WZ-497 derived from the stems of Aster tataricus L. f. The structures of these α-pyrones with absolute configurations were determined by comprehensive spectroscopic analysis and computational calculations. All isolated compounds were tested for various bioactivities, including tyrosinase inhibitory activity. The results showed that neuropyrones A-C displayed potent inhibitory effects on tyrosinase with IC50 values of 0.38 ± 0.07, 0.49 ± 0.06, and 0.12 ± 0.01 mM, respectively, which were comparable to that of the positive control, kojic acid (IC50 = 0.14 ± 0.021 mM). A molecular docking study revealed the interaction between 3 and the His263, His85, Val283, Asn260, Phe264, and Val248 residues of tyrosinase.

New Dibenzo-α-pyrone Derivatives with α-Glucosidase Inhibitory Activities from the Marine-Derived Fungus Alternaria alternata.

Three new dibenzo-α-pyrone derivatives, alternolides A-C (1-3), and seven known congeners (4-10) were isolated from the marine-derived fungus of Alternaria alternata LW37 assisted by the one strain-many compounds (OSMAC) strategy. The structures of 1-3 were established by extensive spectroscopic analyses, and their absolute configurations were determined by modified Snatzke's method and electronic circular dichroism (ECD) calculations. Compounds 6 and 7 showed good 1,1-diphenyl-2-picrylhydrazyl (DPPH) antioxidant scavenging activities with IC50 values of 83.94 ± 4.14 and 23.60 ± 1.23 µM, respectively. Additionally, 2, 3 and 7 exhibited inhibitory effects against α-glucosidase with IC50 values of 725.85 ± 4.75, 451.25 ± 6.95 and 6.27 ± 0.68 µM, respectively. The enzyme kinetics study indicated 2 and 3 were mixed-type inhibitors of α-glucosidase with Ki values of 347.0 and 108.5 µM, respectively. Furthermore, the interactions of 2, 3 and 7 with α-glucosidase were investigated by molecular docking.

2-(2-(Dimethylamino)vinyl)-4H-pyran-4-ones as Novel and Convenient Building-Blocks for the Synthesis of Conjugated 4-Pyrone Derivatives.

A straightforward approach for the construction of the new class of conjugated pyrans based on enamination of 2-methyl-4-pyrones with DMF-DMA was developed. 2-(2-(Dimethylamino)vinyl)-4-pyrones are highly reactive substrates that undergo 1,6-conjugate addition/elimination or 1,3-dipolar cycloaddition/elimination followed by substitution of the dimethylamino group without ring opening. This strategy includes selective transformations leading to conjugated and isoxazolyl-substituted 4-pyrone structures. The photophysical properties of the prepared 4-pyrones were determined in view of further design of novel merocyanine fluorophores. A solvatochromism was found for enamino-substituted 4-pyrones accompanied by a strong increase in fluorescence intensity in alcohols. The prepared conjugated structures demonstrated valuable photophysical properties, such as a large Stokes shift (up to 204 nm) and a good quantum yield (up to 28%).

Computational Analysis of Triazole-Based Kojic Acid Analogs as Tyrosinase Inhibitors by Molecular Dynamics and Free Energy Calculations.

Molecular docking, molecular dynamics (MD) simulations and the linear interaction energy (LIE) method were used here to predict binding modes and free energy for a set of 1,2,3-triazole-based KA analogs as potent inhibitors of Tyrosinase (TYR), a key metalloenzyme of the melanogenesis process. Initially, molecular docking calculations satisfactorily predicted the binding mode of evaluated KA analogs, where the KA part overlays the crystal conformation of the KA inhibitor into the catalytic site of TYR. The MD simulations were followed by the LIE method, which reproduced the experimental binding free energies for KA analogs with an r2 equal to 0.97, suggesting the robustness of our theoretical model. Moreover, the van der Waals contributions performed by some residues such as Phe197, Pro201, Arg209, Met215 and Val218 are responsible for the binding recognition of 1,2,3-triazole-based KA analogs in TYR catalytic site. Finally, our calculations provide suitable validation of the combination of molecular docking, MD, and LIE approaches as a powerful tool in the structure-based drug design of new and potent TYR inhibitors.

Fused-ring α-pyrones from intramolecular C-H activation and their lipids-lowering activity associated with LXR-IDOL-LDLR axis regulation.

Lipids-lowering is considered as the most effective approach to decrease the risk of Atherosclerotic cardiovascular disease (ASCVD), of which atherosclerosis is the most common cause. Natural products containing a unique type of α-pyrone was reported to suppress atherosclerosis in which α-pyrone might be considered as an important pharmacore. In this study, an efficient one-pot intramolecular C-H activation strategy was applied to the synthesis of potentially bioactive α-pyrone derivatives. As the result, three different scaffolds were quickly and conveniently generated, including thiophenes, pyrrole and indole derivatives. Among of them, eight α-pyrone derivatives showed potential effects to promote the uptake of LDL in HepG2 cells. Active unique α-pyrones compounds exhibiting potent in vitro and in vivo lipids-lowering effects, and a novel mechanism associated with the regulation of LXR-IDOL-LDLR axis, the new pathway targeted pharmacologically to control plasma cholesterol levels, were disclosed firstly in this study.

Experimental and Computational Studies on the Biotransformation of Pseudopyronines with Human Cytochrome P450 CYP4F2.

The secondary metabolite pseudopyronine B, isolated from Pseudomonas mosselii P33, was biotransformed by human P450 enzymes, heterologously expressed in the fission yeast Schizosaccharomyces pombe. Small-scale studies confirmed that both CYP4F2 and CYP4F3A were capable of oxidizing the substrate, with the former achieving a higher yield. In larger-scale studies using CYP4F2, three new oxidation products were obtained, the structures of which were elucidated by UV-vis, 1D and 2D NMR, and HR-MS spectroscopy. These corresponded to hydroxylated, carboxylated, and ester derivatives (1-3) of pseudopyronine B, all of which had been oxidized exclusively at the ω-position of the C-6 alkyl chain. In silico homology modeling experiments highlighted key interactions between oxygen atoms of the pyrone ring and two serine residues and a histidine residue of CYP4F2, which hold the substrate in a suitable orientation for oxidation at the terminus of the C-6 alkyl chain. Additional modeling studies with all three pseudopyronines revealed that the seven-carbon alkyl chain of pseudopyronine B was the perfect length for oxidation, with the terminal carbon lying close to the heme iron. The antibacterial activity of the substrates and three oxidation products was also assessed, revealing that oxidation at the ω-position removes all antimicrobial activity. This study both increases the range of known substrates for human CYF4F2 and CYP4F3A enzymes and demonstrates their utility in producing additional natural product derivatives.

In vivo characterization of key iridoid biosynthesis pathway genes in catnip (Nepeta cataria).

MAIN CONCLUSION: Using virus-induced gene silencing, we demonstrated that the enzymes GES, ISY, and MLPL are responsible for nepetalactone biosynthesis in Nepeta cataria. Nepetalactone is the main iridoid that is found in the Nepeta genus and is well-known for its psychoactive effect on house cats. Moreover, there is a burgeoning interest into the effect of nepetalactone on insects. Although the enzymes for nepetalactone biosynthesis have been biochemically assayed in vitro, validation of the role that these enzymes have in planta has not been demonstrated. Virus-induced gene silencing (VIGS) is a silencing method that relies on transient transformation and is an approach that has been particularly successful when applied to a variety of non-model plants. Here, we use a recently designed visual-marker dependent VIGS system to demonstrate that the nepetalactone biosynthetic enzymes GES, ISY, and MLPL impact nepetalactone biosynthesis in Nepeta cataria.

Ocellatuperoxides A-F, Uncommon Anti-Tumoral γ-Pyrone Peroxides from a Photosynthetic Mollusk Placobranchus ocellatus.

Six new pairs of γ-pyrone polypropionate enantiomers with an unusual peroxyl bridge at the side chain, namely (±)-ocellatuperoxides A-F (1-6), were isolated and characterized from the South China Sea photosynthetic mollusk Placobranchus ocellatus. Extensive spectroscopic analysis, single crystal X-ray diffraction analysis, ECD- (electronic circular dichroism) comparison, and TDDFT (time-dependent density functional theory) ECD computation were used to determine the structures and absolute configurations of new compounds. In a cell viability assay, several compounds showed considerable anti-tumoral effects on human non-small cell lung cancer cells A549 with Gefitinib (7.4 µM) and Erlotinib (2.1 µM) as positive controls. Further RNA-sequencing analysis and gene expression evaluation indicated that the anti-tumoral activity of the most effective compound 3 was associated with the regulation of several important genes, such as FGFR1 and HDAC5.

A Nanobody-Based Immunoassay for Detection of Ustilaginoidins in Rice Samples.

Ustilaginoidins are a class of bis-naphtho-γ-pyrone mycotoxins produced by the pathogen Villosiclava virens of rice false smut, which has recently become one of the most devastating diseases in rice-growing regions worldwide. In this research, the nanobody phage display library was established after an alpaca was immunized with the hemiustilaginoidin F-hapten coupled with bovine serum albumin (BSA). Heterologous antigen selection and combing trypsin with competition alternant elution methods were performed for nanobody screening. Two nanobodies, namely, Nb-B15 and Nb-C21, were selected for the establishment of indirect competitive enzyme-linked immunosorbent assays (ic-ELISAs). For Nb-B15 and Nb-C21, their IC₅₀ values were 11.86 µg/mL and 11.22 µg/mL, and the detection ranges were at 3.41-19.98 µg/mL and 1.17-32.13 µg/mL, respectively. Two nanobodies had a broad spectrum to quantify the contents of total ustilaginoidins in rice samples according to cross-reactivity. The recognition mechanisms of Nb-B15 and Nb-C21 against ustilaginoidin A were elucidated by molecular modeling and docking. The key amino acid sites for the binding of Nb-B15 or Nb-C21 to ustilaginoidin A were mainly located in the FR1 and CDR1 regions. As Nb-B15 was superior to Nb-C21 in the aspects of protein expression, ELISA titer, and tolerance to organic solvents, it was selected for application in the detection of actual contaminated rice samples. The total ustilaginoidin contents of rice samples were analyzed by Nb-B15-based ic-ELISA and HPLC-DAD, between which the results were found to be consistent. The developed immunoassay based on the nanobody from the alpaca can be employed as a rapid and effective method for detection of total utilaginoidins in contaminated rice samples.

Phelliribsin B: A New Styrylpyrone Derivative from the Medicinal Fungus Phellinus ribis.

A novel styrylpyrone derivative, named phelliribsin B (1), as well as four biogenetically related known compounds, phellifuropyranone A (2), inoscavin C (3), inoscavin A (4), and inoscavin D (5) were separated and purified from the medicinal fungus Phellinus ribis. The structure of phelliribsin B was determined by spectroscopic analysis, and the absolute configuration was assigned by experimental and calculated ECD data. Additionally, the plausible biosynthetic pathway of 1 was also proposed. Compound 1 showed moderately cytotoxic activity against HepG2 and SKOV-3 tumor cell lines with IC50 values of 32.71 and 57.89 µM, respectively. Based on the results of cytotoxicity against HepG2 tumor cells, the structure-activity relationship of compounds 1-4 with similar skeletons was discussed. The styrylpyrone derivatives with similar skeletons have moderately cytotoxic activity and have the potential to play an important role in the anti-tumor treatment.

Thermorubin Biosynthesis Initiated by a Salicylate Synthase Suggests an Unusual Conversion of Phenols to Pyrones.

Thermorubin is a tetracyclic naphthoisocoumarin natural product that demands investigation due to its novel mechanism of bacterial protein synthesis inhibition and its unusual structural features. In this work, we describe the identification of the biosynthetic cluster responsible for thermorubin from the sequenced Laceyella sacchari producer species and its confirmation via heterologous production in Escherichia coli. Based on an in-depth annotation of the cluster, we propose a biosynthetic pathway that accounts for the formation of the unique, nonterminal pyrone. Additionally, the expression and use of salicylate synthase TheO enabled testing of the stability properties of this extremophile-derived enzyme. TheO displayed rapid kinetics and a remarkably robust secondary structure, converting chorismate to salicylate with a KM of 109 ± 12 µM, kcat of 9.17 ± 0.36 min-1, and catalytic efficiency (kcat/KM) of 84 ± 9 nM-1 min-1, and retained significant activity up to 50 °C. These studies serve as the basis for continued biosynthetic investigations and bioinspired synthetic approaches.

Cytospones E-J from the endophytic fungus Cytospora rhizophorae.

Cytospones E-J (1-6), six unreported α-pyrone derivatives, together with six known ones (7-12) were isolated from the solid culture of the endophytic fungus Cytospora rhizophorae A761, an endophytic fungus from Gynochthodes officinalis. The structures of the unreported compounds were unambiguously elucidated through spectroscopic analyses (1D, 2D NMR and HRESIMS), and their absolute configurations were assigned by single-crystal X-ray diffraction (Cu Kα) analyses. Furthermore, cytospones E-J were evaluated for anti-inflammatory and α-glucosidase inhibitory activities.

Resveratrol-Maltol and Resveratrol-Thiophene Hybrids as Cholinesterase Inhibitors and Antioxidants: Synthesis, Biometal Chelating Capability and Crystal Structure.

New resveratrol-thiophene and resveratrol-maltol hybrids were synthesized as cholinesterase inhibitors and antioxidants. As with photostability experiments, biological tests also found remarkable differences in the properties and behavior of thiophene and maltol hybrids. While resveratrol-thiophene hybrids have excellent inhibitory and antioxidant properties (similar to the activity of reference drug galantamine), maltols have been proven to be weaker inhibitors and antioxidants. The molecular docking of selected active ligands gave insight into the structures of docked enzymes. It enabled the identification of interactions between the ligand and the active site of both cholinesterases. The maltols that proved to be active cholinesterase inhibitors were able to coordinate Fe3+ ion, forming complexes of 1:1 composition. Their formation constants, determined by spectrophotometry, are very similar, lgK = 11.6-12.6, suggesting that Fe3+ binds to the common hydroxy-pyranone moiety and is hardly affected by the other aromatic part of the ligand. Accordingly, the characteristic bands in their individual absorption spectra are uniformly red-shifted relative to those of the free ligands. The crystal structures of two new resveratrol-maltol hybrids were recorded, giving additional information on the molecules' intermolecular hydrogen bonds and packing. In this way, several functionalities of these new resveratrol hybrids were examined as a necessary approach to finding more effective drugs for complicated neurodegenerative diseases.

Bioactive Monoterpenes and Polyketides from the Ascidian-Derived Fungus Diaporthe sp. SYSU-MS4722.

There has been a tremendous increase in the rate of new terpenoids from marine-derived fungi being discovered, while new monoterpenes were rarely isolated from marine-derived fungi in the past two decades. Three new monoterpenes, diaporterpenes A-C (1-3), and one new α-pyrones, diaporpyrone A (6), along with nine known polyketides 4, 5, and 7-13 were isolated from the ascidian-derived fungus Diaporthe sp. SYSU-MS4722. Their planar structures were elucidated based on extensive spectroscopic analyses (1D and 2D NMR and HR-ESIMS). The absolute configurations of 1 and 3 were identified by an X-ray crystallographic diffraction experiment using Cu-Ka radiation, and those of compound 2 were assigned by calculating NMR chemical shifts and ECD spectra. It afforded an example of natural epimers with different physical properties, especially crystallization, due to the difference in intermolecular hydrogen bonding. Compounds 9, 10, and 13 showed moderate total antioxidant capacity (0.82 of 9; 0.70 of 10; 0.48 of 13) with Trolox (total antioxidant capacity: 1.0) as a positive control, and compounds 5 and 7 showed anti-inflammatory activity with IC50 values of 35.4 and 40.8 µM, respectively (positive control indomethacin: IC50 = 35.8 µM).

Discovery of Flavonoids as Novel Inhibitors of ATP Citrate Lyase: Structure-Activity Relationship and Inhibition Profiles.

ATP citrate lyase (ACLY) is a key enzyme in glucolipid metabolism and its aberrantly high expression is closely associated with various cancers, hyperlipemia and atherosclerotic cardiovascular diseases. Prospects of ACLY inhibitors as treatments of these diseases are excellent. To date, flavonoids have not been extensively reported as ACLY inhibitors. In our study, 138 flavonoids were screened and 21 of them were subjected to concentration-response curves. A remarkable structure-activity relationship (SAR) trend was found: ortho-dihydroxyphenyl and a conjugated system maintained by a pyrone ring were critical for inhibitory activity. Among these flavonoids, herbacetin had a typical structure and showed a non-aggregated state in solution and a high inhibition potency (IC50 = 0.50 ± 0.08 µM), and therefore was selected as a representative for the ligand-protein interaction study. In thermal shift assays, herbacetin improved the thermal stability of ACLY, suggesting a direct interaction with ACLY. Kinetic studies determined that herbacetin was a noncompetitive inhibitor of ACLY, as illustrated by molecular docking and dynamics simulation. Together, this work demonstrated flavonoids as novel and potent ACLY inhibitors with a remarkable SAR trend, which may help design high-potency ACLY inhibitors. In-depth studies of herbacetin deepened our understanding of the interactions between flavonoids and ACLY.