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Fungi are important sources for the discovery of natural products. During the last decades, technological progress and the increasing number of sequenced genomes facilitated the exploration of new secondary metabolites. Among those, polyketides represent a structurally diverse group with manifold biological activities. In this study, we successfully used genome mining and genetic manipulation for functional proof of a polyketide biosynthetic gene cluster from the filamentous fungus Penicillium crustosum. Gene activation in the native host and heterologous expression in Aspergillus nidulans led to the identification of the xil cluster, being responsible for the formation of the 6-methyl-2-pyrone derivative xylariolide D. Feeding with 13C-labeled precursors supported the hypothesis of chain branching during the backbone formation catalyzed by a highly reducing fungal polyketide synthase. A cytochrome P450-catalyzed hydroxylation converts the PKS product to the final metabolite. This proved that just two enzymes are required for the biosynthesis of xylariolide D. [ABSTRACT FROM AUTHOR]

It is of great significance to synergistically modulate d -band centers (ε d) of multiple transition metals (TMs) for designing and developing highly efficient hybridized-electrocatalysts. Herein, the downshift in Co ε d is realized by synthesizing Co single atoms with low nitrogen-coordination number (CoN 2) at high T during the pyrolysis step while upshifting Ru ε d is achieved via decreasing Ru precursor mass to anchor Ru nanoclusters instead of nanoparticles on CoN 2 −containing carbon substrates (CoN 2 /Ru−NC). In our strategy, the downshift in Co ε d leads to a favorable affinity between Co sites of CoN 2 /Ru−NC and H* for desorption of H 2 ; additionally, upshifting Ru ε d helps the adsorption of OH* intermediates on Ru nanoclusters for CoN 2 /Ru−NC to promote water dissociation toward accelerating alkaline producing H 2. In consequence, CoN 2 /Ru−NC demonstrates an outstanding activity with an ultralow overpotential of ∼ 9.0 mV at 10.0 mA cm−2, which suppresses activities of Pt/C (24.0 mV) and most previously-reported Ru-based electrocatalysts. [Display omitted] • Our strategy anchors Ru nanoclusters on CoN 2 −containing carbon substrates. • Downshifting Co d -band center (ε d) activates Co atoms as catalytical sites. • Upshifting Ru ε d promotes H 2 O dissociation on Ru nanoclusters for alkaline HER. • This work provides a new way to design efficient electrocatalyst by modulating ε d. [ABSTRACT FROM AUTHOR]

Cytochrome P450 monooxygenases (P450s) are considered nature's most versatile catalysts and play a crucial role in regio- and stereoselective oxidation reactions on a broad range of organic molecules. The oxyfunctionalisation of unactivated carbon-hydrogen (C-H) bonds, in particular, represents a key step in the biosynthesis of many natural products as it provides substrates with increased reactivity for tailoring reactions. In this study, we investigated the function of the P450 enzyme TraB in the terrestric acid biosynthetic pathway. We firstly deleted the gene coding for the DNA repair subunit protein Ku70 by using split marker-based deletion plasmids for convenient recycling of the selection marker to improve gene targeting in Penicillium crustosum. Hereby, we reduced ectopic DNA integration and facilitated genetic manipulation in P. crustosum. Afterward, gene deletion in the Δku70 mutant of the native producer P. crustosum and heterologous expression in Aspergillus nidulans with precursor feeding proved the involvement of TraB in the formation of crustosic acid by catalysing the essential hydroxylation reaction of viridicatic acid. Key points: •Deletion of Ku70 by using split marker approach for selection marker recycling. •Functional identification of the cytochrome P450 enzyme TraB. •Fulfilling the reaction steps in the terrestric acid biosynthesis. [ABSTRACT FROM AUTHOR]

Background: Knee osteoarthritis (KOA) is one of the most common musculoskeletal disorders. Acupotomy may be effective for KOA, but the evidence is limited. This trial aims to determine the effectiveness and safety of acupotomy for KOA.Methods/design: This is a parallel-group, assessor-blinded randomized controlled trial. Two hundred patients with KOA will be recruited and randomly assigned to two groups (group A or group D) in a 1:1 ratio. Patients in group A will receive acupotomy and topical diclofenac diethylamine for 4 weeks, while patients in group D will receive topical diclofenac diethylamine alone for 4 weeks. The primary outcome will be the response rate-the proportion of patients who achieve the minimal clinically important improvement in pain and function at week 4 compared with baseline. Secondary outcomes will include pain, function, quality of life, the use of rescue medicine (loxoprofen sodium), and adverse events at weeks 4, 8, and 24 after randomization. Besides, joint fluid and serum will be collected to assess the level of inflammatory cytokines, like TNF-α, IL-1β, and MMP-3.Discussion: This study will contribute to a better understanding of the effectiveness and safety of acupotomy in combination with topical nonsteroidal anti-inflammatory drugs. If the hypothesis is confirmed, acupotomy may be recommended as adjunctive therapy for patients with KOA. Results of the study will be of great importance for the guidelines of clinical therapy.Trial Registration: Chinese Clinical Trial Registry ChiCTR2100043005 Registered on 4 February 2021. [ABSTRACT FROM AUTHOR]

Accumulation of two benzoyl esters in Aspergillus ustus after feeding with alcohols was reported 30 years ago. To the best of our knowledge, the biosynthesis for these esters has not been elucidated prior to this study. Here, we demonstrate that these compounds are artifical products of the phenethyl polyketide ustethylin A biosynthestic pathway. In addition, four aditional benzoyl esters with different methylation levels were also isolated and identified as shunt products. Feeding experiments provided evidence that the enzyme-bound polyketide acyl intermediates are hijacked by externally fed MeOH or EtOH, leading to the formation of the benzoyl esters. [ABSTRACT FROM AUTHOR]

2,5-Diketopiperazines are the smallest cyclic peptides comprising two amino acids connected via two peptide bonds. They can be biosynthesized in nature by two different enzyme families, either by nonribosomal peptide synthetases or by cyclodipeptide synthases. Due to the stable scaffold of the diketopiperazine ring, they can serve as precursors for further modifications by different tailoring enzymes, such as methyltransferases, prenyltransferases, oxidoreductases like cyclodipeptide oxidases, 2-oxoglutarate-dependent monooxygenases and cytochrome P450 enzymes, leading to the formation of intriguing secondary metabolites. Among them, cyclodipeptide synthase-associated P450s attracted recently significant attention, since they are able to catalyse a broader variety of astonishing reactions than just oxidation by insertion of an oxygen. The P450-catalysed reactions include hydroxylation at a tertiary carbon, aromatisation of the diketopiperazine ring, intramolecular and intermolecular carbon-carbon and carbon-nitrogen bond formation of cyclodipeptides and nucleobase transfer reactions. Elucidation of the crystal structures of three P450s as cyclodipeptide dimerases provides a structural basis for understanding the reaction mechanism and generating new enzymes by protein engineering. This review summarises recent publications on cyclodipeptide modifications by P450s. Key Points • Intriguing reactions catalysed by cyclodipeptide synthase-associated cytochrome P450s • Homo- and heterodimerisation of diketopiperazines • Coupling of guanine and hypoxanthine with diketopiperazines [ABSTRACT FROM AUTHOR]

Covering: up to April 2020 Fungal benzene carbaldehydes with salicylaldehydes as predominant representatives carry usually hydroxyl groups, prenyl moieties and alkyl side chains. They are found in both basidiomycetes and ascomycetes as key intermediates or end products of various biosynthetic pathways and exhibit diverse biological and pharmacological activities. The skeletons of the benzene carbaldehydes are usually derived from polyketide pathways catalysed by iterative fungal polyketide synthases. The aldehyde groups are formed by direct PKS releasing, reduction of benzoic acids or oxidation of benzyl alcohols. [ABSTRACT FROM AUTHOR]

NotF from Aspergillus sp. MF297-2 and BrePT from Aspergillus versicolor catalyze a reverse C2-prenylation of brevianamide F in the biosynthetic pathway of brevianamides and notoamides. NotF was reported to use only brevianamide F as substrate while BrePT demonstrated broad substrate promiscuity. With high identity at amino acid level, it is interesting to reinvestigate the catalytic activities of these two prenyltransferases in vitro toward 14 cyclodipeptides. Product identification of the in vitro assays by MS proved that NotF and BrePT share similar catalytic ability and substrate promiscuity. [ABSTRACT FROM AUTHOR]

FgaPT2 from Aspergillus fumigatus catalyzes a Friedel-Crafts alkylation at C-4 of l-tryptophan and is involved in the biosynthesis of the ergot alkaloids fumigaclavines. Several tryptophan-containing cyclic dipeptides had also been prenylated by FgaPT2, but the turnover rate ( k) was low. Here, we report the generation of FgaPT2 mutants by saturation mutagenesis at the amino acid residue Arg244 to improve its catalytic efficiency toward cyclic dipeptides. Thirteen mutated enzymes demonstrated up to 76-fold higher turnover number toward seven cyclic dipeptides than the non-mutated FgaPT2. More importantly, the mutated enzymes exhibited different preferences toward these substrates. This study provides a convenient approach for creation of new biocatalysts for production of C4-prenylated cyclic dipeptides. [ABSTRACT FROM AUTHOR]

The purpose of this article was to explore the influences of alkalis types on the cellulose/Mn 3 O 4 composites via a sonochemistry method. In this study, cellulose/Mn 3 O 4 composites were successfully fabricated using four types of alkalis (urea (CO(NH 2 ) 2 ), hexamethylenetetramine ((CH 2 ) 6 N 4 , HMT), NaOH, and KOH) by an environmentally-friendly sonochemistry method. The phase, shape, thermal stability, and the formation mechanism of the cellulose composites were researched in detail. Experimental results demonstrated that the types of alkalis played an important role in the phase, shape, dispersion, and thermal stability of cellulose/Mn 3 O 4 composites. By thermal treatment of cellulose/Mn 3 O 4 composites at 600 °C for 3 h in air, the Mn 3 O 4 crystals were obtained. This novel method reported here maybe has a guiding significance for the synthesis of manganese oxide materials and other metal oxides using cellulose as template. [ABSTRACT FROM AUTHOR]

7-DMATS from Aspergillus fumigatus and SirD from Leptosphaeria maculans catalyse a C 7-prenylation of l -tryptophan and an O -prenylation of l -tyrosine in nature, respectively. SirD was reported to catalyse the C 7-prenylation of l -tryptophan and some derivatives thereof in vitro. We report here the O-prenylation of tyrosine and O - or N -prenylation of its derivatives by 7-DMATS. These results provide experimental evidence for the close relationship of tyrosine O - and tryptophan C 7-prenyltransferases regarding their substrate and catalytic promiscuity. [ABSTRACT FROM AUTHOR]

Highlights: [•] Hybrids from cellulose and silver were successfully prepared. [•] The hydrothermal method was introduced. [•] Holocellulose was used as the reactant. [•] The reaction parameters have influence on the hybrids. [•] Fructose and glucose as reducing reagents have been comparing studied. [ABSTRACT FROM AUTHOR]

Highlights: [•] Hybrids from lignocelluloses and Ag were successfully prepared. [ • ] Environmentally-friendly sonochemistry method was introduced. [•] The reaction parameters have influence on the hybrids. [•] Reducing reagents favored the growth of Ag nanostructures. [ABSTRACT FROM AUTHOR]

A big challenge in organic synthesis is to reach a high regioselectivity. Enzymes catalyze usually highly regiospecific reactions and can function as ideal biocatalysts for such purposes. Some secondary metabolite enzymes can even use distinctly different unnatural substrates and expand therefore their potential usage in chemoenzymatic synthesis. We report here the acceptance of benzyl diphosphate as an alkyl donor by the fungal dimethylallyl transferase FgaPT2 and the regiospecific enzymatic benzylation of tryptophan and several analogues. [ABSTRACT FROM AUTHOR]

Prenylated indole alkaloids derived from L-tryptophan are widely distributed in nature and show diverse biological and pharmacological activities, usually distinct from their non-prenylated precursors. Prenyltransferases catalyze the transfer reactions of prenyl moieties onto the indole nucleus and contribute largely to the structural diversity of these compounds. In this study, we demonstrate the acceptance of cyclo- L-homotryptophan- D-valine, an unnatural cyclic dipeptide, by eight prenyltransferases of the dimethylallyltryptophan synthase superfamily. Seven products with one prenyl moiety at each position of the indole nucleus and one diprenylated derivative were isolated from enzyme assays of cyclo- L-homotryptophan- D-valine with dimethylallyl diphosphate. To the best of our knowledge, this is the first report for production of seven monoprenylated products from one substrate by one-step reactions. [ABSTRACT FROM AUTHOR]

Highlights: [•] Cellulose/TiO2 hybrids were prepared by hydrothermal method. [•] The experimental factors have influence on the phase and shape of hybrids. [•] Cellulose/TiO2 hybrids possess a high antimicrobial activity. [•] TiO2 was obtained by calcination the hybrids. [ABSTRACT FROM AUTHOR]

Abstract: The aim of this study was to investigate the synthesis of inorganic materials using cellulose as a template by thermal treatment of the precursor. Cellulose-based composites have been successfully fabricated by an efficient microwave-assisted method. The influences of heating time on the phases and shape of the precursor were investigated. Mn2O3 materials were obtained by thermal treatment of the precursor at 600°C for 3h in air. The morphology of cellulose composites was preserved after thermal transformation to form Mn2O3. The products were characterized by X-ray powder diffraction (XRD), Fourier transform infrared spectrometry (FTIR), energy-dispersive X-ray spectra (EDS), scanning electron microscopy (SEM), thermogravimetric analysis (TGA), and differential thermal analysis (DTA). A rapid microwave-assisted method with the thermal post-treatment provides a promising route for the fabrication of inorganic materials using natural polymer as a template. [Copyright &y& Elsevier]

Abstract: By means of a simultaneous microwave-assisted method and a simple chemical reaction, cellulose/AgCl nanocomposites have been successfully synthesized using cellulose solution and AgNO3 in N,N-dimethylacetamide (DMAc) solvent. The cellulose solution was firstly prepared by the dissolution of the microcrystalline cellulose and lithium chloride (LiCl) in DMAc. DMAc acts as both a solvent and a microwave absorber. LiCl was used as the reactant to fabricate AgCl crystals. The effects of the heating time and heating temperature on the products were studied. This method is based on the simultaneous formation of AgCl nanoparticles and precipitation of the cellulose, leading to a homogeneous distribution of AgCl nanoparticles in the cellulose matrix. The experimental results confirmed the formation of cellulose/AgCl nanocomposites with high-purity, good thermal stability and antimicrobial activity. This rapid, green and environmentally friendly microwave-assisted method opens a new window to the high value-added applications of biomass. [Copyright &y& Elsevier]

AuaA is a membrane-bound farnesyltransferase from the myxobacterium Stigmatella aurantiaca involved in the biosynthesis of aurachins. Like other known membrane-bound aromatic prenyltransferases, AuaA contains two conserved aspartate-rich motifs. Several amino acids in the first motif NXxxDxxxD were proposed to be responsible for prenyl diphosphate binding via metal ions like Mg. Site-directed mutagenesis experiments demonstrated in this study that asparagine, but not the arginine residue in NRxxDxxxD, is important for the enzyme activity of AuaA, differing from the importance of NQ or ND residues in the NQxxDxxxD or NDxxDxxxD motifs observed in some membrane-bound prenyltransferases. The second motif of known membrane-bound prenyltransferases was proposed to be involved in the binding of their aromatic substrates. KDIxDxEGD, also found in AuaA, had been previously speculated to be characteristic for binding of flavonoids or homogenisate. Site-directed mutagenesis experiments with AuaA showed that KDIxDxEGD was critical for the enzyme activity. However, this motif is very likely not specific for flavonoid or homogenisate prenyltransferases, because none of the tested flavonoids was accepted by AuaA or its mutant R53A in the presence of farnesyl, geranyl or dimethylallyl diphosphate. [ABSTRACT FROM AUTHOR]

Abstract: Bionanocomposites with the combination of natural polymers and inorganic nanoparticles may induce unique properties and exhibit promising functions for different applications. Herein, we report a hydrothermal route to the preparation of cellulose/CaCO3 bionanocomposites using the cellulose solution, Ca(NO3)2·4H2O and Na2SiO3·9H2O. The cellulose solution was previously prepared by the dissolution of microcrystalline cellulose in NaOH–urea aqueous solution. The urea also acts as the CO3 2− source for the synthesis of CaCO3. The influences of several reaction parameters, such as the heating time, the heating temperature, and the types of additives on the products were investigated by X-ray powder diffraction, Fourier transform infrared spectrometry, scanning electron microscopy, thermogravimetric analysis, and differential thermal analysis. The experimental results demonstrated that the hydrothermal conditions had an effect on the morphology of the bionanocomposites. Cytotoxicity experiments indicated that the cellulose/CaCO3 bionanocomposites had good biocompatibility, so that the bionanocomposites could be ideal candidate for practical biomedical applications. [Copyright &y& Elsevier]

Abstract: Fabrication of biomass materials by a microwave-assisted method in ionic liquids allows the high value-added applications of biomass by combining three major green chemistry principles: using environmentally preferable solvents, using an environmentally friendly method, and making use of renewable biomass materials. Herein, we report a rapid and green microwave-assisted method for the synthesis of the cellulose/calcium silicate nanocomposites in ionic liquids and recycled ionic liquids. These calcium silicate nanoparticles or nanosheets as prepared were homogeneously dispersed in the cellulose matrix. The experimental results confirm that the ionic liquids can be used repeatedly. Of course, the slight differences were also observed using ionic liquids and recycled ionic liquids. Compared with other conventional methods, the rapid, green, and environmentally friendly microwave-assisted method in ionic liquids opens a new window to the high value-added applications of biomass. [Copyright &y& Elsevier]

Abstract: A facile microwave-assisted method was developed to fabricate cellulose–silver nanocomposites by reducing silver nitrate in ethylene glycol (EG). EG acts as a solvent, a reducing reagent, and a microwave absorber in the whole system, thus no additional reductant is needed. The influences of the heating time and heating temperature on the products were investigated. The products were characterized by X-ray diffraction (XRD), Fourier transform infrared (FT-IR), and scanning electron microscope (SEM). The thermal stability of cellulose–silver nanocomposites in nitrogen and air was studied using thermogravimetric analysis (TG) and differential scanning calorimetric analysis (DSC). Also, the cellulose–silver nanocomposites possess a high antimicrobial activity against the model microbes Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive). It is expected that the cellulose–silver nanocomposites are a promising material for the application in the biomedical field. [Copyright &y& Elsevier]

Abstract: Preparation of cellulose-based nanocomposites by microwave-assisted ionic liquid method allows the high value-added applications of cellulose by combining three major green chemistry principles: using environmentally preferable solvents, environmentally friendly method and renewable biomaterials. In this paper, we report the microwave-assisted ionic liquid method for the fast controlled synthesis of the cellulose/calcium silicate nanocomposites by using the microcrystalline cellulose, Ca(NO3)2·4H2O and Na2SiO3·9H2O in ethylene glycol. The calcium silicate nanoparticles were homogeneously dispersed in the cellulose matrix. The experimental results showed that the additive of ionic liquid favored the composite of cellulose and calcium silicate. The weight loss of nanocomposites was decreased with the increasing ionic liquid concentrations. The influences of heating times, heating temperatures, and ionic liquid concentrations on the products were investigated. This method is fast, environmentally friendly and suitable for the large-scale production of cellulose-based nanocomposites. [Copyright &y& Elsevier]

Abstract: A simple, rapid, and efficient microwave-assisted synthesis of cellulose–silver nanocomposites with silver nanoparticles homogeneously dispersed in the cellulose matrix using cellulose solution, AgNO3 and ascorbic acid in N,N-dimethylacetamide (DMAc) is reported. The cellulose solution was prepared by the dissolution of microcrystalline cellulose in a solvent system of lithium chloride (LiCl)/DMAc. The effects of the microwave heating times and ascorbic acid concentration on the nanocomposites were investigated. The microstructure, size, morphology, and thermal properties of these nanocomposites were analyzed with X-ray diffraction (XRD), Fourier transform infrared (FT-IR), thermogravimetric analysis (TG), differential scanning calorimetric analysis (DSC), and scanning electron microscopy (SEM). The results revealed that the ascorbic acid concentration played an important role in the phase of the nanocomposites. This work provided a promising way to prepare cellulose–silver nanocomposites with good dispersity. [ABSTRACT FROM AUTHOR]

Abstract: Cellulose–calcium silicate nanocomposites with calcium silicate nanoparticles homogeneously dispersed in the cellulose matrix have been successfully synthesized using cellulose solution, Ca(NO3)2·4H2O and Na2SiO3·9H2O in ethanol/water mixed solvents at room temperature for 24h. The cellulose solution was previously prepared by the dissolution of cellulose in a solvent system of N,N-dimethylacetamide (DMAc)/lithium chloride (LiCl). The feeding order had an influence on the morphology of the cellulose–calcium silicate nanocomposites and the size of the calcium silicate particles. The cellulose in nanocomposites showed cellulose type II crystalline structure. The products were characterized by X-ray powder diffraction (XRD), thermogravimetric analysis (TG), differential scanning calorimetric analysis (DSC), Fourier transform infrared spectrometry (FT-IR), energy-dispersive X-ray spectra (EDS), and scanning electron microscopy (SEM). [Copyright &y& Elsevier]

The article presents a study regarding the function and role of the putative prenyltransferase gene SirD in the biosynthesis of the phytotoxin sirodesmin PL in Leptosphaeria maculans. It says that SirD was identified in the gene cluster which encodes the biosynthesis of sirodesmin PL, whose gene products was 449 aa. The methods used include sequence analysis, DNA isolation, and fungal culture. Results of the study show higher substrate flexibility of SirD compared with 7-DMATS.

Abstract: A series of putative indole prenyltransferase genes could be identified in the genome sequences of different fungal strains including Aspergillus fumigatus and Neosartorya fischeri. The gene products show significant sequence similarities to dimethylallyltryptophan synthases from different fungi. We have cloned and overexpressed seven of these genes, fgaPT1, fgaPT2, ftmPT1, ftmPT2, 7-dmats, cdpNPT and anaPT in Escherichia coli and Saccharomyces cerevisiae. The overproduced enzymes were characterised biochemically. Three additional indole prenyltransferases, DmaW-Cs, TdiB and MaPT were also identified and characterised in the last years. Sequence analysis and comparison with known aromatic prenyltransferases as well as biochemical investigation revealed that these enzymes belong to a group of aromatic prenyltransferases. The characterised prenyltransferases are soluble proteins, catalyse different prenyl transfer reactions on indole moieties of various substrates and do not require divalent metal ions for their prenyl transfer reactions. In addition, indole prenyltransferases carry tryptophan aminopeptidase activity, which strengths their relationship in the evolution. These properties differ clearly from membrane-bound aromatic prenyltransferases from different sources and soluble prenyltransferases from bacteria. All of the indole prenyltransferases accepted only dimethylallyl diphosphate as prenyl donor. On the other hand, they showed broad substrate specificity towards their aromatic substrates. Diverse simple tryptophan derivatives and tryptophan-containing cyclic dipeptides were accepted by these enzymes, providing a strategy for convenient production of biologically active substances, e.g. by chemoenzymatic synthesis. [Copyright &y& Elsevier]

Summary: Recently, five indole prenyltransferases from Aspergillus fumigatus have been proven biochemically to be responsible for prenylations of diverse substrates. In this study, we show peptidase activities of 7-DMATS, FgaPT1, CdpNPT, and FtmPT1, with preference for linear peptides containing a tryptophanyl moiety at the N terminus. Testing of 31 peptides revealed that these enzymes shared similar substrate specificity and accepted H-L-Trp-L-Ala-OH and H-L-Trp-Gly-OH as best substrates for aminopeptidase activity. By using H-L-Trp-Gly-OH as substrate, Km values at 350, 380, 300, and 420 μM and enzymatic rate constants kcat/Km at 0.51, 0.24, 0.53, and 0.14 mM−1s−1 were determined for 7-DMATS, FgaPT1, CdpNPT, and FtmPT1, respectively. In contrast to prenyltransferase activities, the aminopeptidase activities were strongly or completely inhibited by EDTA. Mn2+ increased the aminopeptidase activities of FtmPT1 and CdpNPT up to 4- and 6-fold, respectively. To the best of our knowledge, this is the first report on the catalytic promiscuity of prenyltransferases. [Copyright &y& Elsevier]

Recently, a gene for a 7-dimethylallyltryptophan synthase (7-DMATS) was identified in Aspergillus fumigatus and its enzymatic function was proven biochemically. In this study, the behaviour of 7-DMATS towards aromatic substrates was investigated and compared with that of the 4-dimethylallyltryptophan synthase FgaPT2 from the same fungus. In total, 24 simple indole derivatives were tested as potential substrates for 7-DMATS. With an exception of 7-methyltryptophan, all of the substances were accepted by 7-DMATS and converted to their prenylated derivatives, indicating a more flexible substrate specificity of 7-DMATS in comparison to that of FgaPT2. The relative activities of 7-DMATS towards these substrates were from 4% to 89% of that of l-tryptophan, much higher than that of FgaPT2. Structural elucidation of the isolated enzymatic products by nuclear magnetic resonance and mass spectrometry analysis proved unequivocally the prenylation at position C7 of the indole ring. Overnight incubation with eight substances showed that the conversion ratios were in the range of 55.9% to 99.7%. This study provided an additional example that prenylated indole derivatives can be effectively produced by using the overproduced and purified 7-DMATS. [ABSTRACT FROM AUTHOR]

Cyclodipeptides (CDPs) build an important class of secondary metabolites with a plethora of pharmacological impact such as antibiotic, cytotoxic, antifungal properties or even as anti‐Alzheimer drug candidates. The core structure of 2,5‐diketopiperazines, a subclass of CDPs, is composed of two 'head‐to‐tail' connected α‐amino acids. Their chemical diversity is broadened by tailoring enzymes as members of their biosynthetic gene clusters, such as CDP‐dimerizing P450s. Recently, a new class of P450 enzymes, termed GymBx, was identified in the biosynthetic pathway of guatyromycines. These enzymes catalyse both an intramolecular C−C bond formation within cyclo‐l‐Tyr‐l‐Tyr (cYY) and a nucleobase transfer reaction, which is unique for CDP‐modifying P450 enzymes. In this study, we report the structures of two members, GymB1 and GymB5, in their unbound states, as well as GymB5 in complex with hypoxanthine and cYY. Structural data of the ternary complex shed light into CDP binding and nucleobase transfer reaction, identified key residues for substrate recognition and modified the enzymatic bifunctionality. By mutagenesis we successfully established a GymB5 variant that strictly switched the chemoselectivity of the reaction towards an intermolecular coupling enzyme. This data set a solid basis for future protein engineering of P450 enzymes to synthesize new CDP‐nucleobase adducts. [ABSTRACT FROM AUTHOR]

Transgenic Nicotiana tabacum cell cultures formed a new, artificial secondary metabolite, i.e. 4-hydroxyben-zoate (4HB), by expression of the bacterial gene ubiC, which encodes chorismate pyruvate-lyase. 4HB was converted in the cells to two different glucosides, i.e. 4-O-(1-rβ-D-gIucosyl)benzoic acid (4HB0G) and 4HB l-rβ-D-glucosyl ester (4HBCOOG). The same metabolization was also observed with exogenously fed 4HB. This glucosylation is catalyzed by constitutively expressed glucosyltransferases which could be detected in cell-free extracts of transgenic and untransformed cells in the same activity. The enzymes forming the two respective glucosides differ in their pH optima, their affinity for 4HB and their regulation. Both in vivo and in cell-free extracts, 4HBOG is the main product at low 4HB concentration, and 4HBCOOG at high 4HB concentration. Whereas 4HB0G is accumulated as an apparently stable secondary metabolite, 4HBCOOG presents a metabolically active form of 4HB. After feeding of [U-14C]4HB, a transient accumulation of 4HBC00G was followed by incorporation of radioactive 4HB into the cell wall, suggesting that 4HBCOOG, but not 4HBOG serves as precursor of cell wall bound 4HB [ABSTRACT FROM AUTHOR]

• Systemic inflammation induces cognitive impairments via unclear mechanisms. • C3/C3a receptor signaling plays a key role in microglia activation and cognition. • LPS selectively induced inhibitory but not excitatory synapse related protein loss. Systemic inflammation induces cognitive impairments via unclear mechanisms. Increasing evidence has suggested complement C3/C3a receptor signaling, a key component of innate immune pathogen defense, plays an important role in cognition and neurodegeneration, whereas its dysfunction is implicated in many neurological disorders. However, it remains unclear whether complement C3/C3a receptor signaling was involved in systemic inflammation-induced cognitive impairments. In the present study, we showed that hippocampal complement C3 levels in astrocytes and C3a receptor expressions in microglia were specifically up-regulated after lipopolysaccharide (LPS) injection. Interestingly, LPS selectively induced inhibitory but not excitatory synapse related protein loss. Notably, C3a receptor antagonist SB290157 trifluoroacetate attenuated LPS-induced hippocampal neuroinflammation and inhibitory synapse related protein loss, contributing to improved cognitive function. In conclusion, our study suggests that complement C3/C3a receptor signaling plays a key role in LPS-induced cognitive impairments, which may serve a therapeutic target for systemic inflammation related cognitive disorders. [ABSTRACT FROM AUTHOR]

Abstract: The purpose of this paper is to explore the synthesis of biomass-based composites by green method in green solution. Cellulose/F-substituted hydroxyapatite (HA) nanocomposites have been successfully fabricated by the microwave-assisted ionic liquids (ILs) method. The effects of the heating time, the ionic liquids concentration, and the NaF concentration on the products were investigated. The experimental results demonstrated that F-substituted HA particles were embedded in the cellulose matrix or dispersed on the surface of cellulose matrix. The XRD results indicated that the cellulose/CaF nanocomposites were obtained at high NaF concentration. The ionic liquids concentration played an important role in the dispersion of particles. The number of F-substituted HA in nanocomposites increased with increasing heating time. This rapid and environmentally friendly microwave-assisted ionic liquids method is provided a promising way to prepare cellulose-based nanocomposites. [Copyright &y& Elsevier]

Abstract: The Ag/calcium silicate nanocomposite with core-shell nanostructure has been successfully synthesized using Ag solution, Ca(NO3)2·4H2O and Na2SiO3·9H2O in ethanol/water mixed solvents at room temperature for 48h. Ag solution was previously prepared by microwave-assisted method in ethylene glycol (EG) at 150°C for 10min. The nanocomposites consisted of Ag core and an amorphous calcium silicate shell. The XRD and EDS results confirmed that the product was the Ag/calcium silicate nanocomposite. The TEM micrographs indicated that the Ag/calcium silicate nanocomposite was core-shell nanoparticles. The effects of Ca(NO3)2·4H2O and Na2SiO3·9H2O concentration on the shells of Ag/calcium silicate nanocomposite were investigated. The products were characterized by X-ray powder diffraction (XRD), transmission electron microscopy (TEM), and energy-dispersive X-ray spectra (EDS). This method is simple, fast and may be extended to the synthesis of the other kinds of core-shell nanocomposites. [Copyright &y& Elsevier]

Abstract: We report the microwave-assisted synthesis of the cellulose-carbonated hydroxyapatite (CHA) nanocomposites with CHA nanostructures dispersed in the cellulose matrix by using the cellulose solution, CaCl2, and NaH2PO4. The cellulose solution was previously prepared by the dissolution of microcrystalline cellulose in NaOH–urea aqueous solution. The influences of the heating time and cellulose concentration on the products were also investigated. The X-ray powder diffraction (XRD) and Fourier transform infrared spectrometry (FT-IR) results indicated that the obtained products were the cellulose–CHA nanocomposites. The scanning electron microscopy (SEM) micrographs showed the CHA nanostructures were dispersed in the cellulose matrix. The thermal stability of the cellulose–CHA nanocomposites in air was investigated using thermogravimetric analysis (TGA) and differential thermal analysis (DTA). This method is simple, fast, low-cost and suitable for large-scale production of cellulose-based nanocomposites. [ABSTRACT FROM AUTHOR]

The cobia Rachycentron canadum, mainly distributed in the warm waters of tropical and subtropical regions around the world, remains a fish of considerable economic importance. Detailed diversity and the number of microsatellite sequences in the cobia genome are still unintelligible. The primary aim of this work was to identify and quantify the miscellaneous SSR sequences in the cobia genome. More than 280,000 sequences were sequenced and screened using next‐generation sequencing technology and microsatellite identification. Perfect mononucleotide repeats, dinucleotide microsatellites, and trinucleotide microsatellites contain (A)10/(T)10, (AC)6/(TG)6, and (AAT)5–32 as the largest number of motifs in each type of microsatellite, respectively. The tetranucleotide and pentanucleotide microsatellites (TTM and PTM) consist of the largest number of motifs of both (ATCT)5–32 and (TCAT)5–31 in TTMs, and (CTCTC)5–9 in PTMs, whereas the hexanucleotide microsatellites are rarely observed in the cobia genome. All c. 38000 sequences of composite microsatellites are extremely diverse, including compound (11.71%), interrupted compound (71.77%), complex (0.45%), and interrupted complex (16.07%). In this study, we developed a convenient and useful recording system for writing down and categorizing diverse composite microsatellite types. This system will provide great support for exploring repeat origins, evolutionary mechanisms, and the application of polymorphic microsatellites. [ABSTRACT FROM AUTHOR]

Prenyltransferases (PTs) from the dimethylallyl tryptophan synthase (DMATS) superfamily are known as efficient biocatalysts and mainly catalyze regioselective Friedel-Crafts alkylation of tryptophan and tryptophan-containing cyclodipeptides (CDPs). They can also use other unnatural aromatic compounds as substrates and play therefore a pivotal role in increasing structural diversity and biological activities of a broad range of natural and unnatural products. In recent years, several prenylated dimeric CDPs have been identified with wide range of bioactivities. In this study, we demonstrate the production of prenylated dimeric CDPs by chemoenzymatic synthesis with a known promiscuous enzyme EchPT1, which uses cyclo-l-Trp-l-Ala as natural substrate for reverse C2-prenylation. High product yields were achieved with EchPT1 for C3-N1′ and C3-C3′ linked dimers of cyclo-l-Trp-l-Trp. Isolation and structural elucidation confirmed the product structures to be reversely C19/C19′-mono- and diprenylated cyclo-l-Trp-l-Trp dimers. Our study provides an additional example for increasing structural diversity by prenylation of complex substrates with known biosynthetic enzymes. Key points: • Chemoenzymatic synthesis of prenylated cyclo-l-Trp-l-Trp dimers • Same prenylation pattern and position for cyclodipeptides and their dimers. • Indole prenyltransferases such as EchPT1 can be widely used as biocatalysts. [ABSTRACT FROM AUTHOR]

Genome mining revealed the presence of two cdps-p450 operons in Saccharopolyspora antimicrobica. Heterologous expression, biochemical characterisation and structure elucidation proved that the two P450 enzymes catalyse distinct regio- and stereospecific dimerizations of cyclo-(L -Trp- L -Trp), which significantly expands the repertoire of diketopiperazine-tailoring enzymes. TtpB1 connects the monomers via C3–C3′, both from the opposite side of H-11/H-11′, while TtpB2 is characterised as the first P450 to mainly catalyse the unusual linkage between N1′ and C3 from the H-11 side. [ABSTRACT FROM AUTHOR]

Mining microbial genomes including those of Streptomyces reveals the presence of a large number of biosynthetic gene clusters. Unraveling this genetic potential has proved to be a useful approach for novel compound discovery. Here, we report the heterologous expression of two similar P450‐associated cyclodipeptide synthase‐containing gene clusters in Streptomyces coelicolor and identification of eight rare and novel natural products, the C3‐guaninyl indole alkaloids guanitrypmycins. Expression of different gene combinations proved that the cyclodipeptide synthases assemble cyclo‐l‐Trp‐l‐Phe and cyclo‐l‐Trp‐l‐Tyr, which are consecutively and regiospecifically modified by cyclodipeptide oxidases, cytochrome P450 enzymes, and N‐methyltransferases. In vivo and in vitro results proved that the P450 enzymes function as key biocatalysts and catalyze the regio‐ and stereospecific 3α‐guaninylation at the indole ring of the tryptophanyl moiety. Isotope‐exchange experiments provided evidence for the non‐enzymatic epimerization of the biosynthetic pathway products via keto–enol tautomerism. This post‐pathway modification during cultivation further increases the structural diversity of guanitrypmycins. [ABSTRACT FROM AUTHOR]

Mining microbial genomes including those of Streptomyces reveals the presence of a large number of biosynthetic gene clusters. Unraveling this genetic potential has proved to be a useful approach for novel compound discovery. Here, we report the heterologous expression of two similar P450‐associated cyclodipeptide synthase‐containing gene clusters in Streptomyces coelicolor and identification of eight rare and novel natural products, the C3‐guaninyl indole alkaloids guanitrypmycins. Expression of different gene combinations proved that the cyclodipeptide synthases assemble cyclo‐l‐Trp‐l‐Phe and cyclo‐l‐Trp‐l‐Tyr, which are consecutively and regiospecifically modified by cyclodipeptide oxidases, cytochrome P450 enzymes, and N‐methyltransferases. In vivo and in vitro results proved that the P450 enzymes function as key biocatalysts and catalyze the regio‐ and stereospecific 3α‐guaninylation at the indole ring of the tryptophanyl moiety. Isotope‐exchange experiments provided evidence for the non‐enzymatic epimerization of the biosynthetic pathway products via keto–enol tautomerism. This post‐pathway modification during cultivation further increases the structural diversity of guanitrypmycins. [ABSTRACT FROM AUTHOR]

The fungal prenyltransferase ShPT from Stereum hirsutum was believed to prenylate 4-hydroxybenzyl alcohol and thereby be involved in the vibralactone biosynthesis. In this study, we demonstrate that hydroxynaphthalenes instead of benzyl alcohol or aldehyde were accepted by ShPT for regular C-prenylation in the presence of both dimethylallyl and geranyl diphosphate. Although the natural substrate of ShPT remains unknown, our results provide one additional prenyltransferase from basidiomycetes, which are less studied, in comparison to those from other sources. Furthermore, this study expands the chemical toolbox for regioselective production of prenylated naphthalene derivatives. Key points: •Basidiomycetous prenyltransferase •Biochemical characterization •A DMATS prenyltransferase prenylating hydroxynaphthalene derivatives [ABSTRACT FROM AUTHOR]

Background. Systemic inflammation impairs cognitive performance, yet the brain networks mediating this process remain to be elucidated. The purpose of the current study was to use resting-state functional magnetic resonance imaging (fMRI) to explore changes in the functional connectivity in a lipopolysaccharide- (LPS-) induced systemic inflammation animal model. Materials and Methods. We used the regional homogeneity (ReHo) method to examine abnormal brain regions between the control and LPS groups and then considered them as seeds of functional connectivity analysis. Results. Compared with the control group, our study showed that (1) LPS impaired mood function, as reflected by a depression-like behavior in the forced swim test; (2) LPS induced significantly increased ReHo values in the anterior cingulate cortex (ACC) and caudate putamen (CPu); (3) the ACC seed showed increased functional connectivity with the retrosplenial cortex, superior colliculus, and inferior colliculus; and (4) the right CPu seed showed increased functional connectivity with the left CPu. Linear regression analysis showed a LPS-induced depression-like behavior which was associated with increased ReHo values in the ACC and right CPu. Moreover, the LPS-induced depression-like behavior was related to increased functional connectivity between the right CPu and left CPu. Conclusion. This is the first study to show that systemic inflammation impairs mood function that is associated with an altered resting-state functional network based on ReHo analysis, providing evidence of the abnormal regional brain spontaneous activity which might be involved in inflammation-related neurobehavioral abnormalities. [ABSTRACT FROM AUTHOR]

In nature, cyclic dipeptide prenyltransferases catalyze regioselective Friedel-Crafts alkylations of tryptophan-containing cyclic dipeptides. This enzyme class, belonging to the dimethylallyl tryptophan synthase superfamily, is known to be flexible toward aromatic prenyl acceptors, while mostly retaining its typical regioselectivity. Ardeemin fumiquinazoline (FQ) (1), a tryptophan-containing cyclic tripeptide derivative, is assembled in Aspergillus fischeri by the non-ribosomal peptide synthetase ArdA and modified by the prenyltransferase ArdB, leading to the pharmaceutically active hexacyclic ardeemin. Therefore, 1 and its enantiomer ent-ardeemin FQ (2) constitute potential substrates for aromatic prenyltransferases. In this study, we investigated the acceptance of both enantiomers by two cyclic dipeptide C2-prenyltransferases BrePT and FtmPT1 and three C3-prenyltransferases CdpNPT, CdpC3PT, and AnaPT. LC-MS analysis of the incubation mixtures and NMR analysis of the isolated products revealed that the stereochemistry at C11 and C14 in 1 and 2 has a strong influence on their acceptance by these enzymes and the regioselectivity of the prenylation reactions. 1 was very well accepted by BrePT, FtmPT1, and CdpNPT, with C2- or C3-prenylated derivatives as predominant products, which fills the prenylation gaps by tryptophan prenyltransferases reported in a previous study. 2 was a poor substrate for all the enzymes and converted with low regioselectivity and mainly prenylated at C6 and C7 of the indole moiety. [ABSTRACT FROM AUTHOR]

The transannular disulfide functions as a key structural element imparting diverse biological activities to epidithiodiketopiperazines (ETPs). Although mechanisms were proposed in previous studies, α,β′‐disulfide formation in ETPs is not well‐determined owing to the failure to identify the hypothetical intermediate. Herein, we characterize the key ortho‐quinone methide (o‐QM) intermediate and prove its involvement in the carbon‐sulfur migration from an α,α′‐ to an α,β′‐disulfide by elucidating pretrichodermamide A biosynthesis, which is catalyzed by a FAD‐dependent thioredoxin oxygenase TdaE harboring a noncanonical CXXQ motif. Biochemical investigations of recombinant TdaE and mutants demonstrated that the construction of the α,β′‐disulfide was initiated by Gln140 triggering proton abstraction for generation of the essential o‐QM intermediate, accompanied by β′‐acetoxy elimination. Subsequent attack on the α,α′‐disulfide by Cys137 led to disulfide migration and spirofuran formation. This study expands the biocatalytic toolbox for transannular disulfide formation and sets the stage for the targeted discovery of bioactive ETPs. [ABSTRACT FROM AUTHOR]

The transannular disulfide functions as a key structural element imparting diverse biological activities to epidithiodiketopiperazines (ETPs). Although mechanisms were proposed in previous studies, α,β′‐disulfide formation in ETPs is not well‐determined owing to the failure to identify the hypothetical intermediate. Herein, we characterize the key ortho‐quinone methide (o‐QM) intermediate and prove its involvement in the carbon‐sulfur migration from an α,α′‐ to an α,β′‐disulfide by elucidating pretrichodermamide A biosynthesis, which is catalyzed by a FAD‐dependent thioredoxin oxygenase TdaE harboring a noncanonical CXXQ motif. Biochemical investigations of recombinant TdaE and mutants demonstrated that the construction of the α,β′‐disulfide was initiated by Gln140 triggering proton abstraction for generation of the essential o‐QM intermediate, accompanied by β′‐acetoxy elimination. Subsequent attack on the α,α′‐disulfide by Cys137 led to disulfide migration and spirofuran formation. This study expands the biocatalytic toolbox for transannular disulfide formation and sets the stage for the targeted discovery of bioactive ETPs. [ABSTRACT FROM AUTHOR]

Cyclodipeptide synthases (CDPSs) comprise normally 200-300 amino acid residues and are mainly found in bacteria. They hijack aminoacyl-tRNAs from the ribosomal machinery for cyclodipeptide formation. In this study, nine new CDPS genes from eight Streptomyces strains were cloned into pET28a vector and expressed in Escherichia coli. Structural elucidation of the isolated products led to the identification of one cyclo-l-Trp-l-Leu, two cyclo-l-Trp-l-Pro, and three cyclo-l-Trp-l-Trp synthases. Other three CDPSs produce cyclo-l-Trp-l-Ala or cyclo-l-Trp-l-Tyr as the major cyclodipeptide. Total product yields of 46 to 211 mg/L E. coli culture were obtained. Our findings represent rare examples of CDPS family derived from actinobacteria that form various tryptophan-containing cyclodipeptides. Furthermore, this study highlights the potential of the microbial machinery for tryptophan-containing cyclodipeptide biosynthesis and provides valid experimental basis for further combination of these CDPS genes with other modification genes in synthetic biology. [ABSTRACT FROM AUTHOR]

Fungal epidithiodiketopiperazines (ETPs) possess large structural diversity and complexity due to modifications of the cyclodipeptide skeleton. Elucidation of the biosynthetic pathway of pretrichodermamide A (1) in Trichoderma hypoxylon revealed a flexible catalytic machinery of multiple enzymes for generating ETP diversity. Seven tailoring enzymes encoded by the tda cluster are involved in 1 biosynthesis, that is, four P450s TdaB and TdaQ for 1,2‐oxazine formation, TdaI for C7′‐hydroxylation, and TdaG for C4, C5‐epoxidation, two methyltransferases TdaH for C6′‐ and TdaO for C7′‐O‐methylation, and a reductase TdaD for furan opening. Gene deletions led to the identification of 25 novel ETPs, including 20 shunt products, indicating the catalytic promiscuity of Tda enzymes. Particularly, TdaG and TdaD accept various substrates and catalyze regiospecific reactions at different stages of 1 biosynthesis. Our study not only uncovers a hidden library of ETP alkaloids, but also helps to understand the hidden chemical diversity of natural products by pathway manipulation. [ABSTRACT FROM AUTHOR]

Fungal epidithiodiketopiperazines (ETPs) possess large structural diversity and complexity due to modifications of the cyclodipeptide skeleton. Elucidation of the biosynthetic pathway of pretrichodermamide A (1) in Trichoderma hypoxylon revealed a flexible catalytic machinery of multiple enzymes for generating ETP diversity. Seven tailoring enzymes encoded by the tda cluster are involved in 1 biosynthesis, that is, four P450s TdaB and TdaQ for 1,2‐oxazine formation, TdaI for C7′‐hydroxylation, and TdaG for C4, C5‐epoxidation, two methyltransferases TdaH for C6′‐ and TdaO for C7′‐O‐methylation, and a reductase TdaD for furan opening. Gene deletions led to the identification of 25 novel ETPs, including 20 shunt products, indicating the catalytic promiscuity of Tda enzymes. Particularly, TdaG and TdaD accept various substrates and catalyze regiospecific reactions at different stages of 1 biosynthesis. Our study not only uncovers a hidden library of ETP alkaloids, but also helps to understand the hidden chemical diversity of natural products by pathway manipulation. [ABSTRACT FROM AUTHOR]

The acceptance of benzyl diphosphate (II) as an alkyl donor by the fungal dimethylallyl transferase FgaPT2 and the regiospecific enzymatic benzylation of tryptophan and analogues are studied. [ABSTRACT FROM AUTHOR]

Ascomycetous fungi are often found in agricultural products and foods as contaminants. They produce hazardous mycotoxins for human and animals. On the other hand, the fungal metabolites including mycotoxins are important drug candidates and the enzymes involved in the biosynthesis of these compounds are valuable biocatalysts for production of designed compounds. One of the enzyme groups are members of the dimethylallyl tryptophan synthase superfamily, which mainly catalyze prenylations of tryptophan and tryptophan-containing cyclodipeptides (CDPs). Decoration of CDPs in the biosynthesis of multiple prenylated metabolites in nature is usually initiated by regiospecific C2-prenylation at the indole ring, followed by second and third ones as well as by other modifications. However, the strict substrate specificity can prohibit the further prenylation of unnatural C2-prenylated compounds. To overcome this, we firstly obtained C4-, C5-, C6-, and C7-prenylated cyclo-l-Trp-l-Pro. These products were then used as substrates for the promiscuous C2-prenyltransferase EchPT1, which normally uses the unprenylated CDPs as substrates. Four unnatural diprenylated cyclo-l-Trp-l-Pro including the unique unexpected N1,C6-diprenylated derivative with significant yields were obtained in this way. Our study provides an excellent example for increasing structural diversity by reprogramming the reaction orders of natural biosynthetic pathways. Furthermore, this is the first report that EchPT1 can also catalyze N1-prenylation at the indole ring. Key points: • Prenyltransferases as biocatalysts for unnatural substrates. • Chemoenzymatic synthesis of designed molecules. • A cyclodipeptide prenyltransferase as prenylating enzyme of already prenylated products. [ABSTRACT FROM AUTHOR]