[Cloning and functional analysis of AcFMO from onion during alliine biosynthesis].

Flavin-containing monooxygenase (FMO) is the key enzyme in the biosynthesis pathway of CSOs with sulfur oxidation. In order to explore the molecular regulatory mechanism of FMO in the synthesis of onion CSOs, based on transcriptome database and phylogenetic analysis, one AcFMO gene that may be involved in alliin synthesis was obtained, the AcFMO had a cDNA of 1 374 bp and encoded 457 amino acids, which was evolutionarily closest to the AsFMO of garlic. Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) indicated that AcFMO was the highest in the flowers and the lowest in the leaf sheaths. The results of subcellular localization showed that the AcFMO gene product was widely distributed throughout the cell A yeast expression vector was constructed, and the AcFMO gene was ecotopically overexpressed in yeast to further study the enzyme function in vitro and could catalyze the synthesis of alliin by S-allyl-l-cysteine. In summary, the cloning and functional identification of AcFMO have important reference value for understanding the biosynthesis of CSOs in onions.

Initial Steps in Methanobactin Biosynthesis: Substrate Binding by the Mixed-Valent Diiron Enzyme MbnBC.

The MbnBC enzyme complex converts cysteine residues in a peptide substrate, MbnA, to oxazolone/thioamide groups during the biosynthesis of copper chelator methanobactin (Mbn). MbnBC belongs to the mixed-valent diiron oxygenase (MVDO) family, of which members use an Fe(II)Fe(III) cofactor to react with dioxygen for substrate modification. Several crystal structures of the inactive Fe(III)Fe(III) form of MbnBC alone and in complex with MbnA have been reported, but a mechanistic understanding requires determination of the oxidation states of the crystallographically observed Fe ions in the catalytically active Fe(II)Fe(III) state, along with the site of MbnA binding. Here, we have used electron nuclear double resonance (ENDOR) spectroscopy to determine such structural and electronic properties of the active site, in particular, the mode of substrate binding to the MV state, information not accessible by X-ray crystallography alone. The oxidation states of the two Fe ions were determined by 15N ENDOR analysis. The presence and locations of both bridging and terminal exogenous solvent ligands were determined using 1H and 2H ENDOR. In addition, 2H ENDOR using an isotopically labeled MbnA substrate indicates that MbnA binds to the Fe(III) ion of the cluster via the sulfur atom of its N-terminal modifiable cysteine residue, with displacement of a coordinated solvent ligand as shown by complementary 1H ENDOR. These results, which underscore the utility of ENDOR in studying MVDOs, provide a molecular picture of the initial steps in Mbn biosynthesis.

Polyphenols from hickory nut reduce the occurrence of atherosclerosis in mice by improving intestinal microbiota and inhibiting trimethylamine N-oxide production.

BACKGROUND: Trimethylamine N-oxide (TMAO), a metabolite produced by intestinal microbiota through metabolizing phosphatidylcholine, choline, l-carnitine and betaine in the diet, has been implicated in the pathogenesis of atherosclerosis (AS). Concurrently, dietary polyphenols have garnered attention for their potential to ameliorate obesity, diabetes and atherosclerosis primarily by modulating the intestinal microbial structure. Hickory (Carya cathayensis) nut, a polyphenol-rich food product favored for its palatability, emerges as a candidate for exploration. HYPOTHESIS/PURPOSE: The relationship between polyphenol of hickory nut and atherosclerosis prevention will be firstly clarified, providing theoretical basis for the discovery of natural products counteracting TMAO-induced AS process in hickory nut. STUDY DESIGN AND METHODS: Employing Enzyme-linked Immunosorbent Assay (ELISA) and histological examination of aortic samples, the effects of total polyphenol extract on obesity index, inflammatory index and pathological changes of atherosclerosis in C57BL/6 J mice fed with high-fat and high choline diet were evaluated. Further, the composition, abundance, and function of mouse gut microbiota were analyzed through 16srDNA sequencing. Concurrently, the levels of TMAO and the expression of key enzymes (CutC and FMO3) involved in its synthesis are quantified using ELISA, Western Blot and Real-Time Quantitative PCR (RT-qPCR). Additionally, targeted metabolomic profiling of the hickory nut polyphenol extract was conducted, accompanied by molecular docking simulations to predict interactions between candidate polyphenols and the CutC/FMO3 using Autodock Vina. Finally, the docking prediction were verified by microscale thermophoresis (MST) . RESULTS: Polyphenol extracts of hickory nut improved the index of obesity and inflammation, and alleviated the pathological changes of atherosclerosis in C57BL/6 J mice fed with high-fat and high-choline diet. Meanwhile, these polyphenol extracts also changed the composition and function of intestinal microbiota, and increased the abundance of microorganisms in mice. Notably, the abundance of intestinal microbiota endowed with CutC gene was significantly reduced, coherent with expression of CutC catalyzing TMA production. Moreover, polyphenol extracts also decreased the expression of FMO3 in the liver, contributing to the reduction of TMAO levels in serum. Furthermore, metabonomic profile analysis of these polyphenol extracts identified 647 kinds of polyphenols. Molecular docking predication further demonstrated that Casuariin and Cinnamtannin B2 had the most potential inhibition on the enzymatic activities of CutC or FMO3, respectively. Notably, MST analysis corroborated the potential for direct interaction between CutC enzyme and available polyphenols such as Corilagin, (-)-Gallocatechin gallate and Epigallocatechin gallate. CONCLUSION: Hickory polyphenol extract can mitigate HFD-induced AS by regulating intestinal microflora in murine models. In addition, TMA-FMO3-TMAO pathway may play a key role in this process. This research unveils, for the inaugural time, the complex interaction between hickory nut-derived polyphenols and gut microbial, providing novel insights into the role of dietary polyphenols in AS prevention.

Nitrogen sources alter ligninase and cellulase activities of thermophilic fungi isolated from compost and vermicompost.

Fungi harboring lignocellulolytic activity accelerate the composting process of agricultural wastes; however, using thermophilic fungal isolates for this process has been paid little attention. Moreover, exogenous nitrogen sources may differently affect fungal lignocellulolytic activity. A total of 250 thermophilic fungi were isolated from local compost and vermicompost samples. First, the isolates were qualitative assayed for ligninase and cellulase activities using Congo red (CR) and carboxymethyl cellulose (CMC) as substrates, respectively. Then, twenty superior isolates harboring higher ligninase and cellulase activities were selected and quantitatively assayed for both enzymes in basic mineral (BM) liquid medium supplemented with the relevant substrates and nitrogen sources including (NH4)2SO4 (AS), NH4NO3 (AN), urea (U), AS + U (1:1), or AN + U (1:1) with final nitrogen concentration of 0.3 g/L. The highest ligninase activities of 99.94, 89.82, 95.42, 96.25, and 98.34% of CR decolorization were recorded in isolates VC85, VC94, VC85, C145, and VC85 in the presence of AS, U, AS + U, AN, and AN + U, respectively. Mean ligninase activity of 63.75% in superior isolates was achieved in the presence of AS and ranked the highest among other N compounds. The isolates C200 and C184 exhibited the highest cellulolytic activity in the presence of AS and AN + U by 8.8 and 6.5 U/ml, respectively. Mean cellulase activity of 3.90 U/mL was achieved in AN + U and ranked the highest among other N compounds. Molecular identification of twenty superior isolates confirmed that all of them are belonging to Aspergillus fumigatus group. Focusing on the highest ligninase activity of the isolate VC85 in the presence of AS, the combination can be recommended as a potential bio-accelerator for compost production.

Extracellular ligninases production and lignin degradation by Paenibacillus polymyxa.

Bacteria represent an attractive source for the isolation and identification of potentially useful microorganisms for lignin depolymerization, a process required for the use of agricultural waste. In this work, ten autochthonous bacteria isolated from straw, cow manure, and composts were characterized for potential use in the biodelignification of the waste. A comparison of the ability to degrade lignin and the efficiency of ligninolytic enzymes was performed in bacteria grown in media with lignin as a sole carbon source (LLM, 3.5g/L lignin-alkali) and in complex media supplemented with All-Ban fiber (FLM, 1.5g/L). Bacterial isolates showed different abilities to degrade lignin, they decreased the lignin concentration from 7.6 to 18.6% in LLM and from 11.1 to 44.8% in FLM. They also presented the activity of manganese peroxidase, lignin peroxidases, and laccases with different specific activities. However, strain 26 identified as Paenibacillus polymyxa by sequencing the 16S rRNA showed the highest activity of lignin peroxidase and the ability to degrade efficiently lignocellulose. In addition, P. polymyxa showed the highest potential (desirability ≥ 0.795) related to the best combination of properties to depolymerize lignin from biomass. The results suggest that P. polymyxa has a coordinated lignin degradation system constituted of lignin peroxidase, manganese peroxidase, and laccase enzymes.

ß-Cryptoxanthin Attenuates Cigarette-Smoke-Induced Lung Lesions in the Absence of Carotenoid Cleavage Enzymes (BCO1/BCO2) in Mice.

High dietary intake of ß-cryptoxanthin (BCX, an oxygenated provitamin A carotenoid) is associated with a lower risk of lung disease in smokers. BCX can be cleaved by ß-carotene-15,15'-oxygenase (BCO1) and ß-carotene-9',10'-oxygenase (BCO2) to produce retinol and apo-10'-carotenoids. We investigated whether BCX has protective effects against cigarette smoke (CS)-induced lung injury, dependent or independent of BCO1/BCO2 and their metabolites. Both BCO1-/-/BCO2-/- double knockout mice (DKO) and wild type (WT) littermates were supplemented with BCX 14 days and then exposed to CS for an additional 14 days. CS exposure significantly induced macrophage and neutrophil infiltration in the lung tissues of mice, regardless of genotypes, compared to the non-exposed littermates. BCX treatment significantly inhibited CS-induced inflammatory cell infiltration, hyperplasia in the bronchial epithelium, and enlarged alveolar airspaces in both WT and DKO mice, regardless of sex. The protective effects of BCX were associated with lower expression of IL-6, TNF-α, and matrix metalloproteinases-2 and -9. BCX treatment led to a significant increase in hepatic BCX levels in DKO mice, but not in WT mice, which had significant increase in hepatic retinol concentration. No apo-10'-carotenoids were detected in any of the groups. In vitro BCX, at comparable doses of 3-OH-ß-apo-10'-carotenal, was effective at inhibiting the lipopolysaccharide-induced inflammatory response in a human bronchial epithelial cell line. These data indicate that BCX can serve as an effective protective agent against CS-induced lung lesions in the absence of carotenoid cleavage enzymes.

Interspecies Comparison of Interaction Energies between Photosynthetic Protein RuBisCO and 2CABP Ligand.

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) functions as the initial enzyme in the dark reactions of photosynthesis, catalyzing reactions that extract CO2 from the atmosphere and fix CO2 into organic compounds. RuBisCO is classified into four types (isoforms I-IV) according to sequence-based phylogenetic trees. Given its size, the computational cost of accurate quantum-chemical calculations for functional analysis of RuBisCO is high; however, recent advances in hardware performance and the use of the fragment molecular orbital (FMO) method have enabled the ab initio analyses of RuBisCO. Here, we performed FMO calculations on multiple structural datasets for various complexes with the 2'-carboxylarabinitol 1,5-bisphosphate (2CABP) ligand as a substrate analog and investigated whether phylogenetic relationships based on sequence information are physicochemically relevant as well as whether novel information unobtainable from sequence information can be revealed. We extracted features similar to the phylogenetic relationships found in sequence analysis, and in terms of singular value decomposition, we identified residues that strongly interacted with the ligand and the characteristics of the isoforms for each principal component. These results identified a strong correlation between phylogenetic relationships obtained by sequence analysis and residue interaction energies with the ligand. Notably, some important residues were located far from the ligand, making comparisons among species using only residues proximal to the ligand insufficient.

Pseudomonas in environmental bioremediation of hydrocarbons and phenolic compounds- key catabolic degradation enzymes and new analytical platforms for comprehensive investigation.

Pollution of the environment with petroleum hydrocarbons and phenolic compounds is one of the biggest problems in the age of industrialization and high technology. Species of the genus Pseudomonas, present in almost all hydrocarbon-contaminated areas, play a particular role in biodegradation of these xenobiotics, as the genus has the potential to decompose various hydrocarbons and phenolic compounds, using them as its only source of carbon. Plasticity of carbon metabolism is one of the adaptive strategies used by Pseudomonas to survive exposure to toxic organic compounds, so a good knowledge of its mechanisms of degradation enables the development of new strategies for the treatment of pollutants in the environment. The capacity of microorganisms to metabolize aromatic compounds has contributed to the evolutionally conserved oxygenases. Regardless of the differences in structure and complexity between mono- and polycyclic aromatic hydrocarbons, all these compounds are thermodynamically stable and chemically inert, so for their decomposition, ring activation by oxygenases is crucial. Genus Pseudomonas uses several upper and lower metabolic pathways to transform and degrade hydrocarbons, phenolic compounds, and petroleum hydrocarbons. Data obtained from newly developed omics analytical platforms have enormous potential not only to facilitate our understanding of processes at the molecular level but also enable us to instigate and monitor complex biodegradations by Pseudomonas. Biotechnological application of aromatic metabolic pathways in Pseudomonas to bioremediation of environments polluted with crude oil, biovalorization of lignin for production of bioplastics, biofuel, and bio-based chemicals, as well as Pseudomonas-assisted phytoremediation are also considered.

Loss of anthocyanidin synthase gene is associated with white flowers of Salvia miltiorrhiza Bge. f. alba, a natural variant of S. miltiorrhiza.

MAIN CONCLUSION: SmANS deletion leads to white flower mutation in Salvia miltiorrhiza. SmANS deletion leads to white flower mutation in Salvia miltiorrhiza. Abstract Salvia miltiorrhiza is an essential traditional Chinese medicine (TCM) with purple flowers, and S. miltiorrhiza Bge. f. alba is a unique intraspecific variation with white flowers. The molecular mechanism of flower color formation in S. miltiorrhiza will provide vital information for the variation and evolution. Here, we performed HPLC, transcriptomic, and re-sequencing analyses of purple-flowered S. miltiorrhiza line 'Zihua105' (ZH105) and white-flowered S. miltiorrhiza Bge. f. alba line 'Baihua18' (BH18). Delphinidin was the most anthocyanidin in ZH105, which become the main different between ZH105 vs. BH18 flowers. Transcriptome analysis revealed 299 differentially expressed genes (DEGs). SmANS, the anthocyanidin synthase gene in the down-stream anthocyanin biosynthesis pathway, was significantly expressed in ZH105 corollas, suggesting it might play a key role in white petal formation. Whole-genome re-sequencing revealed that a 6.75 kb segment located on chromosome 5, which contains the complete sequence of the SmANS genes, was lost in BH18 and another S. miltiorrhiza Bge. f. alba line. In contrast, the other five purple-flowered S. miltiorrhiza lines both possessed this segment. Further molecular marker identification also confirmed that wild S. miltiorrhiza Bge. f. alba lines lost regions that contained a complete or important part of SmANS sequences. Subsequently, the research showed that the deletion mutant of SmANS genes resulted in the natural white flower color variant of S. miltiorrhiza.

Hypochlorous acid facilitates inducible nitric oxide synthase subunit dissociation: The link between heme destruction, disturbance of the zinc-tetrathiolate center, and the prevention by melatonin.

Inducible nitric oxide synthase (iNOS) is a zinc-containing hemoprotein composed of two identical subunits, each containing a reductase and an oxygenase domain. The reductase domain contains binding sites for NADPH, FAD, FMN, and tightly bound calmodulin and the oxygenase domain contains binding sites for heme, tetrahydrobiopterin (H4B), and l-arginine. The enzyme converts l-arginine into nitric oxide (NO) and citrulline in the presence of O2. It has previously been demonstrated that myeloperoxidase (MPO), which catalyzes formation of hypochlorous acid (HOCl) from hydrogen peroxide (H2O2) and chloride (Cl-), is enhanced in inflammatory diseases and could be a potent scavenger of NO. Using absorbance spectroscopy and gel filtration chromatography, we investigated the role of increasing concentrations of HOCl in mediating iNOS heme destruction and subsequent subunit dissociation and unfolding. The results showed that dimer iNOS dissociation between 15 and 100 µM HOCl was accompanied by loss of heme content and NO synthesis activity. The dissociated subunits-maintained cytochrome c and ferricyanide reductase activities. There was partial unfolding of the subunits at 300 µM HOCl and above, and the subunit unfolding transition was accompanied by loss of reductase activities. These events can be prevented when the enzyme is preincubated with melatonin prior to HOCl addition. Melatonin supplementation to patients experiencing low NO levels due to inflammatory diseases may be helpful to restore physiological NO functions.

Deficiency of PSRC1 accelerates atherosclerosis by increasing TMAO production via manipulating gut microbiota and flavin monooxygenase 3.

Maladaptive inflammatory and immune responses are responsible for intestinal barrier integrity and function dysregulation. Proline/serine-rich coiled-coil protein 1 (PSRC1) critically contributes to the immune system, but direct data on the gut microbiota and the microbial metabolite trimethylamine N-oxide (TMAO) are lacking. Here, we investigated the impact of PSRC1 deletion on TMAO generation and atherosclerosis. We first found that PSRC1 deletion in apoE-/- mice accelerated atherosclerotic plaque formation, and then the gut microbiota and metabolites were detected using metagenomics and untargeted metabolomics. Our results showed that PSRC1 deficiency enriched trimethylamine (TMA)-producing bacteria and functional potential for TMA synthesis and accordingly enhanced plasma betaine and TMAO production. Furthermore, PSRC1 deficiency resulted in a proinflammatory colonic phenotype that was significantly associated with the dysregulated bacteria. Unexpectedly, hepatic RNA-seq indicated upregulated flavin monooxygenase 3 (FMO3) expression following PSRC1 knockout. Mechanistically, PSRC1 overexpression inhibited FMO3 expression in vitro, while an ERα inhibitor rescued the downregulation. Consistently, PSRC1-knockout mice exhibited higher plasma TMAO levels with a choline-supplemented diet, which was gut microbiota dependent, as evidenced by antibiotic treatment. To investigate the role of dysbiosis induced by PSRC1 deletion in atherogenesis, apoE-/- mice were transplanted with the fecal microbiota from either apoE-/- or PSRC1-/-apoE-/- donor mice. Mice that received PSRC1-knockout mouse feces showed an elevation in TMAO levels, as well as plaque lipid deposition and macrophage accumulation, which were accompanied by increased plasma lipid levels and impaired hepatic cholesterol transport. Overall, we identified PSRC1 as an atherosclerosis-protective factor, at least in part, attributable to its regulation of TMAO generation via a multistep pathway. Thus, PSRC1 holds great potential for manipulating the gut microbiome and alleviating atherosclerosis.

22R- but not 22S-hydroxycholesterol is recruited for diosgenin biosynthesis.

Diosgenin is an important compound in the pharmaceutical industry and it is biosynthesized in several eudicot and monocot species, herein represented by fenugreek (a eudicot), and Dioscorea zingiberensis (a monocot). Formation of diosgenin can be achieved by the early C22,16-oxidations of cholesterol followed by a late C26-oxidation. This study reveals that, in both fenugreek and D. zingiberensis, the early C22,16-oxygenase(s) shows strict 22R-stereospecificity for hydroxylation of the substrates. Evidence against the recently proposed intermediacy of 16S,22S-dihydroxycholesterol in diosgenin biosynthesis was also found. Moreover, in contrast to the eudicot fenugreek, which utilizes a single multifunctional cytochrome P450 (TfCYP90B50) to perform the early C22,16-oxidations, the monocot D. zingiberensis has evolved two separate cytochrome P450 enzymes, with DzCYP90B71 being specific for the 22R-oxidation and DzCYP90G6 for the C16-oxidation. We suggest that the DzCYP90B71/DzCYP90G6 pair represent more broadly conserved catalysts for diosgenin biosynthesis in monocots.

Unraveling the metabolic pathway of choline-TMA-TMAO: Effects of gypenosides and implications for the therapy of TMAO related diseases.

Trimethylamine-N-oxide (TMAO) has emerged as a promising new therapeutic target for the treatment of central nervous system diseases, atherosclerosis and other diseases. However, its origin in the brain is unclear. Gynostemma pentaphyllum (Thunb.) Makino can reduce the increase of TMAO level caused by a high fat diet. But its effective chemical composition and specific mechanism have not been reported. The study confirmed that TMA was more easily to penetrate blood brain barrier than TMAO, the MAO enzyme was partly involved in the transformation of the TMA in brain, which further supplemented the choline-TMA-TMAO pathway. Based on the above metabolic pathway, using multi-omics approaches, such as microbiodiversity, metagenomics and lipidomics, it was demonstrated that the reduction of plasma TMAO levels by gypenosides did not act on FMO3 and MAO in the pathway, but remodeled the microbiota and affected the trimethylamine lyase needed in the conversion of choline to TMA in intestinal flora. At the same time, gypenosides interfered with enzymes associated with TCA and lipid metabolism, thus affecting TMAO and lipid metabolism. Considering the bidirectional transformation of phosphatidycholine and choline, lipid metabolism and TMAO metabolism could affected each other to some extent. In conclusion, our study revealed the intrinsic correlation between long-term application of gypenosides to lipid reduction and nervous system protection, and explained why gypenosides were used to treat brain diseases, even though they had a poor ability to enter the brain. Besides, it provided a theoretical basis for clinical application of gypenosides and the development of new drugs.

Facile and label-free fluorescence strategy for evaluating the influence of bioactive ingredients on FMO3 activity via supramolecular host-guest reporter pair.

Screening inhibitors of flavin monooxygenase 3 (FMO3) is very important for treating trimethylamine N-oxide (TMAO) derived thrombotic diseases. Herein, focusing on Xuefu Zhuyu decoction (XFZYD) as a Chinese traditional medicine with antithrombotic efficacy, a facile and label-free fluorescence strategy was developed for evaluating the influence of the bioactive ingredients in XFZYD on FMO3 activity through indicator displacement assay. To this end, the optimized supramolecular host-guest (p-sulfonatocalix[4]arene-oxazine 1) reporter pair and FMO3 catalytic system were exploited to determine the influence of the bioactive compounds in XFZYD on the conversion from TMA to TMAO. From the nine compounds tested, naringin, paeoniflorin, ß-ecdysterone, 18ß-glycyrrhizic acid, amygdalin, albiflorin, and saikosaponin A downregulated FMO3 activity and reduced TMAO biosynthesis. Moreover, molecular docking was successfully applied to simulate the optimal conformation of a receptor-ligand complex between FMO3 and all tested compounds except for ß-ecdysterone. Therefore, this approach provides a novel and promising strategy for screening FMO3 inhibitors from Chinese traditional medicine by supramolecular sensing.

MEASUREMENTS OF SEASONAL VARIATIONS OF RADIOACTIVITY DISTRIBUTIONS IN RIVERINE SOIL SEDIMENT OF ADO-ODO OTA, SOUTH-WEST NIGERIA: PROBABILISTIC APPROACH USING MONTE CARLO.

The radioactivity levels were measured using a hand-held gamma-ray survey meter and NaI (Tl) based gamma spectroscopy to evaluate the seasonal variation of radioactivity levels in the riverine area of Ado-Odo Ota. The measured iso-dose map reported higher gamma dose rate of 79 nGy/h, approximately 34% higher than the world average of 59 nGy/h. The values for U-238, Th-232 and K-40 activity levels ranged between 29.9 and 21.6; 103.2 and 31.2; 802.2 and 233.5 with mean values of 26.1, 55.6 and 499.3 Bq/kg, respectively. According to the mean, 5th and 95th percentiles of the probabilities using the Monte Carlo simulation, the Radium equivalent activities and the absorbed dose rates are within their respective recommended limits of 370 Bq/kg and 84 nGy/h. This study could be used to monitor dose rates and radiological risks for the areas covering the small area (Ado-Odo Ota) to the larger area (West African Region) as baseline data.

Do methanotrophs drive phosphorus mineralization in soil ecosystem?

Experiments were carried out to elucidate linkage between methane consumption and mineralization of phosphorous (P) from different P sources. The treatments were (i) no CH4 + no P amendment (absolute control), (ii) with CH4 + no P amendment (control), (iii) with CH4 + inorganic P as Ca3(PO4)2, and (iv) with CH4 + organic P as sodium phytate. P sources were added at 25 µg P·(g soil)-1. Soils were incubated to undergo three repeated CH4 feeding cycles, referred to as feeding cycle I, feeding cycle II, and feeding cycle III. CH4 consumption rate k (µg CH4 consumed·(g soil)-1·day-1) was 0.297 ± 0.028 in no P amendment control, 0.457 ± 0.016 in Ca3(PO4)2, and 0.627 ± 0.013 in sodium phytate. Rate k was stimulated by 2 to 6 times over CH4 feeding cycles and followed the trend of sodium phytate > Ca3(PO4)2 > no P amendment control. CH4 consumption stimulated P solubilization from Ca3(PO4)2 by a factor of 2.86. Acid phosphatase (µg paranitrophenol released·(g soil)-1·h-1) was higher in sodium phytate than the no P amendment control. Abundance of 16S rRNA and pmoA genes increased with CH4 consumption rates. The results of the study suggested that CH4 consumption drives mineralization of unavailable inorganic and organic P sources in the soil ecosystem.

Effect of benzothiadiazole treatment on quality and anthocyanin biosynthesis in plum fruit during storage at ambient temperature.

BACKGROUND: Plums tend to experience a reduction in fruit quality due to ripening and they deteriorate quickly during storage at room temperature. Benzothiadiazole (BTH) is a plant elicitor capable of inducing disease resistance in many crops. In this study, the effect of BTH treatment on fruit ripening, fruit quality, and anthocyanin biosynthesis in 'Taoxingli' plum was investigated. RESULTS: The results showed that BTH treatment could accelerate fruit ripening without affecting the incidence of fruit decay or the shelf life. Benzothiadiazole treatment improved the quality and consumer acceptability of 'Taoxingli' plums during storage by increasing the sweetness, red color formation, and the concentration of healthy antioxidant compounds. The BTH treatment could also effectively promote the biosynthesis of anthocyanin by enhancing the enzyme activities of phenylalanine ammonia-lyase (PAL), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), and uridine diphosphate flavonoid 3-O-glucosyltransferase (UFGT) and up-regulating the gene expressions of PsPAL, PsCHI, PsDFR, PsANS, and PsUFGT during storage. CONCLUSION: Benzothiadiazole treatment could be a potential postharvest technology for improving fruit quality and consumer acceptability in harvested plum fruit. © 2020 Society of Chemical Industry.

Physiological and metabolic analysis of winter jujube after postharvest treatment with calcium chloride and a composite film.

BACKGROUND: Ziziphus jujuba Miller cv. Dongzao is extremely susceptible to reddening, browning, nutritional loss, and perishability after harvest. In this study, we evaluated the mechanisms of calcium chloride and chitosan/nano-silica composite film treatments on the quality, especially in reddening, by physiological and metabolomic assays. RESULTS: The treatment delayed the decline of phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), and chalcone isomerase (CHI) activities. Meanwhile, the treated groups retarded the increases in anthocyanin and quercetin contents by inhibiting the gene expressions of flavonol synthase (ZjFLS), dihydroflavonol 4-reductase (ZjDFR), and anthocyanidin synthase (ZjANS), while promoting leucoanthocyanidin reductase (ZjLAR) expression, which leads to retardation of fruit reddening. Anthocyanins were found to be responsible for post-harvest winter jujube reddening through principal component analysis. Results from the technique for order preference by similarity to an ideal solution indicated that the treated group delayed the decline of the quality of 'Dongzao' and extended its shelf life. CONCLUSION: The treatment induced the heightening of flavonoids metabolism. They enhanced the nutritional value and the ability to resist stress by delaying the decline of PAL, CHS, and CHI activities. Meanwhile, the treated groups retarded the increase in anthocyanin and quercetin contents by inhibiting the gene expressions of ZjFLS, ZjDFR, and ZjANS and promoting ZjLAR expression, which leads to retardation of fruit reddening. Anthocyanins are responsible for post-harvest winter jujube reddening. Coating treatment effectively delayed the decline of winter jujube quality. © 2020 Society of Chemical Industry.

[Regulatory effect of the lipid metabolic pathways of TMAO, FMO3 and FXR on compound stress-induced ED in rats and mechanisms of Yimusake intervention].

OBJECTIVE: To study of the regulatory effects of the lipid metabolic pathways of trimethylamine-N-oxide (TMAO), flavin-containingmonooxidase 3 (FMO3) and farnesoid X receptor (FXR) on compound stress-induced ED (CSED) rats and the mechanisms of Yimusake Tablets (YMSK) intervention. METHODS: Based on the results of metabonomics analysis, we determined the concentration of TMAO in the serum of the rats in the normal control (n = 30), the CSED model control (n = 30) and the YMSK intervention group (intragastrical administration of YMSK at 250 mg/kg once daily for 2－3 weeks after modeling, n = 30) by nuclear magnetic resonance (NMR) spectroscopy test. We also detected the expressions of the FMO3, FXR1 and FXR2 proteins in the liver tissue of the three groups of rats by Western blot. RESULTS: The serum TMAO level was significantly elevated in the CSED model control compared with that in the normal control group (ï¼»46.64 ± 5.16ï¼½ vs ï¼»34.98 ± 3.69ï¼½ µg/mL, P < 0.01) but remarkably decreased after YMSK intervention (ï¼»39.63 ± 4.81ï¼½ µg/mL) in comparison with that in the CSED model control group (P < 0.01). The rats in the CSED model control group, compared with the normal controls, showed significantly upregulated expressions of FMO3 (1.75 ± 0.90 vs 0.86 ± 0.62, P < 0.01),FXR1 (1.29 ± 0.38 vs 0.78 ± 0.25, P < 0.01) and FXR2 in the liver tissue (1.90 ± 0.63 vs 0.42 ± 0.27, P < 0.01), but all the three expressions were markedly decreased after YMSK intervention (FMO3: 1.05 ± 0.38, P < 0.05; FXR1: 1.07 ± 0.42, P < 0.05; FXR2: 1.04 ± 0.46, P < 0.01) as compared with those in the CSED model control group. CONCLUSIONS: The lipid metabolic pathways of TMAO, FMO3 and FXR underwent significant changes in the rat model of compound stress-induced ED, which could be improved by YMSK intervention, suggesting that YMSK may play an important role in protecting erectile function by regulating the lipid metabolic pathways of TMAO, FMO3 and FXR.

Engineering CrtW and CrtZ for improving biosynthesis of astaxanthin in Escherichia coli.

This study engineered ß-carotene ketolase CrtW and ß-carotene hydroxylase CrtZ to improve biosynthesis of astaxanthin in Escherichia coli. Firstly, crtW was randomly mutated to increase CrtW activities on conversion from ß-carotene to astaxanthin. A crtW* mutant with A6T, T105A and L239M mutations has improved 5.35-fold astaxanthin production compared with the wild-type control. Secondly, the expression levels of crtW* and crtZ on chromosomal were balanced by simultaneous modulation RBS regions of their genes using RBS library. The strain RBS54 selected from RBS library, directed the pathway exclusively towards the desired product astaxanthin as predominant carotenoid (99%). Lastly, the number of chromosomal copies of the balanced crtW-crtZ cassette from RBS54 was increased using a Cre-loxP based technique, and a strain with 30 copies of the crtW*-crtZ cassette was selected. This final strain DL-A008 had a 9.8-fold increase of astaxanthin production compared with the wild-type control. Fed-batch fermentation showed that DL-A008 produced astaxanthin as predominant carotenoid (99%) with a specific titer of 0.88 g·L-1 without addition of inducer. In conclusion, through constructing crtW mutation, balancing the expression levels between crtW* and crtZ, and increasing the copy number of the balanced crtW*-crtZ cassette, the activities of ß-carotene ketolase and ß-carotene hydroxylase were improved for conversion of ß-carotene to astaxanthin with higher efficiency. The series of conventional and novel metabolic engineering strategies were designed and applied to construct the astaxanthin hetero-producer strain of E. coli, possibly offering a general approach for the construction of stable hetero-producer strains for other natural products.