Mapping quantitative trait loci associated with self-(in)compatibility in goji berries (Lycium barbarum).

BACKGROUND: Goji (Lycium barbarum L.) is a perennial deciduous shrub widely distributed in arid and semiarid regions of Northwest China. It is highly valued for its medicinal and functional properties. Most goji varieties are naturally self-incompatible, posing challenges in breeding and cultivation. Self-incompatibility is a complex genetic trait, with ongoing debates regarding the number of self-incompatible loci. To date, no genetic mappings has been conducted for S loci or other loci related to self-incompatibility in goji. RESULTS: We used genome resequencing to create a high-resolution map for detecting de novo single-nucleotide polymorphisms (SNP) in goji. We focused on 229 F1 individuals from self-compatible '13-19' and self-incompatible 'new 9' varieties. Subsequently, we conducted a quantitative trait locus (QTL) analysis on traits associated with self-compatibility in goji berries. The genetic map consisted of 249,327 SNPs distributed across 12 linkage groups (LGs), spanning a total distance of 1243.74 cM, with an average interval of 0.002 cM. Phenotypic data related to self-incompatibility, such as average fruit weight, fruit rate, compatibility index, and comparable compatibility index after self-pollination and geitonogamy, were collected for the years 2021-2022, as well as for an extra year representing the mean data from 2021 to 2022 (2021/22). A total of 43 significant QTL, corresponding to multiple traits were identified, accounting for more than 11% of the observed phenotypic variation. Notably, a specific QTL on chromosome 2 consistently appeared across different years, irrespective of the relationship between self-pollination and geitonogamy. Within the localization interval, 1180 genes were annotated, including Lba02g01102 (annotated as an S-RNase gene), which showed pistil-specific expression. Cloning of S-RNase genes revealed that the parents had two different S-RNase alleles, namely S1S11 and S2S8. S-genotype identification of the F1 population indicated segregation of the four S-alleles from the parents in the offspring, with the type of S-RNase gene significantly associated with self-compatibility. CONCLUSIONS: In summary, our study provides valuable insights into the genetic mechanism underlying self-compatibility in goji berries. This highlights the importance of further positional cloning investigations and emphasizes the importance of integration of marker-assisted selection in goji breeding programs.

Selenium Treatment Ameliorates the Adverse Effects Caused by Dynamin Gene Knockdown in Bombyx mori.

Our previous research reported the influence of 50 µM selenium (Se) on the cytosolization (endocytosis) pathway, which in turn stimulates the growth and development of Bombyx mori. Lately, dynamin is recognized as one of the key proteins in endocytosis. To explore the underlying mechanisms of Se impact, the dynamin gene was knocked down by injecting siRNAs (Dynamin-1, Dynamin-2, and Dynamin-3). This was followed by an analysis of the target gene and levels of silk protein genes, as well as growth and developmental indices, Se-enrichment capacity, degree of oxidative damage, and antioxidant capacity of B. mori. Our findings showed a considerable decrease in the relative expression of the dynamin gene in all tissues 24 h after the interference and a dramatic decrease in the silkworm body after 48 h. RNAi dynamin gene decreased the silkworm body weight, cocoon shell weight, and the ratio of cocoon. In the meantime, malondialdehyde level increased and glutathione level and superoxide dismutase/catalase activities decreased. 50 µM Se markedly ameliorated these growth and physiological deficits as well as decreases in dynamin gene expression. On the other hand, there were no significant effects on fertility (including produced eggs and laid eggs) between the interference and Se treatments. Additionally, the Se content in the B. mori increased after the dynamin gene interference. The dynamin gene was highly expressed in the silk gland and declined significantly after interference. Among the three siRNAs (Dynamin-1, Dynamin-2, and Dynamin-3), the dynamin-2 displayed the highest interference effects to target gene expression. Our results demonstrated that 50 µM Se was effective to prevent any adverse effects caused by dynamin knockdown in silkworms. This provides practical implications for B. mori breeding industry.

Asparagus saponins: effective natural beneficial ingredient in functional foods, from preparation to applications.

Asparagus species is recognized as a perennial herb with several valuable functional ingredients, and has been widely used as medicine and food since ancient times. Among its main chemical constituents, saponins play a vital role in the health benefits and biological activities including anti-cancer, antioxidant, immunomodulatory, anti-microbial, anti-inflammatory, and hypoglycemic. This review summarizes the preparation methods, structure and classification, biological functions, as well as the food and non-food applications of asparagus saponins, with a special emphasis on its anti-cancer effects in vitro and in vivo. Further, the main challenges and limitations of the current research trends in asparagus saponins are highlighted after a detailed analysis of the recent research information. This review bridges the gap between bioactive components and human health and aids current research on functional and health-promoting foods and medicinal application of Asparagus saponins.

Evolutionary research trend of Polygonatum species: a comprehensive account of their transformation from traditional medicines to functional foods.

With the advances in Polygonatum research, there is a huge interest in harnessing the valuable functional ingredients of this genus with the potential for functional foods. This review emphasizes the different aspects of Ploygonatum based research starting from its bioactive compounds, their structural characterization, various extraction methods, as well as biological activities. In view of its integral use as an essential medicinal plant, our review emphasizes on its promising food applications both as an ingredient and as a whole food, and its improved health benefits with potential for agricultural and environmental relevance are also discussed. As we collated the recent research information, we present the main challenges and limitations of the current research trend in this area which can upgrade the further expansion of Polygonatum-related research that will strengthen its economic and accessible nutritional value in the food and health industries. By highlighting the need for the unattended species, this review not only fills existing research gaps, but also encourages the researchers to find new avenues for the natural production of bio-based functional materials and the development of highly functional and health-promoting foods for disease prevention and treatment.

Lycium barbarum (Goji) as functional food: a review of its nutrition, phytochemical structure, biological features, and food industry prospects.

Lycium genus (Goji berry) is recognized as a good source of homology of medicine and food, with various nutrients and phytochemicals. Lately, numerous studies have focused on the chemical constituents and biological functions of the L. barbarum L., covering phytochemical and pharmacological aspects. We aim to provide exclusive data on the nutrients of L. barbarum L. fruits and phytochemicals, including their structural characterization, the evolution of extraction, and purification processes of different phytochemicals of L. barbarum L. fruit while placing greater emphasis on their wide-ranging health effects. This review also profitably offers innovative approaches for the food industry and industrial applications of L. barbarum L. and addresses some current situations and problems in the development of L. barbarum L. in deep processing products, which can provide clues for the sustainable development of L. barbarum L. industry.

Deciphering the toxicity mechanism of haloquinolines on Chlorella pyrenoidosa using QSAR and metabolomics approaches.

The hazardous potential of haloquinolines (HQLs) is becoming an issue of great concern due to its wide and long-term usage in many personal care products. We examined the growth inhibition, structure-activity relationship, and toxicity mechanism of 33 HQLs on Chlorella pyrenoidosa using the 72-h algal growth inhibition assay, three-dimensional quantitative structure-activity relationship (3D-QSAR), and metabolomics. We found that the IC50 (half maximal inhibitory concentration) values for 33 compounds ranged from 4.52 to > 150 mg·L-1, most tested compounds were toxic (1 mg·L-1 < IC50 < 10 mg·L-1) or harmful (10 mg·L-1 < IC50 < 100 mg·L-1) for the aquatic ecosystem. Hydrophobic properties of HQLs dominate their toxicity. Halogen atoms with large volume appear at the 2, 3, 4, 5, 6, and 7-positions of the quinoline ring to significantly increase the toxicity. In algal cells, HQLs can block diverse carbohydrates, lipids, and amino acid metabolism pathways, thereby resulting in energy usage, osmotic pressure regulation, membrane integrity, oxidative stress disorder, thus fatally damaging algal cells. Therefore, our results provide insight into the toxicity mechanism and ecological risk of HQLs.

Updates on Plant-Based Protein Products as an Alternative to Animal Protein: Technology, Properties, and Their Health Benefits.

Plant-based protein products, represented by "plant meat", are gaining more and more popularity as an alternative to animal proteins. In the present review, we aimed to update the current status of research and industrial growth of plant-based protein products, including plant-based meat, plant-based eggs, plant-based dairy products, and plant-based protein emulsion foods. Moreover, the common processing technology of plant-based protein products and its principles, as well as the emerging strategies, are given equal importance. The knowledge gap between the use of plant proteins and animal proteins is also described, such as poor functional properties, insufficient texture, low protein biomass, allergens, and off-flavors, etc. Furthermore, the nutritional and health benefits of plant-based protein products are highlighted. Lately, researchers are committed to exploring novel plant protein resources and high-quality proteins with enhanced properties through the latest scientific and technological interventions, including physical, chemical, enzyme, fermentation, germination, and protein interaction technology.

Preparation and Characterization of Eugenol Incorporated Pullulan-Gelatin Based Edible Film of Pickering Emulsion and Its Application in Chilled Beef Preservation.

The purpose of this study was to develop a composite film composed of eugenol Pickering emulsion and pullulan-gelatin, and to evaluate its preservation effect on chilled beef. The prepared composite film was comprehensively evaluated in terms of the stability of emulsion, the physical properties of the film, and an analysis of freshness preservation for chilled beef. The emulsion size (296.0 ± 10.2 nm), polydispersity index (0.457 ± 0.039), and potential (20.1 ± 0.9 mV) proved the success of emulsion. At the same time, the films displayed good mechanical and barrier properties. The index of beef preservation also indicated that eugenol was a better active ingredient than clove essence oil, which led to the rise of potential of hydrogen, chroma and water content, and effectively inhibited microbial propagation, protein degradation and lipid oxidation. These results suggest that the prepared composites can be used as promising materials for chilled beef preservation.

Unveiling of dietary and gut-microbiota derived B vitamins: Metabolism patterns and their synergistic functions in gut-brain homeostasis.

Nutrition-gut cross-talk holds a vital position in sustaining intestinal function, and micronutrient metabolism has emerged as the foremost metabolic pathway to preserve gut homeostasis. Among micronutrients, B vitamins have evolved prior to DNA/RNA and are known for their vital roles for major evolutionary transitions in extant organisms. Despite their universal requirement and critical role, not all the three domains of life are endowed with a natural ability for de novo B vitamins synthesis. The human gut microbiome constitutes prototrophs and auxotroph which are entirely dependent on dietary intake and gut microbial production of B vitamins. The syntrophic metabolism involving cross-feeding of B vitamins and community-wide exchange between commensal bacteria elicit important changes in the diversity and composition of the human gut microbiome. Hereto, we discuss the B-vitamins sharing among prototrophic and auxotrophic gut bacteria, their absorption in small intestine and transport in distal gut, functional role in relation to the gut homeostasis and symptoms linked to their deficiency. We also briefly explore their potential involvement as psychobiotics in brain energetic metabolism (kynurenines/tryptophan pathway) for neurological functions and highlight their deficiency related malfunctioning.

Exploration of walnut components and their association with health effects.

Traditionally, walnuts have occupied an imperative position in the functional food market with consistently recognized nutritious and functional properties. In the past years, the lipid profile of walnuts has brought much scientific attention via linking a cascade of biological attributes and health-promoting effects. Over time, researchers have focused on diversified composition (polyphenols and vitamins) of different parts of walnut (flower, pellicle, and kernel) and emphasized their physiological significance. Consequently, a plethora of reports has emerged on the potential role of walnut consumption against a series of diseases including cancer, gut dysbiosis, cardiovascular, and neurodegenerative diseases. Therefore, we accumulated the updated data on composition and classification, extraction methods, and utilization of different parts of walnuts as well as associated beneficial effects under in vivo and clinical studies. Altogether, this review summarized the ameliorative effects of a walnut-enriched diet in chronic diseases which can be designated to the synergistic or individual effects of walnut components mainly through anti-oxidative and anti-inflammatory role.

Hydrogen sulfide treatment retrieves the inhibition of growth and development characteristics in silkworm (Bombyx mori) via phosphoacetyl glucosamine mutase gene knock down.

Phosphoacetyl glucosamine mutase (PGM) is the key gene for glycolysis of important metabolic pathways in silkworm, and H2 S (7.5 µM) can promote the growth and development of silkworm. Herein, we used body cavity injection of small-interfering RNA (siRNA) to interfere with the PGM gene in H2 S-treated silkworms. After RNA interference (RNAi), we investigated the growth and development of the silkworm. H2 S treatment could significantly recover the inhibition of body weight, cocoon weight, cocoon shell weight, and cocoon shell ratio by knocking down PGM gene in silkworm, without significant effects on eggs laying and production, and then analyzed the mRNA expression of PGM gene. The interference of siRNA significantly decreased the expression of targeted PGM gene and was concentrated in 48 h followed by gradual recovery. Three interference fragments also showed different interference effects, and siRNA of PGM-3 exerted the highest interference effect to the target gene expression. Fat body had the highest mRNA expression of PGM gene, and the best interference effect was observed after siRNA injection. The results showed that the gene based on H2 S treatment may have an important impact on the growth and development of silkworm by affecting its metabolic pathway.

Ultrasound-Assisted Preparation of Maillard Reaction Products Derived from Hydrolyzed Soybean Meal with Meaty Flavor in an Oil-In-Water System.

In the present work, we prepared Maillard reaction products (MRPs) derived from enzyme hydrolyzed soybean meal with ultrasound assistance in an oil-(oxidized lard)-in-water system (UEL-MRPs) or oil-free system (UN-MRPs), and the effect of ultrasound on the properties of the obtained MRPs was evaluated. The analysis of fatty acids in lard with different treatments showed that ultrasound can generate more unsaturated fatty acids in the aqueous phase. The UV-Vis absorbances of UEL-MRPs, UN-MRPs, and MRPs obtained in an oil-in-water system (EL-MRPs) and MRPs obtained in an oil-free system (N-MRPs) at 294 and 420 nm indicated that ultrasound could increase the amount of Maillard reaction intermediates and melanoids in the final products of the Maillard reaction. This was in line with the result obtained from color change determination-that ultrasound can darken the resultant MRPs. Volatile analysis showed ultrasound can not only increase the number of volatile substances, but also greatly increase the composition of volatile substances in UEL-MRPs and UN-MRPs, especially the composition of those contributing to the flavor of the MRPs, such as oxygen-containing heterocycles, sulfur-containing compounds, and nitrogen-containing heterocycles. Descriptive sensory evaluation revealed that UN-MRPs and UEL-MRPs had the highest scores in total acceptance, ranking in the top two, and UEL-MRPs had the strongest meaty flavor among these four kinds of MRPs. Furthermore, the measurements of antioxidant activities, including DPPH radical-scavenging activity, hydroxyl radical scavenging ability, and ferric ion reducing antioxidant power, were conducted, showing that UN-MRPs exhibited the highest antioxidant activity among all the MRPs.

Colorimetric biosensing of nopaline synthase terminator using Fe3O4@Au and hemin-functionalized reduced graphene oxide.

In this paper, we present a simple and label-free colorimetric biosensor for detection of the nopaline synthase (NOS) terminator in genetically modified (GM) plants. The "signal on" colorimetric biosensor was developed using a nanocomposite consisted of gold nanoparticles doped magnetic Fe3O4 nanoparticles (Fe3O4@Au NP), capture probe DNA (cDNA), and hemin-functionalized reduced graphene oxide nanosheets (H-GN). The nanocomposite was successfully prepared by means of Au-S bonds and the strong π interactions between cDNA and H-GN. The sensing approach is based on the excellent peroxidase-mimicking activity of H-GN and its different electrostatic interactions with single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). In presence of the target NOS, the cDNA in the nanocomposite will hybridize with its complementary sequence, and form dsDNA structure. Due to the weak π interactions between dsDNA and H-GN, a portion of H-GN will be released from the surface of Fe3O4@Au NPs and transferred into solution. After magnetic separation was performed, the supernatant was incubated with 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H2O2. The released H-GN can catalyze the oxidation reaction of TMB and turn the colorless solution blue. This "signal-on" colorimetric biosensor shows a broad linear range of 0.5-100 nM for the target NOS, with a 0.19 nM detection limit. The application of the biosensor for determination of NOS segments in samples of GM and non-GM tomatoes shows that it can discriminate between GM and non-GM plants. The reliability of the method for samples of NOS-spiked GM tomato suggests satisfactory recoveries in the range of 93.6%-94.2%.

Icariside II inhibits tumorigenesis via inhibiting AKT/Cyclin E/ CDK 2 pathway and activating mitochondria-dependent pathway.

Cervical cancer contributes largely in women cancer-related mortality. Herein, Icariside II, a flavonoid extracted from edible and pharmaceutical plant Epimedium brevicornum Maxim, exhibited significant anticancer activity on cervical cancer. At first, it was observed that Icariside II inhibited Hela cell proliferation at IC50 (9.2 µM) and the growth of Hela-originated xenografts in BALB/c nude mice. Next, we studied the underlying mechanisms of Icariside II from the aspects of cell growth and cell death. As for cell growth, Icariside II arrested cell cycle at G0/G1 phase through AKT/Cyclin E/CDK 2 from transcriptional and translational levels. As for cell death, Flow Cytometry and Immunofluorescence showed that Icariside II promoted cell death in a dose-dependet manner. And, Icariside II turned to activate the mitochondria-dependent pathway Caspase 9/Caspase 3 much more significantly than death receptor pathway Caspase 8/Caspase 3. Taken together, Icariside II presented anticancer effect on cervical cancer both in vitro and in vivo. Our study provides the evidence that Icariside II can be used as a suitable novel agent in cervical cancer treatment.

Riboflavin-overproducing lactobacilli for the enrichment of fermented soymilk: insights into improved nutritional and functional attributes.

The influence of riboflavin (B2)-overproducing lactobacilli on the antioxidant status, isoflavone conversion, off-flavor reduction, amino acid profile, and viscosity of B2-bio-enriched fermented soymilk was investigated. Results showed that B2 in fermented soymilk was notably increased from 0.2 to 3.8 µg/mL for Lactobacillus fermentum UFG169 and to 1.9 µg/mL for Lactobacillus plantarum UFG10. The apparent viscosity significantly changed with rising acidity and agglutination of protein. The off-flavor volatile substances (hexanal and nonanal) were significantly reduced in fermented soymilk. Furthermore, a large amount of glucoside form isoflavones was deglycosylated into bioactive aglycones after 4 h up to 32 h. B2 content and isoflavones significantly improved the antioxidant status of soymilk. Partial least squares regression analysis correlated the strain activity and fermentation time with the improved nutritional and functional soymilk qualities. This study demonstrated the strategy for strain development for B2-bio-enriched fermentation to extend the health-promoting benefits of soymilk and soy-related foods. KEY POINTS: • B2-enriched fermentation enhanced the nutrition and functional status of soymilk. • Fermentation time significantly affected the apparent viscosity of fermented soymilk. • Off-flavor volatile substances were significantly reduced or even diminished. • Increased B2and bioactive isoflavones contributed to improved antioxidant potential.

Calcium ion assisted fluorescence determination of microRNA-167 using carbon dots-labeled probe DNA and polydopamine-coated Fe3O4 nanoparticles.

A selective and sensitive fluorescence biosensor is described for determination of microRNA-167 using fluorescent resonant energy transfer (FRET) strategy. The FRET system comprises carbon dots (CDs, donor) labeled with probe DNA (pDNA) and polydopamine (PDA)-coated Fe3O4 nanoparticles (Fe3O4@PDA NPs, acceptor). The CDs-pDNA can be absorbed onto the surface of Fe3O4@PDA NPs because of the strong π interaction between pDNA and PDA. With the enhanced adsorption ability of Fe3O4@PDA NPs by Ca2+, the fluorescence intensity of CDs at 445 nm (excitation at 360 nm) is quenched. In presence of microRNA-167, the hybridized complex of CDs-pDNA-microRNA-167 will be released from the surface of Fe3O4@PDA NPs due to the weak π interaction of the complex and PDA. This results in the fluorescence recovery of CDs. By application of twice-magnetic separation, the biosensor shows a wide linear range of 0.5-100 nM to microRNA-167 with a 76 pM detection limit. The method was applied to the determination of microRNA-167 in samples of total microRNA extractions from A. thaliana seedlings, and the recoveries ranged from 96.4 to 98.3%.

Evaluation of the Metabolic Effects of Hydrogen Sulfide on the Development of Bombyx mori (Lepidoptera: Bombycidae), Using Liquid Chromatography-Mass Spectrometry-Based Metabolomics.

Hydrogen sulfide (H2S) is a highly poisonous gas with an unpleasant smell of rotten eggs. Previous studies of H2S have primarily focused on its effects on mammalian nervous and respiratory systems. In this study, silkworm developmental parameters and changes in metabolites in response to H2S exposure were investigated using a hemolymph metabolomic approach, based on liquid chromatography-mass spectrometry (LC-MS). The developmental parameters, body weight, cocoon weight, cocoon shell weight, and cocoon shell ratio, were noticeably increased following H2S exposure, with the greatest effects observed at 7.5-µM H2S. Metabolites upregulated under H2S exposure (7.5 µM) were related to inflammation, and included (6Z, 9Z, 12Z)-octadecatrienoic acid, choline phosphate, and malic acid, while hexadecanoic acid was downregulated. Identified metabolites were involved in biological processes, including pyrimidine, purine, and fatty acid metabolism, which are likely to affect silk gland function. These results demonstrate that H2S is beneficial to silkworm development and alters metabolic pathways related to spinning function and inflammation. The present study provides new information regarding the potential functions of H2S in insects and metabolic pathways related to this phenomenon.

Antibacterial Activity and Mechanism of Ginger Essential Oil against Escherichia coli and Staphylococcus aureus.

Though essential oils exhibit antibacterial activity against food pathogens, their underlying mechanism is understudied. We extracted ginger essential oil (GEO) using supercritical CO2 and steam distillation. A chemical composition comparison by GC-MS showed that the main components of the extracted GEOs were zingiberene and α-curcumene. Their antibacterial activity and associated mechanism against Staphylococcus aureus and Escherichia coli were investigated. The diameter of inhibition zone (DIZ) of GEO against S. aureus was 17.1 mm, with a minimum inhibition concentration (MIC) of 1.0 mg/mL, and minimum bactericide concentration (MBC) of 2.0 mg/mL. For E. coli, the DIZ was 12.3 mm with MIC and MBC values of 2.0 mg/mL and 4.0 mg/mL, respectively. The SDS-PAGE analysis revealed that some of the electrophoretic bacterial cell proteins bands disappeared with the increase in GEO concentration. Consequently, the nucleic acids content of bacterial suspension was raised significantly and the metabolic activity of bacteria was markedly decreased. GEO could thus inhibit the expression of some genes linked to bacterial energy metabolism, tricarboxylic acid cycle, cell membrane-related proteins, and DNA metabolism. Our findings speculate the bactericidal effects of GEO primarily through disruption of the bacterial cell membrane indicating its suitability in food perseveration.

Cytokinin is involved in TPS22-mediated selenium tolerance in Arabidopsis thaliana.

Background and Aims: Excess selenium (Se) is toxic to plants, but relatively little is known about the regulatory mechanism of plant Se tolerance. This study explored the role of the TPS22 gene in Se tolerance in Arabidopsis thaliana. Methods: Arabidopsis wild type and XVE mutant seeds were grown on half-strength MS media containing Na2SeO3 for screening of the Se-tolerant mutant tps22. The XVE T-DNA-tagged genomic sequence in tps22 was identified by TAIL-PCR. The TPS22 gene was transformed into the mutant tps22 and wild type plants using the flower infiltration method. Wild type, tps22 mutant and transgenic seedlings were cultivated on vertical plates for phenotype analysis, physiological index measurement and gene expression analysis. Key Results: We identified an Arabidopsis Se-tolerant mutant tps22 from the XVE pool lines, and cloned the gene which encodes the terpenoid synthase (TPS22). TPS22 was downregulated by Se stress, and loss-of-function of TPS22 resulted in decreased Se accumulation and enhanced Se tolerance; by contrast, overexpression of TPS22 showed similar traits to the wild type under Se stress. Further analysis revealed that TPS22 mediated Se tolerance through reduction of Se uptake and activation of metabolism detoxification, which decreased transcription of high-affinity transporters PHT1;1, PHT1;8 and PHT1;9 and significantly increased transcription of selenocysteine methyltransferase (SMT), respectively. Moreover, loss-of-function of TPS22 resulted in reduced cytokinin level and repression of cytokinin signalling components AHK3 and AHK4, and upregulation of ARR3, ARR15 and ARR16. Exogenous cytokinin increased transcription of PHT1;1, PHT2;1 and SMT and decreased Se tolerance of the tps22 mutant. In addition, enhanced Se resistance of the tps22 mutant was associated with glutathione (GSH). Conclusions: Se stress downregulated TPS22, which reduced endogenous cytokinin level, and then affected the key factors of Se uptake and metabolism detoxification. This cascade of events resulted in reduced Se accumulation and enhanced Se tolerance.

Chemoenzymatic synthesis of 3'-phosphoadenosine-5'-phosphosulfate coupling with an ATP regeneration system.

3'-Phosphoadenosine-5'-phosphosulfate (PAPS) is the obligate cosubstrate and source of the sulfonate group in the chemoenzymatic synthesis of heparin, a commonly used anticoagulant drug. Previously, using ATP as the substrate, we had developed a one-pot synthesis to prepare PAPS with 47% ATP conversion efficiency. During the reaction, 47% of ATP was converted into the by-product, ADP. Here, to increase the conversion ratio of ATP to PAPS, an ATP regeneration system was developed to couple with PAPS synthesis. In the ATP regeneration system, the chemical compound, monopotassium phosphoenolpyruvate (PEP-K+), was synthesized and used as the phospho-donor. By using 3-bromopyruvic acid as the starting material, the total yield of PEP-K+ synthesis was over 50% at low cost. Then, the enzyme PykA from Escherichia coli was overexpressed, purified, and used to convert the by-product ADP into ATP. When coupled the ATP regeneration system with PAPS synthesis, the higher ratio of PEP-K+ to ADP was associated with higher ATP conversion efficiency. By using the ATP regeneration system, the conversion ratio of ATP to PAPS was increased to 98% as determined by PAMN-HPLC analysis, and 5 g of PAPS was produced in 1 L of the reaction mixture. Furthermore, the chemoenzymatic synthesized PAPS was purified and freeze-dried without observed decomposition. However, the powdery PAPS was more unstable than the PAPS sodium salt in aqueous solution at ambient temperature. This developed chemoenzymatic approach of PAPS production will contribute to the synthesis of heparin, in which PAPS is necessary as the individual sulfo-donor.