Transcriptome analysis of Pueraria candollei var. mirifica for gene discovery in the biosyntheses of isoflavones and miroestrol

International audienceBackground: Pueraria candollei var. mirifica, a Thai medicinal plant used traditionally as a rejuvenating herb, is known as a rich source of phytoestrogens, including isoflavonoids and the highly estrogenic miroestrol and deoxymiroestrol. Although these active constituents in P. candollei var. mirifica have been known for some time, actual knowledge regarding their biosynthetic genes remains unknown.Results: Miroestrol biosynthesis was reconsidered and the most plausible mechanism starting from the isoflavonoid daidzein was proposed. A de novo transcriptome analysis was conducted using combined P. candollei var. mirifica tissues of young leaves, mature leaves, tuberous cortices, and cortex-excised tubers. A total of 166,923 contigs was assembled for functional annotation using protein databases and as a library for identification of genes that are potentially involved in the biosynthesis of isoflavonoids and miroestrol. Twenty-one differentially expressed genes from four separate libraries were identified as candidates involved in these biosynthetic pathways, and their respective expressions were validated by quantitative real-time reverse transcription polymerase chain reaction. Notably, isoflavonoid and miroestrol profiling generated by LC-MS/MS was positively correlated with expression levels of isoflavonoid biosynthetic genes across the four types of tissues. Moreover, we identified R2R3 MYB transcription factors that may be involved in the regulation of isoflavonoid biosynthesis in P. candollei var. mirifica. To confirm the function of a key-isoflavone biosynthetic gene, P. candollei var. mirifica isoflavone synthase identified in our library was transiently co-expressed with an Arabidopsis MYB12 transcription factor (AtMYB12) in Nicotiana benthamiana leaves. Remarkably, the combined expression of these proteins led to the production of the isoflavone genistein.Conclusions: Our results provide compelling evidence regarding the integration of transcriptome and metabolome as a powerful tool for identifying biosynthetic genes and transcription factors possibly involved in the isoflavonoid and miroestrol biosyntheses in P. candollei var. mirifica

Pre-formulation and delivery strategies for the development of bacteriocins as next generation antibiotics

peer-reviewedBacteriocins, a class of antimicrobial peptide produced by bacteria, may offer a potential alternative to traditional antibiotics, an important step towards mitigating the ever increasing antimicrobial resistance crisis. They are active against a range of clinically relevant Gram-positive and Gram-negative bacteria. Bacteriocins have been discussed in the literature for over a century. Although they are used as preservatives in food, no medicine based on their antimicrobial activity exists on the market today. In order to formulate them into clinical antibiotics, pre-formulation studies on their biophysical and physicochemical properties that will influence their activity in vivo and their stability during manufacture must be elucidated. Thermal, pH and enzymatic stability of bacteriocins are commonly studied and regularly reported in the literature. Solubility, permeability and aggregation properties on the other hand are less frequently reported for many bacteriocins, which may contribute to their poor clinical progression. Promising cytotoxicity studies report that bacteriocins exhibit few cytotoxic effects on a variety of mammalian cell lines, at active concentrations. This review highlights the lack of quantitative data and in many cases even qualitative data, on bacteriocins’ solubility, stability, aggregation, permeability and cytotoxicity. The formulation strategies that have been explored to date, proposed routes of administration, trends in in vitro/in vivo behaviour and efforts in clinical development are discussed. The future promise of bacteriocins as a new generation of antibiotics may require tailored local delivery strategies to fulfil their potential as a force to combat antimicrobial-resistant bacterial infections

Identification of Genes Involved in Flocculation by Whole Genome Sequencing of Thauera aminoaromatica Strain MZ1T Floc-defective Mutants

Thauera aminoaromatica MZ1T, a floc-forming bacterium isolated from an industrial activated sludge wastewater treatment plant, overproduces exopolysaccharide (EPS) leading to viscous bulking. This phenomenon results in poor sludge settling and dewatering during the clarification process. To identify genes responsible for bacterial flocculation, a whole genome phenotypic sequencing technique was applied. Genomic DNA of MZ1T flocculation-deficient mutants were subjected to massively parallel sequencing. The resultant high-quality reads were assembled and compared to the reference genome of the wild type genome. We identified nine nonsynonymous mutations and one nonsense mutation putatively involved in EPS biosynthesis. Complementation of the nonsense mutation located in an EPS deacetylase gene restored the flocculating phenotype. The FTIR spectra of EPS isolated from the wild-type showed reduced C=O peak of the N-acetyl group at 1665 cm-1 as compared to the spectra of MZ1T floc-deficient mutant EPS, suggesting that the WT EPS was partially deacetylated. Gene expression analysis also demonstrated the deacetylase gene transcript increased before flocculation occurred. The results suggest that the deacetylation of MZ1T EPS is crucial for flocculation. The information obtained from this study will be useful for preventing viscous bulking and wastewater treatment system failure, and may have potential applications in the biotechnology sector for the controlled removal of cells

Flocculation-Related Gene Identification by Whole-Genome Sequencing of Thauera aminoaromatica MZ1T Floc-Defective Mutants

This article discusses the identification of genes responsible for bacterial flocculation using a whole-genome phenotypic-sequencing technique

Metatranscriptomics Reveals Sequential Expression of Genes Involved in the Production of Melanogenesis Inhibitors by the Defined Microbial Species in Fermented Unpolished Black Rice

Fermentation of food is a metabolic process through the action of enzymes from certain microorganisms. Although roles of the microbial community in the fermented food were investigated using metatranscriptomic approach in terms of flavors, but no study has been reported so far on the function of the microorganisms on producing compounds with a melanogenesis inhibition activity.</jats:p

DNA metabarcoding to unravel plant species composition in selected herbal medicines on the National List of Essential Medicines (NLEM) of Thailand

AbstractTraditional medicines are widely traded across the globe and have received considerable attention in the recent past, with expectations of heightened demand in the future. However, there are increasing global concerns over admixture, which can affect the quality, safety, and efficacy of herbal medicinal products. In this study, we aimed to use DNA metabarcoding to identify 39 Thai herbal products on the Thai National List of Essential Medicines (NLEM) and assess species composition and admixture. Among the products, 24 samples were in-house-prepared formulations, and 15 samples were registered formulations. In our study, DNA metabarcoding analysis using ITS2 and rbcL barcode regions were employed to identify herbal ingredients mentioned in the products. The nuclear region, ITS2, was able to identify herbal ingredients in the products at the genus- and family-levels in 55% and 63% of cases, respectively. The chloroplast gene, rbcL, enabled genus- and family-level identifications in 58% and 73% of cases, respectively. In addition, plant species were detected in larger numbers (Family identified, absolute %) in registered herbal products than in in-house-prepared formulations. The level of fidelity increases concerns about the reliability of the products. This study highlights that DNA metabarcoding is a useful analytical tool when combined with advanced chemical techniques for the identification of plant species in highly processed, multi-ingredient herbal products.</jats:p

Role of Dietary Defatted Rice Bran in the Modulation of Gut Microbiota in AOM/DSS-Induced Colitis-Associated Colorectal Cancer Rat Model

Defatted rice bran (DRB) is a by-product of rice bran derived after the oil extraction. DRB contains several bioactive compounds, including dietary fiber and phytochemicals. The supplementation with DRB manifests chemopreventive effects in terms of anti-chronic inflammation, anti-cell proliferation, and anti-tumorigenesis in the azoxymethane (AOM) and dextran sodium sulfate (DSS)-induced colitis-associated colorectal cancer (CRC) model in rats. However, little is known about its effect on gut microbiota. Herein, we investigated the effect of DRB on gut microbiota and short chain fatty acid (SCFA) production, colonic goblet cell loss, and mucus layer thickness in the AOM/DSS-induced colitis-associated CRC rat model. The results suggested that DRB enhanced the production of beneficial bacteria (Alloprevotella, Prevotellaceae UCG-001, Ruminococcus, Roseburia, Butyricicoccus) and lessened the production of harmful bacteria (Turicibacter, Clostridium sensu stricto 1, Escherichia–Shigella, Citrobacter) present in colonic feces, mucosa, and tumors. In addition, DRB also assisted the cecal SCFAs (acetate, propionate, butyrate) production. Furthermore, DRB restored goblet cell loss and improved the thickness of the mucus layer in colonic tissue. These findings suggested that DRB could be used as a prebiotic supplement to modulate gut microbiota dysbiosis, which decreases the risks of CRC, therefore encouraging further research on the utilization of DRB in various nutritional health products to promote the health-beneficial bacteria in the colon.</jats:p

Role of Dietary Defatted Rice Bran in the Modulation of Gut Microbiota in AOM/DSS-Induced Colitis-Associated Colorectal Cancer Rat Model

Defatted rice bran (DRB) is a by-product of rice bran derived after the oil extraction. DRB contains several bioactive compounds, including dietary fiber and phytochemicals. The supplementation with DRB manifests chemopreventive effects in terms of anti-chronic inflammation, anti-cell proliferation, and anti-tumorigenesis in the azoxymethane (AOM) and dextran sodium sulfate (DSS)-induced colitis-associated colorectal cancer (CRC) model in rats. However, little is known about its effect on gut microbiota. Herein, we investigated the effect of DRB on gut microbiota and short chain fatty acid (SCFA) production, colonic goblet cell loss, and mucus layer thickness in the AOM/DSS-induced colitis-associated CRC rat model. The results suggested that DRB enhanced the production of beneficial bacteria (Alloprevotella, Prevotellaceae UCG-001, Ruminococcus, Roseburia, Butyricicoccus) and lessened the production of harmful bacteria (Turicibacter, Clostridium sensu stricto 1, Escherichia&ndash;Shigella, Citrobacter) present in colonic feces, mucosa, and tumors. In addition, DRB also assisted the cecal SCFAs (acetate, propionate, butyrate) production. Furthermore, DRB restored goblet cell loss and improved the thickness of the mucus layer in colonic tissue. These findings suggested that DRB could be used as a prebiotic supplement to modulate gut microbiota dysbiosis, which decreases the risks of CRC, therefore encouraging further research on the utilization of DRB in various nutritional health products to promote the health-beneficial bacteria in the colon