Microbial Conversion of Protopanaxadiol-Type Ginsenosides by the Edible and Medicinal Mushroom Schizophyllum commune: A Green Biotransformation Strategy.
ABSTRACT: Previous studies have shown that many kinds of microorganisms, including bacteria, yeasts, and filamentous fungi, can convert parent ginsenosides into minor ginsenosides. However, most microorganisms used for ginsenoside transformations may not be safe for food consumption and drug development. In this study, 24 edible and medicinal mushrooms were screened by high-performance liquid chromatography analyses for their ability to microbiologically transform protopanaxadiol (PPD)-type ginsenosides. We observed that the degradation of ginsenosides by Schizophyllum commune was inhibited by high concentrations of sugar in the culture medium. However, the inhibition was avoided by maintaining sugar concentration below 15 g L-1. S. commune showed a strong ability to convert PPD-type ginsenosides (Rb1, Rc, Rb2, and Rd) into minor ginsenosides (F2, C-O, C-Y, C-Mc1, C-Mc, and C-K). The production and bioconversion rates of minor ginsenosides were significantly higher than those previously reported by food microorganisms. The fermentation process is efficient, nontoxic, eco-friendly, and economical, and the required biotransformation systems are readily available. This is the first report about the biotransformation of major ginsenosides into minor ginsenosides through fermentation by edible and medicinal mushrooms. Our results provide a green biodegradation strategy in transformation of ginsenosides using edible and medicinal mushrooms.
Project description:Background:Ginsenosides are known as the principal pharmacological active constituents in Panax medicinal plants such as Asian ginseng, American ginseng, and Notoginseng. Some ginsenosides, especially the 20(R) isomers, are found in trace amounts in natural sources and are difficult to chemically synthesize. The present study provides an approach to produce such trace ginsenosides applying biotransformation through Escherichia coli modified with relevant genes. Methods:Seven uridine diphosphate glycosyltransferase (UGT) genes originating from Panax notoginseng, Medicago sativa, and Bacillus subtilis were synthesized or cloned and constructed into pETM6, an ePathBrick vector, which were then introduced into E. coli BL21star (DE3) separately. 20(R)-Protopanaxadiol (PPD), 20(R)-protopanaxatriol (PPT), and 20(R)-type ginsenosides were used as substrates for biotransformation with recombinant E. coli modified with those UGT genes. Results:E. coli engineered with GT95 syn selectively transfers a glucose moiety to the C20 hydroxyl of 20(R)-PPD and 20(R)-PPT to produce 20(R)-CK and 20(R)-F1, respectively. GTK1- and GTC1-modified E. coli glycosylated the C3-OH of 20(R)-PPD to form 20(R)-Rh2. Moreover, E. coli containing p2GT95synK1, a recreated two-step glycosylation pathway via the ePathBrich, implemented the successive glycosylation at C20-OH and C3-OH of 20(R)-PPD and yielded 20(R)-F2 in the biotransformation broth. Conclusion:This study demonstrates that rare 20(R)-ginsenosides can be produced through E. coli engineered with UTG genes.
Project description:BACKGROUND: Minor ginsenosides, those having low content in ginseng, have higher pharmacological activities. To obtain minor ginsenosides, the biotransformation of American ginseng protopanaxadiol (PPD)-ginsenoside was studied using special ginsenosidase type-I from Aspergillus niger g.848. METHODS: DEAE (diethylaminoethyl)-cellulose and polyacrylamide gel electrophoresis were used in enzyme purification, thin-layer chromatography and high performance liquid chromatography (HPLC) were used in enzyme hydrolysis and kinetics; crude enzyme was used in minor ginsenoside preparation from PPD-ginsenoside; the products were separated with silica-gel-column, and recognized by HPLC and NMR (Nuclear Magnetic Resonance). RESULTS: The enzyme molecular weight was 75 kDa; the enzyme firstly hydrolyzed the C-20 position 20-O-?-D-Glc of ginsenoside Rb1, then the C-3 position 3-O-?-D-Glc with the pathway Rb1?Rd?F2?C-K. However, the enzyme firstly hydrolyzed C-3 position 3-O-?-D-Glc of ginsenoside Rb2 and Rc, finally hydrolyzed 20-O-L-Ara with the pathway Rb2?C-O?C-Y?C-K, and Rc?C-Mc1?C-Mc?C-K. According to enzyme kinetics, K m and V max of Michaelis-Menten equation, the enzyme reaction velocities on ginsenosides were Rb1 > Rb2 > Rc > Rd. However, the pure enzyme yield was only 3.1%, so crude enzyme was used for minor ginsenoside preparation. When the crude enzyme was reacted in 3% American ginseng PPD-ginsenoside (containing Rb1, Rb2, Rc, and Rd) at 45°C and pH 5.0 for 18 h, the main products were minor ginsenosides C-Mc, C-Y, F2, and C-K; average molar yields were 43.7% for C-Mc from Rc, 42.4% for C-Y from Rb2, and 69.5% for F2 and C-K from Rb1 and Rd. CONCLUSION: Four monomer minor ginsenosides were successfully produced (at low-cost) from the PPD-ginsenosides using crude enzyme.
Project description:Biotransformation for increasing the pharmaceutical effect of ginsenosides is getting more and more attractions. Strain Cellulosimicrobium sp. TH-20 isolated from ginseng soil samples was identified to produce enzymes contributing to its excellent biotransformation activity against ginsenosides, the main active components of ginseng. Based on phylogenetic tree and homology analysis, the strain can be designated as Cellulosimicrobium sp. Genome sequencing was performed using the Illumina Miseq to explore the functional genes involved in ginsenoside transformation. The draft genome of Cellulosimicrobium sp. TH-20 encoded 3450 open reading frames, 51 tRNA, and 9 rRNA. All ORFs were annotated using NCBI BLAST with non-redundant proteins, Gene Ontology, Cluster of Orthologous Gene, and Kyoto Encyclopedia of Genes and Genomes databases. A total of 11 genes were selected based on the functional annotation analysis. These genes are relevant to ginsenoside biotransformation, including 6 for beta-glucosidase, 1 for alpha-N-arabinofuranosidase, 1 for alpha-1,6-glucosidase, 1 for endo-1,4-beta-xylanase, 1 for alpha-L-arabinofuranosidase, and 1 for beta-galactosidase. These glycosidases were predicted to catalyze the hydrolysis of sugar moieties attached to the aglycon of ginsenosides and led to the transformation of PPD-type and PPT-type ginsenosides.
Project description:Natural coumarins are present in remarkable amounts as secondary metabolites in edible and medicinal plants, where they display interesting bioactivities. Considering the wide enzymatic arsenal of filamentous fungi, studies on the biotransformation of coumarins using these microorganisms have great importance in green chemical derivatization. Several reports on the biotransformation of coumarins using fungi have highlighted the achievement of chemical analogs with high selectivity by using mild and ecofriendly conditions. Prompted by the enormous pharmacological, alimentary, and chemical interest in coumarin-like compounds, this study evaluated the biotransformation of nine coumarin scaffolds using Cunninghamella elegans ATCC 10028b and Aspergillus brasiliensis ATCC 16404. The chemical reactions which were catalyzed by the microorganisms were highly selective. Among the nine studied coumarins, only two of them were biotransformed. One of the coumarins, 7-hydroxy-2,3-dihydrocyclopenta[c]chromen-4(1H)-one, was biotransformed into the new 7,9-dihydroxy-2,3-dihydrocyclopenta[c]chromen-4(1H)-one, which was generated by selective hydroxylation in an unactivated carbon. Our results highlight some chemical features of coumarin cores that are important to biotransformation using filamentous fungi.
Project description:Background:In the current phytochemical research on ginseng, the differentiation and structural identification of ginsenosides isomers remain challenging. In this paper, a two-dimensional mass spectrometry (2D-MS) method was developed and combined with statistical analysis for the direct differentiation, identification, and relative quantification of protopanaxadiol (PPD)-type ginsenoside isomers. Methods:Collision-induced dissociation was performed at successive collision energy values to produce distinct profiles of the intensity fraction (IF) and ratio of intensity (RI) of the fragment ions. To amplify the differences in tandem mass spectra between isomers, IF and RI were plotted against collision energy. The resulting data distributions were then used to obtain the parameters of the fitted curves, which were used to evaluate the statistical significance of the differences between these distributions via the unpaired t test. Results:A triplet and two pairs of PPD-type ginsenoside isomers were differentiated and identified by their distinct IF and RI distributions. In addition, the fragmentation preference of PPD-type ginsenosides was determined on the basis of the activation energy. The developed 2D-MS method was also extended to quantitatively determine the molar composition of ginsenoside isomers in mixtures of biotransformation products. Conclusion:In comparison with conventional mass spectrometry methods, 2D-MS provides more direct insights into the subtle structural differences between isomers and can be used as an alternative approach for the differentiation of isomeric ginsenosides and natural products.
Project description:Ginseng is an increasingly popular ingredient in supplements for healthcare products and traditional medicine. Heat-processed ginsengs, such as red ginseng or black ginseng, are regarded as more valuable for medicinal use when compared to white ginseng due to some unique less polar ginsenosides that are produced during heat-treatment. Although ginseng leaf contains abundant ginsenosides, attention has mostly focused on ginseng root; relatively few publications have focused on ginseng leaf. Raw ginseng leaf was steamed nine times to make black ginseng leaf using a process that is similar to that used to produce black ginseng root. Sixteen ginsenosides were analyzed during each steaming while using high-performance liquid chromatography (HPLC). The contents of ginsenosides Rd and Re decreased and the less polar ginsenosides (F2, Rg3, Rk2, Rk3, Rh3, Rh4, and protopanaxatriol) enriched during steam treatment. After nine cycles of steaming, the contents of the less polar ginsenosides F2, Rg3, and Rk2 increased by 12.9-fold, 8.6-fold, and 2.6-fold, respectively. Further, we found that the polar protopanaxadiol (PPD) -type ginsenosides are more likely to be converted from ginsenoside Rg3 to ginsenosides Rk1 and Rg5 via dehydration from Rg3, and from ginsenoside Rh2 to ginsenosides Rk2 and Rh3 through losing an H2O molecule than to be completely degraded to the aglycones PPD during the heat process. This study suggests that ginseng leaves can be used to produce less polar ginsenosides through heat processes, such as steaming.
Project description:Ginseng (Panax ginseng) and its bioactive components, ginsenosides, are popular medicinal herbal products, exhibiting various pharmacological effects. Despite their advocated use for medication, the long cultivation periods of ginseng roots and their low ginsenoside content prevent mass production of this compound. Yeast Saccharomyces cerevisiae was engineered for production of protopanaxadiol (PPD), a type of aglycone characterizing ginsenoside. PPD-producing yeast cell factory was further engineered by obtaining a balance between enzyme expressions and altering cofactor availability. Different combinations of promoters (PGPD, PCCW12, and PADH2) were utilized to construct the PPD biosynthetic pathway. Rerouting the redox metabolism to improve NADPH availability in the engineered S. cerevisiae also increased PPD production. Combining these approaches resulted in more than an 11-fold increase in PPD titer over the initially constructed strain. The series of metabolic engineering strategies of this study provides a feasible approach for the microbial production of PPD and development of microbial platforms producing other industrially-relevant terpenoids.
Project description:Some differences have been reported in the biotransformation of ginsenosides, probably due to the types of materials used such as ginseng, enzymes, and microorganisms. Moreover, most microorganisms used for transforming ginsenosides do not meet food-grade standards. We investigated the statistical conversion rate of major ginsenosides in ginsenosides model culture during fermentation by lactic acid bacteria (LAB) to estimate possible pathways.Ginsenosides standard mix was used as a model culture to facilitate clear identification of the metabolic changes. Changes in eight ginsenosides (Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, and Rg2) during fermentation with six strains of LAB were investigated.In most cases, the residual ginsenoside level decreased by 5.9-36.8% compared with the initial ginsenoside level. Ginsenosides Rb1, Rb2, Rc, and Re continuously decreased during fermentation. By contrast, Rd was maintained or slightly increased after 1 d of fermentation. Rg1 and Rg2 reached their lowest values after 1-2 d of fermentation, and then began to increase gradually. The conversion of Rd, Rg1, and Rg2 into smaller deglycosylated forms was more rapid than that of Rd from Rb1, Rb2, and Rc, as well as that of Rg1 and Rg2 from Re during the first 2 d of fermentation with LAB.Ginsenosides Rb1, Rb2, Rc, and Re continuously decreased, whereas ginsenosides Rd, Rg1, and Rg2 increased after 1-2 d of fermentation. This study may provide new insights into the metabolism of ginsenosides and can clarify the metabolic changes in ginsenosides biotransformed by LAB.
Project description:Recent studies have indicated that a major contributor to the innate immune enhancing properties of some medicinal plants is derived from the cell wall components of bacteria colonizing these plants. The purpose of the current study was to assess if the bacteria present within edible and medicinal mushrooms substantially contribute to the innate immune stimulating potential of these mushrooms. Whole mushrooms from thirteen types of edible fungi and individual parts from Agaricus bisporus were analyzed for in vitro macrophage activation as well as bacterial lipopolysaccharides (LPS) content, cell load, and community composition. Substantial variation between samples was observed in macrophage activation (over 500-fold), total bacterial load (over 200-fold), and LPS content (over 10 million-fold). Both LPS content (? = 0.832, p < 0.0001) and total bacterial load (? = 0.701, p < 0.0001) correlated significantly with macrophage activation in the whole mushroom extracts. Extract activity was negated by treatment with NaOH, conditions that inactivate LPS and other bacterial components. Significant correlations between macrophage activation and total bacterial load (? = 0.723, p = 0.0001) and LPS content (? = 0.951, p < 0.0001) were also observed between different tissues of Agaricus bisporus. Pseudomonas and Flavobacterium were the most prevalent genera identified in the different tissue parts and these taxa were significantly correlated with in vitro macrophage activation (? = 0.697, p < 0.0001 and ? = 0.659, p = 0.0001, respectively). These results indicate that components derived from mushroom associated bacteria contribute substantially to the innate immune enhancing activity exhibited by mushrooms and may result in similar therapeutic actions as reported for ingestion of bacterial preparations such as probiotics.
Project description:To investigate the ability of bacteria in the intestinal microbiome to convert naturally occurring primary ginsenosides in red ginseng extract to active secondary ginsenosides.Anti-proliferative ginsenoside activity was tested using mouse lung cancer LM1 cells. Permeabilities were evaluated in Caco-2 cell monolayers. Systemic exposure of secondary ginsenosides was determined in A/J mice. 16S rRNA gene pyrosequencing was used to determine membership and abundance of bacteria in intestinal microbiome.Secondary ginsenoside C-K exhibited higher anti-proliferative activity and permeability than primary ginsenosides. Significant amounts of secondary ginsenosides (F2 and C-K) were found in blood of A/J mice following oral administration of primary ginsenoside Rb1. Because mammalian cells did not hydrolyze ginsenoside, we determined the ability of bacteria to hydrolyze ginsenosides and found that Rb1 underwent stepwise hydrolysis to Rd, F2, and then C-K. Formation of F2 from Rd was the rate-limiting step in the biotransformation of Rb1 to C-K.Conversion to F2 is the rate-limiting step in bioactivation of primary ginsenosides by A/J mouse intestinal microbiome, whose characterization reveals the presence of certain bacterial families capable of enabling the formation of F2 and C-K in vivo.