Comparative metabolomic and transcriptomic analysis of Saccharomyces cerevisiae W303a and CEN.PK2-1C.

Saccharomyces cerevisiae is a health microorganism closely related to human life, especially in food and pharmaceutical industries. S. cerevisiae W303a and CEN.PK2-1C are two commonly used strains for synthetic biology-based natural product production. Yet, the metabolomic and transcriptomic differences between these two strains have not been compared. In this study, metabolomics and transcriptomics were applied to analyze the differential metabolites and differential expression genes (DEGs) between W303a and CEN.PK2-1C cultured in YPD and SD media. The growth rate of W303a in YPD medium was the lowest compared with other groups. When cultured in YPD medium, CEN.PK2-1C produced more phenylalanine than W303a; when cultured in SD medium, W303a produced more phospholipids than CEN.PK2-1C. Transcriptomic analysis revealed that 19 out of 22 genes in glycolysis pathway were expressed at higher levels in CEN.PK2-1C than that in W303a no matter which media were used, and three key genes related to phenylalanine biosynthesis including ARO9, ARO7 and PHA2 were up-regulated in CEN.PK2-1C compared with W303a when cultured in YPD medium, whereas seven DEGs associated with phospholipid biosynthesis were up-regulated in W303a compared with CEN.PK2-1C when cultured in SD medium. The high phenylalanine produced by CEN.PK2-1C and high phospholipids produced by W303a indicated that CEN.PK2-1C may be more suitable for synthesis of natural products with phenylalanine as precursor, whereas W303a may be more appropriate for synthesis of phospholipid metabolites. This finding provides primary information for strain selection between W303a and CEN.PK2-1C for synthetic biology-based natural product production.

New Glycosyltransferases in Panax notoginseng Perfect Main Ginsenosides Biosynthetic Pathways.

Ginsenosides, the main bioactive ingredients of the Panax genus, are dammarane or oleanane triterpenoids with glycosylated modifications at C3/C6/C20 hydroxyls or C28 carboxyl, and their diverse glycosylation pattern has attracted great attention. However, the biosynthesis of some important saponins is still unclear. In this study, six UGTs were characterized, two of which were novel. PnUGT71A3 catalyzes not only the C6 hydroxyl glycosylation of protopanaxatriol (PPT) and F1 to form Rh1 and Rg1, respectively, but also the C20 hydroxyl glycosylation of protopanaxadiol (PPD)-type Rg3 to generate Rd. Especially, PnUGT94M1 is UDP-ß-l-rhamnose (UDP-Rha)-dependent, regioselectively catalyzing the C2' hydroxyl rhamnosylation of C6 glucose of the PPT-type ginsenosides Rg1 and Rh1 to generate ginsenosides Re and Rg2, respectively. Site-directed mutagenesis showed that His21, Asp120, Ser363, and Pro372 are key residues, and the triple mutant (G344S/G345S/L346T) highly improved the activity toward Rg1 and Rh1. The findings in this study, perfect main ginsenosides biosynthetic pathways in the Panax genus, expand the biocatalyst toolbox for ginsenoside production and show that the PSPG motif is one of the options to modify UGTs to improve their activities.

Ginsenosides in Panax genus and their biosynthesis.

Ginsenosides are a series of glycosylated triterpenoids which belong to protopanaxadiol (PPD)-, protopanaxatriol (PPT)-, ocotillol (OCT)- and oleanane (OA)-type saponins known as active compounds of Panax genus. They are accumulated in plant roots, stems, leaves, and flowers. The content and composition of ginsenosides are varied in different ginseng species, and in different parts of a certain plant. In this review, we summarized the representative saponins structures, their distributions and the contents in nearly 20 Panax species, and updated the biosynthetic pathways of ginsenosides focusing on enzymes responsible for structural diversified ginsenoside biosynthesis. We also emphasized the transcription factors in ginsenoside biosynthesis and non-coding RNAs in the growth of Panax genus plants, and highlighted the current three major biotechnological applications for ginsenosides production. This review covered advances in the past four decades, providing more clues for chemical discrimination and assessment on certain ginseng plants, new perspectives for rational evaluation and utilization of ginseng resource, and potential strategies for production of specific ginsenosides.

Notoginsenoside Ft1 activates both glucocorticoid and estrogen receptors to induce endothelium-dependent, nitric oxide-mediated relaxations in rat mesenteric arteries.

Panax notoginseng (Burk.) F.H. Chen has been used traditionally for the treatment of cardiovascular diseases. Notoginsenoside Ft1 (Ft1) is a bioactive saponin from the leaves of P. notoginseng. Experiments were designed to determine whether or not Ft1 is an endothelium-dependent vasodilator. Rat mesenteric arteries were suspended in organ chambers for the measurement of isometric tension during phenylephrine-induced contractions. The cyclic guanosine monophosphate (cGMP) level was assessed using enzyme immunoassay. The phosphorylation and protein expressions of endothelial nitric oxide synthase (eNOS), glucocorticoid receptors (GR), estrogen receptors beta (ERß), protein kinase B (Akt) and extracellular signal-regulated kinase 1/2 (ERK1/2) were determined by Western blotting. The localization of GR and ERß were determined by immunofluorescence staining. Ft1 caused endothelium-dependent relaxations, which were abolished by l-NAME (inhibitor of nitric oxide synthases) and ODQ (inhibitor of soluble guanylyl cyclase). Ft1 increased the cGMP level in rat mesenteric arteries. GR and ERß were present in the endothelial layer and their antagonism by RU486 and PHTPP, respectively, inhibited Ft1-induced endothelium-dependent relaxations and phosphorylations of eNOS, Akt and ERK1/2. Inhibition of phosphoinositide-3-kinase (PI3K) by wortmannin and ERK1/2 by U0126 reduced Ft1-evoked relaxations and eNOS phosphorylation. Taken in conjunction, the present findings suggest that Ft1 stimulates endothelial GRs and ERßs with subsequent activation of the PI3K/Akt and ERK1/2 pathways in rat mesenteric arteries. This results in phosphorylation of eNOS and the release of NO, which activates soluble guanylyl cyclase in the vascular smooth muscle cells leading to relaxations.

Notoginsenoside Ft1 promotes angiogenesis via HIF-1α mediated VEGF secretion and the regulation of PI3K/AKT and Raf/MEK/ERK signaling pathways.

Notoginsenoside Ft1 (Ft1) is a saponin isolated from Panax notoginseng, which has been used traditionally for the treatment of trauma injuries in East Asia. Here we show that Ft1 is a novel stimulator of angiogenesis. The results show that Ft1 induces proliferation, migration, and tube formation in cultured human umbilical vein endothelial cells (HUVECs). Ft1 increases translocalization of hypoxia-inducible factor-1α (HIF-1α) from cytoplasm to nuclei, where it binds to the vascular endothelial growth factor (VEGF) promoter, increasing the expression of VEGF mRNA and the subsequent secretion of the growth factor. Ft1 induces the activation of PI3K/AKT and Raf/MEK/ERK signaling pathways. Pharmacological inhibition with LY294002, wortmanin or PD98059 reduces Ft1-induced angiogenesis, indicating the important role played by these pathways. In addition, Ft1 induces phosphorylation of the mammalian target of rapamycin (mTOR), and siRNA-mediated mTOR knockdown decreases tube formation, proliferation, transport of HIF-1α into nuclei and VEGF mRNA expression in response to Ft1. Finally, in vivo, Ft1 promotes the formation of blood vessels in Matrigel plug and wound healing in mice. Taken together, the present results reveal that Ft1 stimulates angiogenesis via HIF-1α-mediated VEGF expression, with PI3K/AKT and Raf/MEK/ERK signaling cascades concurrently participating in the process.