ABSTRACT
Both natural ginsenoside F2 and unnatural ginsenoside 3β,20S-Di-O-Glc-DM were reported to exhibit anti-tumor activity. Traditional approaches for producing them rely on direct extraction from Panax ginseng, enzymatic catalysis or chemical synthesis, all of which result in low yield and high cost. Metabolic engineering of microbes has been recognized as a green and sustainable biotechnology to produce natural and unnatural products. Hence we engineered the complete biosynthetic pathways of F2 and 3β,20S-Di-O-Glc-DM in Saccharomyces cerevisiae via the CRISPR/Cas9 system. The titers of F2 and 3β,20S-Di-O-Glc-DM were increased from 1.2 to 21.0 mg/L and from 82.0 to 346.1 mg/L at shake flask level, respectively, by multistep metabolic engineering strategies. Additionally, pharmacological evaluation showed that both F2 and 3β,20S-Di-O-Glc-DM exhibited anti-pancreatic cancer activity and the activity of 3β,20S-Di-O-Glc-DM was even better. Furthermore, the titer of 3β,20S-Di-O-Glc-DM reached 2.6 g/L by fed-batch fermentation in a 3 L bioreactor. To our knowledge, this is the first report on demonstrating the anti-pancreatic cancer activity of F2 and 3β,20S-Di-O-Glc-DM, and achieving their de novo biosynthesis by the engineered yeasts. Our work presents an alternative approach to produce F2 and 3β,20S-Di-O-Glc-DM from renewable biomass, which lays a foundation for drug research and development.
ABSTRACT
The present study was designed to perform structural modifications of of neobavaisoflavone (NBIF), using an in vitro enzymatic glycosylation reaction, in order to improve its water-solubility. Two novel glucosides of NBIF were obtained from an enzymatic glycosylation by UDP-glycosyltransferase. The glycosylated products were elucidated by LC-MS, HR-ESI-MS, and NMR analysis. The HPLC peaks were integrated and the concentrations in sample solutions were calculated. The MTT assay was used to detect the cytotoxic activity of compounds in cancer cell lines. Based on the spectroscopic analyses, the two novel glucosides were identified as neobavaisoflavone-4'-O-β-D-glucopyranoside (1) and neobavaisoflavone-4', 7-di-O-β-D-glucopyranoside (2). Additionally, the water-solubilities of compounds 1 and 2 were approximately 175.1- and 4 031.9-fold higher than that of the substrate, respectively. Among the test compounds, only NBIF exhibited weak cytotoxicity against four human cancer cell lines, with IC values ranging from 63.47 to 72.81 µmol·L. These results suggest that in vitro enzymatic glycosylation is a powerful approach to structural modification, improving water-solubility.
Subject(s)
Humans , Antineoplastic Agents , Metabolism , Pharmacology , Bacillus , Cell Line, Tumor , Colorimetry , Drug Screening Assays, Antitumor , Glucosides , Chemistry , Glycosyltransferases , Metabolism , Isoflavones , Chemistry , Molecular Structure , SolubilityABSTRACT
As an important kind of plant secondary metabolites and widely distributed in the plant kingdom, triter-penoid saponins have a variety of biological activities. The constitution and content of triterpenoid saponin are deter-mined by some key enzymes and their expressing level in triterpenoid saponins biosynthesis pathway. So, it is very important to illuminate the molecular mechanism of triterpenoid saponins biosynthetic pathway. In recent years, illu-mination of the entire biosynthetic pathway especially the confirmation and cloning of the key enzymes, such as squa-lene synthase, squalene epoxidase, cytochrome P450 monooxygenase and UDP-glycosyltransferase, had become one of the hot spots by many scholars. In this paper, the entire biosynthetic pathway and some kinds of key enzymes in-volved in the synthesis of carbon skeleton, and its oxidation, and glycation were reviewed for further demonstrating the biosynthetic pathway of triterpenoid saponins and providing a theoretical basis for artificial biosynthesis.