Combinatorial biosynthesis of sapogenins and saponins in Saccharomyces cerevisiae using a C-16α hydroxylase from Bupleurum falcatum.

The saikosaponins comprise oleanane- and ursane-type triterpene saponins that are abundantly present in the roots of the genus Bupleurum widely used in Asian traditional medicine. Here we identified a gene, designated CYP716Y1, encoding a cytochrome P450 monooxygenase from Bupleurum falcatum that catalyzes the C-16α hydroxylation of oleanane- and ursane-type triterpenes. Exploiting this hitherto unavailable enzymatic activity, we launched a combinatorial synthetic biology program in which we combined CYP716Y1 with oxidosqualene cyclase, P450, and glycosyltransferase genes available from other plant species and reconstituted the synthesis of monoglycosylated saponins in yeast. Additionally, we established a culturing strategy in which applying methylated ß-cyclodextrin to the culture medium allows the sequestration of heterologous nonvolatile hydrophobic terpenes, such as triterpene sapogenins, from engineered yeast cells into the growth medium, thereby greatly enhancing productivity. Together, our findings provide a sound base for the development of a synthetic biology platform for the production of bioactive triterpene sapo(ge)nins.

Synergistic and cytotoxic action of indole alkaloids produced from elicited cell cultures of Catharanthus roseus.

CONTEXT: Catharanthus roseus (L.) G. Don (Apocynaceae) is a medicinal plant that produces more than 130 alkaloids, with special attention given to the production of the anti-hypertensive monomeric indole alkaloids, serpentine and ajmalicine, and the antitumor dimeric alkaloids, vinblastine and vincristine. OBJECTIVE: This study evaluated the cytotoxic activity of the indole alkaloid-enriched bioactive extract obtained from suspension cultured-cells of C. roseus elicited with methyl jasmonate (MJ) and cyclodextrins (CDs) in three cell lines: JURKAT E.6 human lymphocytic leukemia, THP-1 human monocytic leukemia and BL 1395 non-tumor human B-cell line. MATERIALS AND METHODS: An indole alkaloid-enriched bioactive extract was obtained from C. roseus cell cultures elicited with MJ and CDs. The indole alkaloids were identified using an HPLC-diode array system coupled to a time-of-flight mass spectrometer using electrospray ionization (ESI) source. The cytotoxic assays were made using the colorimetric assay 2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-S-[(phenylamino)carbonyl]-2 tetrazolium hydroxide (XTT). RESULTS: Four indole alkaloids were identified (catharanthine, ajmalicine, tabersonine and lochnericine) but only catharanthine and ajmalicine were quantified. The concentration of the indole alkaloid-enriched bioactive extract that inhibited cell growth by 50% was 211 and 210 ng/mL for the JURKAT E.6 and THP-1 cell lines, respectively. DISCUSSION AND CONCLUSION: The results confirm that the powerful antitumor activity of this indole alkaloid-enriched bioactive extract is not due to the effect of a single compound but depends on the synergistic action of the four compounds identified.

Kinetic characterization of a basic peroxidase from garlic (Allium sativum L.) cloves.

Peroxidases catalyze the reduction of H(2)O(2) by taking electrons from a variety of compounds from the secondary metabolism including flavonoids and lignin precursors. This work describes the purification and kinetic characterization of a basic peroxidase from garlic cloves using quercetin and p-coumaric acid, flavonoid and phenolic compounds found in garlic cloves. The high catalytic efficiency shown by this basic peroxidase in the oxidation of quercetin at acidic pH suggests good adaptation of this enzyme, involved in quercetin catabolism in the acidic physiological pH conditions of the vacuoles, where it is presumably located. Likewise, garlic peroxidase showed similar oxidation rates for hydroxycinnamyl (p-coumaric) and sinapyl-type structures, which suggests its involvement in the cross-coupling reactions that occur in the cell wall during lignification. On the other hand, the high affinity of this enzyme for H(2)O(2) would be in accordance with the oxidation of both flavonoid and phenolic compounds to regulate H(2)O(2) levels in tissues/organelles, where this peroxidase is expressed.