Long-term stable cell growth and production of vindoline, catharanthine, and ajmalicine of cambial meristematic cells (CMCs) from Catharanthus roseus were observed after 2 years of culture. C. roseus CMCs were treated with β-cyclodextrin (β-CD) and methyl jasmonate (MeJA) individually or in combination and were cultured both in conventional Erlenmeyer flasks (100, 250, and 500 mL) and in a 5-L stirred hybrid airlift bioreactor. CMCs of C. roseus cultured in the bioreactor showed higher yields of vindoline, catharanthine, and ajmalicine than those cultured in flasks. CMCs of C. roseus cultured in the bioreactor and treated with 10 mM β-CD and 150 μM MeJA gave the highest yields of vindoline (7.45 mg/L), catharanthine (1.76 mg/L), and ajmalicine (58.98 mg/L), concentrations that were 799, 654, and 426 % higher, respectively, than yields of CMCs cultured in 100-mL flasks without elicitors. Quantitative reverse transcription (RT)-PCR showed that β-CD and MeJA upregulated transcription levels of genes related to the biosynthesis of terpenoid indole alkaloids (TIAs). This is the first study to report that β-CD induced the generation of NO, which plays an important role in mediating the production of TIAs in C. roseus CMCs. These results suggest that β-CD and MeJA can enhance the production of TIAs in CMCs of C. roseus, and thus, CMCs of C. roseus have significant potential to be an industrial platform for production of bioactive alkaloids.

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Artemisinin has been used in the production of “artemisinin combination therapies” for the treatment of malaria. Feeding of precursors has been proven to be one of the most effective methods to enhance artemisinin production in plant cultured cells. At the current paper, the biosynthesis of artemisinin (ART) and its four analogs from dihydroartemisinic acid (DHAA) in suspension-cultured cells of Artemisia annua were investigated. ARTs were detected by HPLC/GC-MS and isolated by various chromatography methods. The structures of four DHAA metabolites, namely, dihydro-epi-deoxyarteannuin B, arteannuin I, arteannuin K, and 3-β-hydroxy-dihydro-epi-deoxyarteannuin B, were elucidated by physicochemical and spectroscopic analyses. The correlation between gene expression and ART content was investigated. The results of RT-PCR showed that DHAA could up-regulate expression of amorpha-4,11-diene synthase gene (ADS), amorpha-4,11-diene C-12 oxidase gene (CYP71AV1), and farnesyl diphosphate synthase gene (FPS) (3.19-, 7.21-, and 2.04-fold higher than those of control group, resp.), which indicated that biosynthesis processes from DHAA to ART were enzyme-mediated.

Vincristine and vinblastine were found by Liquid Chromatography-Mass Spectrometry (LC-MS) in Catharanthus roseus cambial meristem cells (CMCs) jointly treated with 0.25 mM vindolineand methyl jasmonate (MeJA), suggesting that C. roseus CMCs contain a complete set of the enzymes which are in response to convert vindoline into vincristine and vinblastine. Based on the facts that the transcript levels of vindoline-biosynthetic genes ( STR, SGD and D4H) were up-regulated instead of being down-regulated by adding itself to the culture, and that the transcriptional factor ORCA3 was up-regulated simultaneously, we further confirmed that the transcription of STR, SGD, D4H was manipulated by ORCA3.