Strictosidine Synthase Coding Gene Expression towards Quinine Biosynthesis and Accumulation: Inconsistency in Cultured Cells and Fresh Tissues of Cinchona ledgeriana

Strictosidine synthase, encoded by the gene STR, facilitates the regeneration of strictosidine, a critical intermediate for the synthesis of many plant alkaloids. The gene has, however, never been studied in Cinchona spp. The plants produce quinine alkaloid used for malaria medication, SARS-CoV-2 treatment and other industrial purposes. Cultured cells can produce the alkaloid but only at a much lower yield than the natural tree. This study describes STR expression and quinine content in various plant materials. Bioinformatic analyses were conducted on nine species of Rubiaceae to obtain reference sequences to design conservative primers for Cinchona ledgeriana STR (ClSTR). ClSTR expression was analyzed using qRT-PCR and quinine content was determined using HPLC. A complete coding sequence (CDS) of ClSTR was deposited in NCBI GenBank under the accession number MK422544.1. ClSTR was expressed in cultured cells, young and mature leaves, and stem bark. The elicited cells have higher expression than the control and they performed since the fourth week. However, the quinine content was greater in older cells. The gene expression in young leaves was superior, but quinine was most abundant in the stem bark. Every cell of C. ledgeriana, in culture or in the plant, expressed ClSTR and was capable of synthesizing the alkaloid quinine. The alkaloid from the leaves of the plant might be translocated and accumulated in the bark. No efflux of alkaloids from the confined cultured cells might contribute in triggering feedback inhibition in the biosynthetic pathway. This study revealed a critical obstacle in cell culture as a means of secondary metabolites production that needs further development of metabolic engineering. © 2021 Friends Science Publishers

Alkaloids Production and Cell Growth of Cinchona ledgeriana Moens: Effects of Fungal Filtrate and Methyl Jasmonate Elicitors

Cinchona alkaloids are known as antimalaria and anti-arrhythmic. Due to the long waiting time to harvest, cell culture technology is a challenge. This study aimed to determine the effects of elicitors, filtrate of two strains of endophytic fungi and methyl jasmonate (MeJA), in cell suspension culture of Cinchona ledgeriana on quinine and quinidine production. The cells were cultured for seven weeks in woody plant (WP) media treated with either of those elicitors in various concentrations. The cells growth was observed and the alkaloids were analyzed by HPLC. Cells treated with MeJA failed to grow that led to the cell biomass insufficiency for alkaloids determination. It indicates that the cells are quite sensitive to even low concentration of MeJA that hampered the growth. Cells treated with the filtrate of Diaporthe sp. M13-Millipore filtered (S2M) gave the least cell biomass but presented the highest content of both alkaloids. Diaporthe sp. strain M-13 is stronger as elicitor than M-23 for this plant species. Filtrate of non-virulent fungi can elevate the biosynthesis of alkaloids. This reconfirms that cultured cells are capable to produce secondary metabolites and the productivity can be increased by using an appropriate elicitor.

Aktivitas NADP(H) Oksidoreduktase pada Kultur Sel Kina (Cinchona ledgeriana Moens) Terelisitasi

Cinchona ledgeriana Moens is an industrial plant producing secondary metabolite quinoline alkaloids. To maintain and moreover, to increase the quinoline production especially quinine, in vitro culture system through cell culture could be a potential alternative. If the use of elicitor in cell culture can increase the production of a secondary metabolite, the activity of the enzymes involved in the biosynthetic pathway of the secondary metabolite in question might be increasing. This study aimed to examine the activity of NADPH oxidoreductase in the elicitated cell culture of C. ledgeriana and to evaluate the correlation between the activity of this enzyme and the level of quinine production. The cell cultures of Cinchona were treated with abscisic acid (ABA) or paclobutrazol (PBZ), combined with sucrose, sorbitol, or mannitol in Wood Plant (WP) media, for 7 weeks on a shaker. The quinine concentration was determined using high-performance liquid chromatography (HPLC) and the enzyme activity was measured using fluorometry. The results showed that the highest enzyme activity was found in the P7M cells (PBZ 7 mg/L + mannitol 5.3 g/L + sucrose 20 g/L), followed by the A3S cells (ABA 3 mg/L + sorbitol 5.3 g/L + sucrose 20 g/L). These results correspond to their production level of the quinine alkaloids. The lowest enzyme activity was found in the cultures without elicitor. The increase of NADP(H) enzyme activity in the P7M and A3S treatments were 13.5 and 8.5%, respectively, compared to that in the control cells. Keywords: elicitation, fluorometry, NADP(H) oxidoreductase, quinoline alkaloid

Localization of Alkaloid and Other Secondary Metabolites in Cinchona ledgeriana Moens: Anatomical and Histochemical Studies on Fresh Tissues and Cultured Cells

Cinchona ledgeriana produces several secondary metabolites. The main quinoline alkaloid, quinine that is widely used as an antimalarial drug, is most commonly extracted from the bark of Cinchona, and its leaves contain several other metabolites. Many studies have revealed that cell culture of Cinchona also produces quinine. Nevertheless, the sites of secondary metabolites accumulation are still elusive. This study is aimed at describing specific anatomical structures where alkaloids and some other secondary metabolites are accumulated as well as their localization in leaves and barks of C. ledgeriana, compared to those found in cultured cells. Fresh leaves and barks, and cells of C. ledgeriana were used for anatomical observation and histochemical tests. It was found that these plant parts have specialized structures, idioblast cells with elliptical- and spherical-shapes, scattered in leaf hypodermis, stem cortex, and secondary phloem. Unspecialized structures such as epidermis and palisade mesophyll tissues were also found accumulating some metabolites. Histochemical tests showed that bark and leaves contained alkaloids, terpenoids, phenolic, and lipophilic compounds. Cultured cells presented positive results for alkaloids and terpenoids.