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

MOLECULAR CLONING, BIOINFORMATION ANALYSIS AND EXPRESSION OF THE STRICTOSIDINE SYNTHASE IN Dendrobium officinale

The enzyme strictosidine synthase (STR, EC: 4.3.3.2) plays a key role in the biosynthetic pathway of terpenoid indole alkaloid (TIA). It catalyzes the condensation of the tryptamine and secologanin to form 3α(S)-strictosidine, which is the common precursor of all TIAs. In this paper, a STR gene designated as DoSTR (GenBank: KX068707) was first cloned and characterized from Dendrobium officinale with rapid amplified cDNA ends method (RACE). DoSTR has a length of 1380bp with 1179bp open reading frame encoding 392 amino acids. BlastP analyses showed that its amino acid sequence was classified into Str_synth superfamily. qRT-PCR showed that DoSTR was expressed in all tissues tested, with a significantly higher level in flower and the lowest in stem. Four different treatments with MeJA, SA, ABA and AgNO3, respectively, could induce the DoSTR expression to a different extent. And the effect of MeJA was the most obvious and transcript level of DoSTR induced by MeJA was 20.7 times greater than that of control at 48 hours after treatment. Furthermore, it was found that DoSTR was localized in vacuole through transient expression in tobacco. The characterization and expression of DoSTR can help in further studying the role of DoSTR in the biosynthesis of TIAs in D. officinale. This study may throw light on the alkaloid biosynthesis pathway of D. officinale.

Short Communication: The impact of Gamma radiation on Tdc and Str gene expressions in Catharanthus roseus regenerated plantlets

Noormohammadi Z, Taban M, Farahani F. 2018. Short Communication: The impact of Gamma radiation on Tdc and Str gene expressions in Catharanthus roseus regenerated plantlets. Biodiversitas 19: 1805-1810. Catharanthus roseus L.G. Don, is the essential medicinal plant with considerable attention. This plant is a rich source of terpenoid indole alkaloids (TIAs). The main alkaloids in C. roseus are vinblastine, vincristine, and ajmalicine. The tryptophan decarboxylase (Tdc) and Strictosidine synthase (Str) are key enzymes in TIA biosynthesis. In the present study, Tdc and Str gene expressions, as well as vinblastine production were evaluated in tissue culture regenerated plantlets in 4 groups; control, 60 Gy irradiation, 50 mg/L putrescine and 60 Gy irradiation + 50mg/L putrescine treatments. The results revealed significant increase in Tdc and Str gene expressions in 60 Gy irradiation + 50mg/L putrescine treated plantlets in comparison with control samples by using qPCR methods. HPLC analysis showed a higher amount of vinblastine in 60 Gy + 59 mg/L putrescine treated plantlets. Gamma radiation and putrescine as elicitor and polyamine, respectively, are able to improve vinblastine production in C. roseus.