Abstract
Ajmalicine is one of the most popular antihypertensive drugs obtained from the root barks of Cathranthus roseus (L.) G. Don and Rauvolfia serpentine (L.) Benth. ex Kurz. It has also potential antimicrobial, cytotoxic, central depressant and antioxidant activities. As the demand for the alkaloid is significantly high, metabolic engineering approaches are being tried to increase its production in both homologous and heterologous systems. The metabolic engineering approach requires knowledge of the metabolic regulation of the alkaloid. For understanding the metabolic regulation, fluxomic analysis is important as it helps in understanding the flux of the alkaloid through the complicated metabolic pathway. The present study was conducted to analyse the flux analysis of the ajmalicine biosynthesis, using a genetically encoded Fluorescent Resonance Energy Transfer FRET-based nanosensor for ajmalicine (FLIP-Ajn). Here, we have silenced six important genes of terpenoid indole alkaloid (TIA), namely G10H, 10HGO, TDC, SLS, STR and SDG, through RNA-mediated gene silencing in different batches of C. roseus suspension cells, generating six silenced cell lines. Monitoring of the ajmalicine level was carried out using FLIP-Ajn in these silenced cell lines, with high spatial and temporal resolution. The study offers the rapid, high throughput real-time measurement of ajmalicine flux in response to the silenced TIA genes, thereby identifying the regulatory gene controlling the alkaloid flux in C. roseus suspension cells. We have reported that the STR gene encoding strictosidine synthase of the TIA pathway could be the regulatory gene of the ajmalicine biosynthesis.
Highlights
Ajmalicine is one of the most important monoterpenoid indole alkaloids
We have reported that the STR gene encoding strictosidine synthase of the terpenoid indole alkaloid (TIA) pathway could be the regulatory gene of the ajmalicine biosynthesis
Six silenced cell lines were generated through Agrobacterium tumefaceins-mediated transformation, wherein the transformed gene-silencing plasmids, pG10Hi, p10HGOi, pTDCi, pSLSi, pSTRi and pSDGi, suppressed the TIA pathway genes G10H, 10-hydroxygeraniol oxidoreductase (10HGO), tryptophan decarboxylase (TDC), secologanin synthase (SLS), STR, and SDG, respectively
Summary
Ajmalicine is one of the most important monoterpenoid indole alkaloids. It is isolated from the root barks of Catharanthus roseus (L.) G. Ex Kurz, where its synthesis occurs through the terpenoid indole alkaloid (TIA) pathway [1,2,3] It is a well-known antihypertensive drug, and has been utilized in the treatment of cardiovascular disorders [1,4]. It is known to exhibit antimicrobial [5], cytotoxic [6], antioxidant [7], and central depressant activities [8] As it increases the cerebral blood flow, leading to improved oxygen bioavailability, it possesses intriguing potential in the treatment of Alzheimer’s disease, because the reduction of cerebral blood flow is a common symptom associated with Alzheimer’s disease and different forms of dementia [9,10].
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