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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.
Ghazala Ambrin; Hayssam M. Ali; Altaf Ahmad. Metabolic Regulation Analysis of Ajmalicine Biosynthesis Pathway in Catharanthus roseus (L.) G. Don Suspension Culture Using Nanosensor. Processes 2020, 8, 589 .
AMA StyleGhazala Ambrin, Hayssam M. Ali, Altaf Ahmad. Metabolic Regulation Analysis of Ajmalicine Biosynthesis Pathway in Catharanthus roseus (L.) G. Don Suspension Culture Using Nanosensor. Processes. 2020; 8 (5):589.
Chicago/Turabian StyleGhazala Ambrin; Hayssam M. Ali; Altaf Ahmad. 2020. "Metabolic Regulation Analysis of Ajmalicine Biosynthesis Pathway in Catharanthus roseus (L.) G. Don Suspension Culture Using Nanosensor." Processes 8, no. 5: 589.
(+)-Catechin is an important antioxidant of green tea (Camelia sinensis (L.) O. Kuntze). Catechin is known for its positive role in anticancerous activity, extracellular matrix degradation, cell death regulation, diabetes, and other related disorders. As a result of enormous interest in and great demand for catechin, its biosynthesis using metabolic engineering has become the subject of concentrated research with the aim of enhancing (+)-catechin production. Metabolic flux is an essential concept in the practice of metabolic engineering as it helps in the identification of the regulatory element of a biosynthetic pathway. In the present study, an attempt was made to analyze the metabolic flux of the (+)-catechin biosynthesis pathway in order to decipher the regulatory element of this pathway. Firstly, a genetically encoded fluorescence resonance energy transfer (FRET)-based nanosensor (FLIP-Cat, fluorescence indicator protein for (+)-catechin) was developed for real-time monitoring of (+)-catechin flux. In vitro characterization of the purified protein of the nanosensor showed that the nanosensor was pH stable and (+)-catechin specific. Its calculated Kd was 139 µM. The nanosensor also performed real-time monitoring of (+)-catechin in bacterial cells. In the second step of this study, an entire (+)-catechin biosynthesis pathway was constructed and expressed in E. coli in two sets of plasmid constructs: pET26b-PT7-rbs-PAL-PT7-rbs-4CL-PT7-rbs-CHS-PT7-rbs-CHI and pET26b-T7-rbs-F3H-PT7-rbs- DFR-PT7-rbs-LCR. The E. coli harboring the FLIP-Cat was transformed with these plasmid constructs. The metabolic flux analysis of (+)-catechin was carried out using the FLIP-Cat. The FLIP-Cat successfully monitored the flux of catechin after adding tyrosine, 4-coumaric acid, 4-coumaroyl CoA, naringenin chalcone, naringenin, dihydroquercetin, and leucocyanidin, individually, with the bacterial cells expressing the nanosensor as well as the genes of the (+)-catechin biosynthesis pathway. Dihydroflavonol reductase (DFR) was identified as the main regulatory element of the (+)-catechin biosynthesis pathway. Information about this regulatory element of the (+)-catechin biosynthesis pathway can be used for manipulating the (+)-catechin biosynthesis pathway using a metabolic engineering approach to enhance production of (+)-catechin.
Habiba Kausar; Ghazala Ambrin; Mohammad K. Okla; Walid Soufan; Abdullah A. Al-Ghamdi; Altaf Ahmad. Metabolic Flux Analysis of Catechin Biosynthesis Pathways Using Nanosensor. Antioxidants 2020, 9, 288 .
AMA StyleHabiba Kausar, Ghazala Ambrin, Mohammad K. Okla, Walid Soufan, Abdullah A. Al-Ghamdi, Altaf Ahmad. Metabolic Flux Analysis of Catechin Biosynthesis Pathways Using Nanosensor. Antioxidants. 2020; 9 (4):288.
Chicago/Turabian StyleHabiba Kausar; Ghazala Ambrin; Mohammad K. Okla; Walid Soufan; Abdullah A. Al-Ghamdi; Altaf Ahmad. 2020. "Metabolic Flux Analysis of Catechin Biosynthesis Pathways Using Nanosensor." Antioxidants 9, no. 4: 288.