Metabolic Flux Analysis of Mammalian Cells in High Cell Density Perfusion Cultures Targetting Real-Time Application

2001 ◽  
Vol 34 (5) ◽  
pp. 137-142
Author(s):  
Richard Biener ◽  
Chetan Goudar ◽  
Renato Meneses ◽  
Maria Ng ◽  
Chun Zhang ◽  
...  
Keyword(s):  
Real Time ◽  
Cell Density ◽  
Metabolic Flux Analysis ◽  
Mammalian Cells ◽  
Metabolic Flux ◽  
High Cell Density ◽  
Flux Analysis ◽  
High Cell ◽  
Perfusion Cultures ◽  
Real Time Application

scholarly journals Metabolic Flux Analysis of Catechin Biosynthesis Pathways Using Nanosensor

Antioxidants ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 288
Author(s):  
Habiba Kausar ◽  
Ghazala Ambrin ◽  
Mohammad K. Okla ◽  
Walid Soufan ◽  
Abdullah A. Al-Ghamdi ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Real Time ◽  
Metabolic Flux Analysis ◽  
Metabolic Flux ◽  
Regulatory Element ◽  
Flux Analysis ◽  
Bacterial Cells ◽  
Biosynthesis Pathway ◽  
Real Time Monitoring ◽  
E Coli

(+)-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.

High cell density perfusion cultures of anchorage-dependent Vero cells in a depth filter perfusion system

1995 ◽  
Vol 17 (3) ◽  
pp. 173-183 ◽  
Author(s):  
Sang Kyo Choi ◽  
Ho Nam Chang ◽  
Gyun Min Lee ◽  
Ik-Hwan Kim ◽  
Duk Jae Oh
Keyword(s):  
Cell Density ◽  
High Cell Density ◽  
Vero Cells ◽  
High Cell ◽  
Perfusion System ◽  
Perfusion Cultures ◽  
Depth Filter Perfusion System
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