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Metabolic Flux Analysis of Catechin Biosynthesis Pathways Using Nanosensor

(+)-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 de...

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Autores principales: Kausar, Habiba, Ambrin, Ghazala, Okla, Mohammad K., Soufan, Walid, Al-Ghamdi, Abdullah A., Ahmad, Altaf
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7222200/
https://www.ncbi.nlm.nih.gov/pubmed/32244268
http://dx.doi.org/10.3390/antiox9040288
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author Kausar, Habiba
Ambrin, Ghazala
Okla, Mohammad K.
Soufan, Walid
Al-Ghamdi, Abdullah A.
Ahmad, Altaf
author_facet Kausar, Habiba
Ambrin, Ghazala
Okla, Mohammad K.
Soufan, Walid
Al-Ghamdi, Abdullah A.
Ahmad, Altaf
author_sort Kausar, Habiba
collection PubMed
description (+)-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 K(d) 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.
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spelling pubmed-72222002020-05-28 Metabolic Flux Analysis of Catechin Biosynthesis Pathways Using Nanosensor Kausar, Habiba Ambrin, Ghazala Okla, Mohammad K. Soufan, Walid Al-Ghamdi, Abdullah A. Ahmad, Altaf Antioxidants (Basel) Article (+)-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 K(d) 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. MDPI 2020-03-31 /pmc/articles/PMC7222200/ /pubmed/32244268 http://dx.doi.org/10.3390/antiox9040288 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Kausar, Habiba
Ambrin, Ghazala
Okla, Mohammad K.
Soufan, Walid
Al-Ghamdi, Abdullah A.
Ahmad, Altaf
Metabolic Flux Analysis of Catechin Biosynthesis Pathways Using Nanosensor
title Metabolic Flux Analysis of Catechin Biosynthesis Pathways Using Nanosensor
title_full Metabolic Flux Analysis of Catechin Biosynthesis Pathways Using Nanosensor
title_fullStr Metabolic Flux Analysis of Catechin Biosynthesis Pathways Using Nanosensor
title_full_unstemmed Metabolic Flux Analysis of Catechin Biosynthesis Pathways Using Nanosensor
title_short Metabolic Flux Analysis of Catechin Biosynthesis Pathways Using Nanosensor
title_sort metabolic flux analysis of catechin biosynthesis pathways using nanosensor
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7222200/
https://www.ncbi.nlm.nih.gov/pubmed/32244268
http://dx.doi.org/10.3390/antiox9040288
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