scholarly journals Discovery and evaluation of a novel step in the flavonoid biosynthesis pathway regulated by F3H gene using a yeast expression system

2019 ◽  
Author(s):  
Rahmatullah Jan ◽  
Sajjad Asaf ◽  
Sanjita Paudel ◽  
Sangkyu Lee ◽  
Kyung-Min Kim
Keyword(s):  
Western Blotting ◽  
Rice Plant ◽  
Expression System ◽  
Plant Secondary Metabolites ◽  
Flavonoid Biosynthesis ◽  
Yeast Expression ◽  
List Type ◽  
Biosynthesis Pathway ◽  
Yeast Expression System ◽  
Flavonoid Biosynthesis Pathway

AbstractKaempferol and quercetin are the essential plant secondary metabolites that confer huge biological functions in the plant defense system. These metabolites are produced in low quantities in plants, therefore engineering microbial factory is a favorable strategy for the production of these metabolites. In this study, biosynthetic pathways for kaempferol and quercetin were constructed in Saccharomyces cerevisiae using naringenin as a substrate. The results elucidated a novel step for the first time in kaempferol and quercetin biosynthesis directly from naringenin catalyzed by flavonol 3-hydroxylase (F3H). F3H gene from rice was cloned into pRS42K yeast episomal plasmid (YEP) vector using BamH1 and Xho1 restriction enzymes. We analyzed our target gene activity in engineered and in empty strains. The results were confirmed through TLC followed by Western blotting, nuclear magnetic resonance (NMR), and LC-MS. TLC showed positive results on comparing both compounds extracted from the engineered strain with the standard reference. Western blotting confirmed lack of Oryza sativa flavonol 3-hydroxylase (OsF3H) activity in empty strains while high OsF3H expression in engineered strains. NMR spectroscopy confirmed only quercetin, while LCMS-MS results revealed that F3H is responsible for naringenin conversion to both kaempferol and quercetin. These results concluded that rice F3H catalyzes naringenin metabolism via hydroxylation and synthesizes kaempferol and quercetin.HighlightsCurrent study is a discovery of a novel step in flavonoid biosynthesis pathway of rice plant.In this study F3H gene from rice plant was functionally expressed in yeast expression system.Results confirmed that, F3H gene is responsible for the canalization of naringenin and converted into kaempferol and quercetin.The results were confirmed through, western blotting, TLC, HPLC and NMR analysis.

scholarly journals Discovery and Validation of a Novel Step Catalyzed by OsF3H in the Flavonoid Biosynthesis Pathway

Biology ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 32
Author(s):  
Rahmatullah Jan ◽  
Sajjad Asaf ◽  
Sanjita Paudel ◽  
Lubna ◽  
Sangkyu Lee ◽  
...  
Keyword(s):  
Western Blotting ◽  
Target Gene ◽  
Plant Secondary Metabolites ◽  
Biosynthesis Pathway ◽  
Biological Functions ◽  
Liquid Chromatography Mass Spectrometry ◽  
Defense System ◽  
Layer Chromatography ◽  
Positive Results ◽  
Flavonoid Biosynthesis Pathway

Kaempferol and quercetin are the essential plant secondary metabolites that confer huge biological functions in the plant defense system. In this study, biosynthetic pathways for kaempferol and quercetin were constructed in Saccharomyces cerevisiae using naringenin as a substrate. OsF3H was cloned into pRS42K yeast episomal plasmid (YEp) vector and the activity of the target gene was analyzed in engineered and empty strains. We confirmed a novel step of kaempferol and quercetin biosynthesis directly from naringenin, catalyzed by the rice flavanone 3-hydroxylase (F3H). The results were confirmed through thin layer chromatography (TLC) followed by western blotting, nuclear magnetic resonance (NMR), and liquid chromatography-mass spectrometry LCMS-MS. TLC showed positive results when comparing both compounds extracted from the engineered strain with the standard reference. Western blotting confirmed the lack of OsF3H activity in empty strains and confirmed high OsF3H expression in engineered strains. NMR spectroscopy confirmed only quercetin, while LCMS-MS results revealed that F3H is responsible for the conversion of naringenin to both kaempferol and quercetin.

scholarly journals A fast and simple LC-MS-based characterization of the flavonoid biosynthesis pathway for few seed(ling)s

2016 ◽  
Vol 16 (1) ◽  
Author(s):  
Benjamin Jaegle ◽  
Miran Kalle Uroic ◽  
Xu Holtkotte ◽  
Christina Lucas ◽  
Andreas Ole Termath ◽  
...  
Keyword(s):  
Flavonoid Biosynthesis ◽  
Biosynthesis Pathway ◽  
Seed Ling ◽  
Flavonoid Biosynthesis Pathway

scholarly journals Temporospatial Flavonoids Metabolism Variation in Ginkgo biloba Leaves

2020 ◽  
Vol 11 ◽  
Author(s):  
Ying Guo ◽  
Tongli Wang ◽  
Fang-Fang Fu ◽  
Yousry A. El-Kassaby ◽  
Guibin Wang
Keyword(s):  
Ginkgo Biloba ◽  
Molecular Mechanisms ◽  
Developmental Stages ◽  
Early Stage ◽  
Sunshine Duration ◽  
Flavonoid Biosynthesis ◽  
Flavonoid Glycosides ◽  
Biosynthesis Pathway ◽  
Flavonoids Biosynthesis ◽  
Flavonoid Biosynthesis Pathway

Ginkgo (Ginkgo biloba L.) is a high-value medicinal tree species characterized by its flavonoids beneficial effects that are abundant in leaves. We performed a temporospatial comprehensive transcriptome and metabolome dynamics analyses of clonally propagated Ginkgo plants at four developmental stages (time: May to August) across three different environments (space) to unravel leaves flavonoids biosynthesis variation. Principal component analysis revealed clear gene expression separation across samples from different environments and leaf-developmental stages. We found that flavonoid-related metabolism was more active in the early stage of leaf development, and the content of total flavonoid glycosides and the expression of some genes in flavonoid biosynthesis pathway peaked in May. We also constructed a co-expression regulation network and identified eight GbMYBs and combining with other TF genes (3 GbERFs, 1 GbbHLH, and 1 GbTrihelix) positively regulated the expression of multiple structural genes in the flavonoid biosynthesis pathway. We found that part of these GbTFs (Gb_11316, Gb_32143, and Gb_00128) expressions was negatively correlated with mean minimum temperature and mean relative humidity, while positively correlated with sunshine duration. This study increased our understanding of the molecular mechanisms of flavonoids biosynthesis in Ginkgo leaves and provided insight into the proper production and management of Ginkgo commercial plantations.

scholarly journals Investigating the Effects of Statins on Cellular Lipid Metabolism Using a Yeast Expression System

PLoS ONE ◽  
2009 ◽  
Vol 4 (12) ◽  
pp. e8499 ◽  
Author(s):  
Agata Leszczynska ◽  
Beata Burzynska ◽  
Danuta Plochocka ◽  
Joanna Kaminska ◽  
Magdalena Zimnicka ◽  
...  
Keyword(s):  
Lipid Metabolism ◽  
Expression System ◽  
Yeast Expression ◽  
Cellular Lipid ◽  
Yeast Expression System

scholarly journals Advantages of yeast-based recombinant protein technology as vaccine products against infectious diseases

2021 ◽  
Vol 913 (1) ◽  
pp. 012099
Author(s):  
C S W Lestari ◽  
G Novientri
Keyword(s):  
Recombinant Protein ◽  
Infectious Diseases ◽  
Recombinant Proteins ◽  
Hansenula Polymorpha ◽  
Expression System ◽  
Cost Effective ◽  
Yeast Expression ◽  
Vaccine Strategy ◽  
Vaccination Programs ◽  
Yeast Expression System

Abstract The yeast expression system is widely used to produce functional recombinant proteins in the biopharmaceutical industry, such as vaccine products. The expression system choices using yeast as the host has many advantages. Various vaccines have been produced commercially using yeast expression systems. This review aims to explore the advantages of the yeast expression system in Saccharomyces cerevisiae, Pichia pastoris, and Hansenula polymorpha, which emphasize vaccine products to prevent human infectious diseases. Selection of the appropriate expression system is carried out by identification at the genetic and fermentation levels, taking into account host features, vectors and expression strategies. We also demonstrate the development of a yeast expression system that can produce recombinant proteins, virus-like particles and yeast surface displays as a novel vaccine strategy against infectious diseases. The recombinant protein produced as a vaccine in the yeast system is cost-effective, immunogenic, and safe. In addition, this system has not introduced new microbe variants in nature that will be safe for the environment. Thus, it has the potential to become a commercial product used in vaccination programs to prevent human infectious diseases.

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