scholarly journals Coactivation of MEP-biosynthetic genes and accumulation of abietane diterpenes in Salvia sclarea by heterologous expression of WRKY and MYC2 transcription factors

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Mariaevelina Alfieri ◽  
Maria Carmela Vaccaro ◽  
Elisa Cappetta ◽  
Alfredo Ambrosone ◽  
Nunziatina De Tommasi ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Heterologous Expression ◽  
Biosynthetic Genes ◽  
Salvia Sclarea ◽  
Abietane Diterpenes

scholarly journals Transcriptome analysis of Pueraria candollei var. mirifica for gene discovery in the biosyntheses of isoflavones and miroestrol

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Nithiwat Suntichaikamolkul ◽  
Kittitya Tantisuwanichkul ◽  
Pinidphon Prombutara ◽  
Khwanlada Kobtrakul ◽  
Julie Zumsteg ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Transcriptome Analysis ◽  
De Novo ◽  
Biosynthetic Genes ◽  
Pueraria Candollei ◽  
Isoflavone Synthase ◽  
Young Leaves ◽  
Plausible Mechanism ◽  
Polymerase Chain ◽  
R2r3 Myb

Abstract Background Pueraria candollei var. mirifica, a Thai medicinal plant used traditionally as a rejuvenating herb, is known as a rich source of phytoestrogens, including isoflavonoids and the highly estrogenic miroestrol and deoxymiroestrol. Although these active constituents in P. candollei var. mirifica have been known for some time, actual knowledge regarding their biosynthetic genes remains unknown. Results Miroestrol biosynthesis was reconsidered and the most plausible mechanism starting from the isoflavonoid daidzein was proposed. A de novo transcriptome analysis was conducted using combined P. candollei var. mirifica tissues of young leaves, mature leaves, tuberous cortices, and cortex-excised tubers. A total of 166,923 contigs was assembled for functional annotation using protein databases and as a library for identification of genes that are potentially involved in the biosynthesis of isoflavonoids and miroestrol. Twenty-one differentially expressed genes from four separate libraries were identified as candidates involved in these biosynthetic pathways, and their respective expressions were validated by quantitative real-time reverse transcription polymerase chain reaction. Notably, isoflavonoid and miroestrol profiling generated by LC-MS/MS was positively correlated with expression levels of isoflavonoid biosynthetic genes across the four types of tissues. Moreover, we identified R2R3 MYB transcription factors that may be involved in the regulation of isoflavonoid biosynthesis in P. candollei var. mirifica. To confirm the function of a key-isoflavone biosynthetic gene, P. candollei var. mirifica isoflavone synthase identified in our library was transiently co-expressed with an Arabidopsis MYB12 transcription factor (AtMYB12) in Nicotiana benthamiana leaves. Remarkably, the combined expression of these proteins led to the production of the isoflavone genistein. Conclusions Our results provide compelling evidence regarding the integration of transcriptome and metabolome as a powerful tool for identifying biosynthetic genes and transcription factors possibly involved in the isoflavonoid and miroestrol biosyntheses in P. candollei var. mirifica.

scholarly journals TDP-l-Megosamine Biosynthesis Pathway Elucidation and Megalomicin A Production in Escherichia coli

2010 ◽  
Vol 76 (12) ◽  
pp. 3869-3877 ◽  
Author(s):  
Mariana Useglio ◽  
Salvador Peirú ◽  
Eduardo Rodríguez ◽  
Guillermo R. Labadie ◽  
John R. Carney ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Heterologous Expression ◽  
Gene Cluster ◽  
Mass Spectrometric ◽  
Mass Spectrometric Analysis ◽  
Spectrometric Analysis ◽  
Biosynthesis Pathway ◽  
Biosynthetic Genes

ABSTRACT In vivo reconstitution of the TDP-l-megosamine pathway from the megalomicin gene cluster of Micromonospora megalomicea was accomplished by the heterologous expression of its biosynthetic genes in Escherichia coli. Mass spectrometric analysis of the TDP-sugar intermediates produced from operons containing different sets of genes showed that the production of TDP-l-megosamine from TDP-4-keto-6-deoxy-d-glucose requires only five biosynthetic steps, catalyzed by MegBVI, MegDII, MegDIII, MegDIV, and MegDV. Bioconversion studies demonstrated that the sugar transferase MegDI, along with the helper protein MegDVI, catalyzes the transfer of l-megosamine to either erythromycin C or erythromycin D, suggesting two possible routes for the production of megalomicin A. Analysis in vivo of the hydroxylation step by MegK indicated that erythromycin C is the intermediate of megalomicin A biosynthesis.

Enhanced biosynthesis of bioactive abietane diterpenes by overexpressing AtDXS or AtDXR genes in Salvia sclarea hairy roots

2014 ◽  
Vol 119 (1) ◽  
pp. 65-77 ◽  
Author(s):  
Mariacarmela Vaccaro ◽  
Nicola Malafronte ◽  
Mariaevelina Alfieri ◽  
Nunziatina De Tommasi ◽  
Antonietta Leone
Keyword(s):  
Hairy Roots ◽  
Salvia Sclarea ◽  
Abietane Diterpenes

scholarly journals Transcriptome analysis of Pueraria candollei var. mirifica for gene discovery in the biosyntheses of isoflavones and miroestrol

2019 ◽  
Author(s):  
Nithiwat Suntichaikamolkul ◽  
Kittiya Tantisuwanichkul ◽  
Pinidphon Prombutara ◽  
Khwanlada Kobtrakul ◽  
Julie Zumsteg ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Transcriptome Analysis ◽  
De Novo ◽  
Biosynthetic Genes ◽  
Pueraria Candollei ◽  
Isoflavone Synthase ◽  
Young Leaves ◽  
Plausible Mechanism ◽  
Polymerase Chain ◽  
R2r3 Myb

Abstract Background: Pueraria candollei var. mirifica, a Thai medicinal plant used traditionally as a rejuvenating herb, is known as a rich source of phytoestrogens, including isoflavonoids and the highly estrogenic miroestrol and deoxymiroestrol. Although these active constituents in P. candollei var. mirifica have been known for some time, actual knowledge regarding their biosynthetic genes remains unknown. Results: Miroestrol biosynthesis was reconsidered and the most plausible mechanism starting from the isoflavonoid daidzein was proposed. A de novo transcriptome analysis was conducted using combined P. candollei var. mirifica tissues of young leaves, mature leaves, tuberous cortices, and cortex-excised tubers. A total of 166,923 contigs was assembled for functional annotation using protein databases and as a library for identification of genes that are potentially involved in the biosynthesis of isoflavonoids and miroestrol. Twenty-one differentially expressed genes from four separate libraries were identified as candidates involved in these biosynthetic pathways, and their respective expressions were validated by quantitative real-time reverse transcription polymerase chain reaction. Notably, isoflavonoid and miroestrol profiling generated by LC-MS/MS was positively correlated with expression levels of isoflavonoid biosynthetic genes across the four types of tissues. Moreover, we identified R2R3 MYB transcription factors that may be involved in the regulation of isoflavonoid biosynthesis in P. candollei var. mirifica. To confirm the function of a key-isoflavone biosynthetic gene, P. candollei var. mirifica isoflavone synthase identified in our library was transiently co-expressed with an Arabidopsis MYB12 transcription factor (AtMYB12) in Nicotiana benthamiana leaves. Remarkably, the combined expression of these proteins led to the production of the isoflavone genistein. Conclusions: Our results provide compelling evidence regarding the integration of transcriptome and metabolome as a powerful tool for identifying biosynthetic genes and transcription factors possibly involved in the isoflavonoid and miroestrol biosyntheses in P. candollei var. mirifica.

scholarly journals Heterologous Expression of Transcription Factor AtWRKY57 Alleviates Salt Stress-Induced Oxidative Damage

2018 ◽  
Vol 12 (1) ◽  
pp. 204-218
Author(s):  
Wei Tang
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Salt Stress ◽  
Heterologous Expression ◽  
Stress Tolerance ◽  
Molecular Mechanisms ◽  
Abiotic Stress Tolerance ◽  
Thiobarbituric Acid Reactive Substance ◽  
Salt Stress Tolerance ◽  
Wrky Transcription Factors

Background:WRKY transcription factors play important roles in the responses to abiotic stresses, seed dormancy, seed germination, developmental processes, secondary metabolism, and senescence in plants. However, molecular mechanisms of WRKY transcription factors-related abiotic stress tolerance have not been fully understood.Methods:In this investigation, transcription factor AtWRKY57 was introduced into cell lines of rice (Oryza sativaL.), tobacco (Nicotiana tabacum), and white pine (Pinus strobesL.) for characterization of its function in salt stress tolerance. The purpose of this investigation is to examine the function of AtWRKY in a broad sample of plant species including monocotyledons, dicotyledons, and gymnosperms.Results:The experimental results demonstrated that heterologous expression of transcription factor AtWRKY57 improves salt stress tolerance by decreasing Thiobarbituric Acid Reactive Substance (TBARS), increasing Ascorbate Peroxidase (APOX) and Catalase (CAT) activity under salt stress. In rice, overexpression of transcription factor AtWRKY57 enhances expression of Ca2+-dependent protein kinase genesOsCPk6andOsCPk19to counteract salt stress.Conclusion:These results indicated that transcription factor AtWRKY57 might have practical application in genetic engineering of plant salt tolerance throughout the plant kingdom.

scholarly journals Secondary metabolite biosynthetic diversity in Arctic Ocean metagenomes

2021 ◽  
Vol 7 (12) ◽  
Author(s):  
Adriana Rego ◽  
Antonio Fernandez-Guerra ◽  
Pedro Duarte ◽  
Philipp Assmy ◽  
Pedro N. Leão ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Arctic Ocean ◽  
High Throughput Sequencing ◽  
Large Fraction ◽  
Environmental Dna ◽  
Gene Clusters ◽  
Biosynthetic Gene Clusters ◽  
Bacterial Isolation ◽  
Biosynthetic Genes ◽  
Peptide Synthetases

Polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) are mega enzymes responsible for the biosynthesis of a large fraction of natural products (NPs). Molecular markers for biosynthetic genes, such as the ketosynthase (KS) domain of PKSs, have been used to assess the diversity and distribution of biosynthetic genes in complex microbial communities. More recently, metagenomic studies have complemented and enhanced this approach by allowing the recovery of complete biosynthetic gene clusters (BGCs) from environmental DNA. In this study, the distribution and diversity of biosynthetic genes and clusters from Arctic Ocean samples (NICE-2015 expedition), was assessed using PCR-based strategies coupled with high-throughput sequencing and metagenomic analysis. In total, 149 KS domain OTU sequences were recovered, 36 % of which could not be assigned to any known BGC. In addition, 74 bacterial metagenome-assembled genomes were recovered, from which 179 BGCs were extracted. A network analysis identified potential new NP families, including non-ribosomal peptides and polyketides. Complete or near-complete BGCs were recovered, which will enable future heterologous expression efforts to uncover the respective NPs. Our study represents the first report of biosynthetic diversity assessed for Arctic Ocean metagenomes and highlights the potential of Arctic Ocean planktonic microbiomes for the discovery of novel secondary metabolites. The strategy employed in this study will enable future bioprospection, by identifying promising samples for bacterial isolation efforts, while providing also full-length BGCs for heterologous expression.

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