Production of Genistein in Amaranthus tricolor var. tristis and Spinacia oleracea by Expression of Glycine max Isoflavone Synthase

Isoflavonoids, the diverse group of secondary metabolites derived from the phenylpropanoid pathway, are distributed predominantly in leguminous plants. It has received considerable attention in recent days due to its health promoting benefits and is known to prevent certain diseases in humans. These isoflavonoids are synthesized from flavonoid intermediates of phenylpropanoid pathway by the enzyme isoflavone synthase. Metabolic engineering of isoflavonoid biosynthesis in non-legume crop plants could offer the health benefits of these compounds in diverse plant species further contributing for crop improvement. The transient expression of heterologous genes in the host is considered as an alternative to stable expression, that can provide a rapid way of studying the pathway engineering for metabolite production and could also act as a production platform for nutraceuticals and biopharmaceuticals. In this study, isoflavone genistein was produced in Amaranthus tricolor var. tristis and Spinacia oleracea by transiently expressing Glycine max isoflavone synthase (GmIFS). The GmIFS gene was cloned in plant expression vector pEarleyGate 102 HA and pEAQ-HT-DEST 3 and transformed into plants by agroinfiltration. The presence of transgene in the agroinfiltrated leaves was confirmed by semiquantitative reverse-transcription polymerase chain reaction. The flavonoid substrate naringenin and isoflavonoid genistein were quantified using high performance liquid chromatography in both wild-type and infiltrated leaf samples of both the plants. The naringenin content varied in the range of 65.5–338.5 nM/g fresh weight, while the accumulation of genistein was observed with varying concentrations from 113 to 182.6 nM/g fresh weight in the agroinfiltrated leaf samples of both A. tricolor var. tristis and S. oleracea. These results indicate that the transient expression of GmIFS gene has led to the synthesis of isoflavonoid genistein in A. tricolor var. tristis and S. oleracea providing an insight that stable expression of this gene could enrich the nutraceutical content in the crop plants. To the best of our knowledge, this is the first report on transient expression of GmIFS gene for the production of genistein in A. tricolor var. tristis and S. oleracea.

Transgenic Rice Expressing Isoflavone Synthase Gene from Soybean Shows Resistance against Blast Fungus (Magnaporthe oryzae)

The isoflavones are a group of plant secondary metabolites primarily synthesized in legumes and are known for their role in improving human health and plant disease resistance. The isoflavones, especially genistein, act as precursors for the production of phytoalexins, which may induce broad-spectrum disease resistance in plants. In the present study, we screened transgenic rice lines expressing the isoflavone synthase (GmIFS1) gene from soybean for rice blast (Magnaporthe oryzae) resistance. Two homozygous transgenic lines (I2 and I10), based on single copy gene integration, were identified. The expression of GmIFS1 in transgenic lines was confirmed by qRT-PCR. Genistein was detected in the transgenic lines using LC-MS/MS. Subsequently, the transgenic lines were evaluated against the rice blast pathogen, isolate YJ54 (race IB-54). The results indicated that more than 60% of the plants in both the lines (I2 and I10) showed resistance against the blast pathogen. The progenies of one of the resistant transgenic lines (I10) also showed more than 65% resistance against rice blast. The resistance of these transgenic lines against rice blast may be attributed to the synthesis of isoflavone (genistein) in rice.

Metabolic Engineering of Isoflavonoid Biosynthesis by Expressing Glycine max Isoflavone Synthase in Allium cepa L. for Genistein Production

Isoflavonoids, the diverse group of secondary metabolites derived from the phenylpropanoid pathway, are distributed predominantly in leguminous plants and play a vital role in promoting human health. Genetic engineering of the metabolite synthesis pathway has turned out to be an attractive approach for the production of various secondary metabolites. In our study, we attempted to produce the isoflavone genistein, a well-known health-promoting metabolite, in Allium cepa L. (onion) by introducing Glycine max Isoflavone synthase (GmIFS). The GmIFS gene was cloned into the pEarleyGate 102 HA vector and transformed into onion by Agrobacterium-mediated and biolistic methods. The presence of GmIFS in transgenic onion was confirmed by PCR, dot blot, and Southern hybridization. Analysis of the transgenic onion calli lines demonstrated that the expression of the GmIFS gene led to the production of isoflavone genistein in in vitro tissues. The biolistic stable transformed calli with transformation efficiency of 73% (62.65 nM/g FW) accumulated more genistein than the Agrobacterium stable transformed calli with transformation efficiency of 56% (42.5 nM/g FW). Overall, heterologous gene expression of GmIFS was demonstrated by modifying the secondary metabolite pathway in onion tissues for the production of isoflavone genistein that can boost up human health with its health-promoting properties.

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

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.

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

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.

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

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.

De novo transcriptome assembly reveals putative biosynthetic genes involved in the biosyntheses of isoflavones and miroestrol in Pueraria candollei var. mirifica

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: 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 candidatesinvolved in these biosyntheticpathways, and their respective expressions were validated by quantitative real-time reverse transcription polymerase chain reaction. Notably, isoflavonoid 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 ( At MYB12) in Nicotiana benthamiana leaves. Remarkably, the combined expression of these proteins ledto the production of the isoflavone genistein. Conclusions: Our results provide compelling evidence regarding the integration of transcriptome and metabolome as a powerful tool for unraveling biosynthetic pathways in plants.

De novo transcriptome assembly reveals putative biosynthetic genes involved in the biosyntheses of isoflavones and miroestrol in Pueraria candollei var. mirifica

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. mirifica have been known for some time, actual knowledge regarding their biosynthetic genes remains unknown. Results 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. The assembly of 166,923 contigs was used for a functional annotation against protein databases, and as a library for the identification of genes possibly involved in the biosynthesis of isoflavonoid and miroestrol. Twenty-one differential expressed genes (DEGs) from four separate libraries were identified as candidates involved in the biosynthesis pathways and had their expressions validated by qRT-PCR. Notably, isoflavonoid profiling generated by LC-MS/MS was positively correlated to the expression levels of isoflavonoid biosynthetic genes across the four types of tissues. In addition, R2R3 MYB transcription factors were also identified that may be involved in the regulation of isoflavonoid biosynthesis in P. candollei var. mirifica. To confirm the function of the key-isoflavone biosynthetic gene, P. candollei var. mirifica isoflavone synthase (PmIFS) identified in our library was transiently co-expressed with the Arabidopsis MYB12 transcription factor (AtMYB12) in Nicotiana benthamiana leaves. Remarkably, the combined expression of those proteins led to the production of isoflavone genistein. Conclusions Our results provide compelling evidence regarding the integration of transcriptome and metabolome as a powerful tool for unraveling biosynthetic pathways in plants.