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

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 <i>Amaranthus tricolor</i> var. <i>tristis</i> and <i>Spinacia oleracea</i> by transiently expressing <i>Glycine max isoflavone synthase</i> (<i>Gm</i><i>IFS</i>). The <i>GmIFS</i> 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 <i>A. tricolor</i> var. <i>tristis</i> and <i>S. oleracea</i>. These results indicate that the transient expression of <i>Gm</i><i>IFS</i> gene has led to the synthesis of isoflavonoid genistein in <i>A. tricolor</i> var. <i>tristis</i> and <i>S. oleracea</i> 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 <i>GmIFS</i> gene for the production of genistein in <i>A. tricolor</i> var. <i>tristis</i> and <i>S. oleracea</i>.