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

Ashwini Malla,Sathishkumar Ramalingam,Balamurugan Shanmugaraj,Ashutosh Sharma

doi:10.3390/plants10112311

Abstract

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.

Highlights

The key enzyme involved in the biosynthesis of isoflavonoids from flavonoid substrates, in the branch of phenylpropanoid pathway, is isoflavone synthase (IFS) (Figure 1), which is natively found in leguminous plants [8,18,19,20,21]
The results showed that the transient expression of Glycine max isoflavone synthase (GmIFS) can produce isoflavone genistein from the naringenin intermediate in these leafy vegetables
The seeds of A. tricolor var. tristis and S. oleracea procured from Tamil Nadu AgriculThe seeds of A. tricolor var. tristis and S. oleracea procured from Tamil Nadu Agricultural University seed center were sown in commercially available soil manure mixture for tural University seed center were sown in commercially available soil manure mixture for germination and kept in greenhouse

Summary

Introduction

Isoflavonoids, a class of natural plant polyphenols attribute to many biological activities such as antioxidant [1], antimutagenic [2], anticarcinogenic [3,4,5], antiproliferative [6,7]. Effects are playing an imperative role in human health and nutrition [8,9,10]. These compounds are naturally present in Papilionoideae, a subfamily of Leguminosae or Fabaceae members [11]. The key enzyme involved in the biosynthesis of isoflavonoids from flavonoid substrates, in the branch of phenylpropanoid pathway, is isoflavone synthase (IFS) (Figure 1), which is natively found in leguminous plants [8,18,19,20,21] Apart from the other vital bioactivities, it is evident that the isoflavonoid genistein in dietary consumption showed a wide spectrum of health protective abilities such as reducing the risk of cardiovascular problems [12], chronic diseases [13], diabetes [14,15,16], cancer [3], osteoporosis [12,14,15,16], obesity [17] and relief from menopausal symptoms [9].

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Discussion

Conclusion