Partial Reconstruction of Flavonoid and Isoflavonoid Biosynthesis in Yeast Using Soybean Type I and Type II Chalcone Isomerases  

Abstract

Flavonoids and isoflavonoids are major plant secondary metabolites that mediate diverse biological functions and exert significant ecological impacts. These compounds play important roles in many essential physiological processes. In addition, flavonoids and isoflavonoids have direct but complex effects on human health, ranging from reducing cholesterol levels and preventing certain cancers to improving women's health. In this study, we cloned and functionally characterized five soybean (Glycine max) chalcone isomerases (CHIs), key enzymes in the phenylpropanoid pathway that produces flavonoids and isoflavonoids. Gene expression and kinetics analysis suggest that the soybean type I CHI, which uses naringenin chalcone as substrate, is coordinately regulated with other flavonoid-specific genes, while the type II CHIs, which use a variety of chalcone substrates, are coordinately regulated with an isoflavonoid-specific gene and specifically activated by nodulation signals. Furthermore, we found that some of the newly identified soybean CHIs do not require the 4#-hydroxy moiety on the substrate for high enzyme activity. We then engineered yeast (Saccharomyces cerevisiae) to produce flavonoid and isoflavonoid compounds. When one of the type II CHIs was coexpressed with an isoflavone synthase, the enzyme catalyzing the first committed step of isoflavonoid biosynthesis, various chalcone substrates added to the culture media were converted to an assortment of isoflavanones and isoflavones. We also reconstructed the flavonoid pathway by coexpressing CHI with either flavanone 3b-hydroxylase or flavone synthase II. The in vivo reconstruction of the flavonoid and isoflavonoid pathways in yeast provides a unique platform to study enzyme interactions and metabolic flux.Flavonoids and isoflavonoids are major plant secondary metabolites that mediate diverse biological functions and exert significant ecological impacts. Approximately 20% of the carbon fixed by photosynthesis is believed to be channeled into the phenylpropanoid pathway, generating the majority of the phenolic compounds found in nature, including flavonoids and isoflavonoids (Weisshaar and Jenkins, 1998). These compounds play important roles in many essential physiological processes. For example, lignins are major cell wall components, flavonols are UV protectants, and anthocyanins are the primary attractants for pollinators in many species . In addition, flavonoids and isoflavonoids have direct but complex effects on human health. For example, resveratrol found in red wine has been shown to double the life span of yeast (Saccharomyces cerevisiae; Howitz et al., 2003), the flavonols and tannins found in green tea can drastically reduce the likelihood of stroke and heart disease, and the isoflavones produced by soybean (Glycine max) can prevent many hormone-dependent cancers and improve women's health (Beecher, 2003). Despite being one of the most studied plant secondary-metabolic pathways, surprising new functions and characteristics of these compounds are still be...

Flavone Synthases from Medicago truncatula Are Flavanone-2-Hydroxylases and Are Important for Nodulation

“…Both QRT-PCR assays and promoter-report expression studies suggested that the plants activated the MtFNSII-2 gene that was normally expressed in other tissues, rather than boosting the constitutively root-expressed MtFNSII-1. One possible reason for this choice could be the coordinate activation of biosynthetic pathway enzymes upon defense (nodulation) signals (Ralston et al, 2005). Enzymes that form a complex together are coordinately regulated to enable proper channeling of metabolites.…”

“…In vivo yeast assays were carried out as previously described with minor modifications (Ralston et al, 2005). Yeast cultures (2 mL) were grown overnight at 30°C in appropriate SD dropout liquid media (CLONTECH) containing 2% (w/v) Glc.…”

“…Aliquots of the above extracts were analyzed on an Agilent 1100 series HPLC system using a Spherisorb ODS-2 reverse-phase C-18 column (5 mm; 250 3 4.6 mm) following previous methods (Akashi et al, 1999;Ralston et al, 2005). Samples were diluted into methanol and then separated using an 18-min linear gradient from 20% methanol:80% 10 mM ammonium acetate, pH 5.6, to 100% methanol at a flow rate of 1 mL min 21 .…”

Bivalent RNA interference to increase isoflavone biosynthesis in soybean (Glycine max)

“…The expression levels of GmFNSII were only approximately one-fifth of the expression of F3H in the vector-control hairy roots. However, since both the genes showed tissue-specific expression patterns and differential transcript levels in all the tissues (Ralston et al 2005;Jiang et al 2010), it was not possible to speculate whether the results would be repeated if different tissues were tested in transgenic plants.…”

“…Importantly, the genes encoding each of these enzymes have been identified (Steele et al 1999;Zabala and Vodkin 2005;Jiang et al 2010). Ralston et al (2005) described the RNA interference (RNAi) of soybean isoflavone synthase genes using the soybean cotyledon system, and this led to enhanced plant susceptibility to Phytophthore sojae in hairy roots. Jiang et al (2010) suggested that RNAi-mediated suppression of isoflavone synthase pathway genes could enhance isoflavone production in soybean.…”