Brian Mcgonigle

Identification and expression of isoflavone synthase, the key enzyme for biosynthesis of isoflavones in legumes

Isoflavones have drawn much attention because of their benefits to human health. These compounds, which are produced almost exclusively in legumes, have natural roles in plant defense and root nodulation. Isoflavone synthase catalyzes the first committed step of isoflavone biosynthesis, a branch of the phenylpropanoid pathway. To identify the gene encoding this enzyme, we used a yeast expression assay to screen soybean ESTs encoding cytochrome P450 proteins. We identified two soybean genes encoding isoflavone synthase, and used them to isolate homologous genes from other leguminous species including red clover, white clover, hairy vetch, mung bean, alfalfa, lentil, snow pea, and lupine, as well as from the nonleguminous sugarbeet. We expressed soybean isoflavone synthase in Arabidopsis thaliana, which led to production of the isoflavone genistein in this nonlegume plant. Identification of the isoflavone synthase gene should allow manipulation of the phenylpropanoid pathway for agronomic and nutritional purposes.

Metabolic engineering to increase isoflavone biosynthesis in soybean seed

Isoflavone levels in Glycine max (soybean) were increased via metabolic engineering of the complex phenylpropanoid biosynthetic pathway. Phenylpropanoid pathway genes were activated by expression of the maize C1 and R transcription factors in soybean seed, which decreased genistein and increased the daidzein levels with a small overall increase in total isoflavone levels. Cosuppression of flavanone 3-hydroxylase to block the anthocyanin branch of the pathway, in conjunction with C1/R expression, resulted in higher levels of isoflavones. The combination of transcription factor-driven gene activation and suppression of a competing pathway provided a successful means of enhancing accumulation of isoflavones in soybean seed.

Transgenic production of epoxy fatty acids by expression of a cytochrome P450 enzyme from Euphorbia lagascae seed.

Seed oils of a number of Asteraceae and Euphorbiaceae species are enriched in 12-epoxyoctadeca-cis-9-enoic acid (vernolic acid), an unusual 18-carbon Δ12-epoxy fatty acid with potential industrial value. It has been previously demonstrated that the epoxy group of vernolic acid is synthesized by the activity of a Δ12-oleic acid desaturase-like enzyme in seeds of the Asteraceae Crepis palaestina and Vernonia galamensis. In contrast, results from metabolic studies have suggested the involvement of a cytochrome P450 enzyme in vernolic acid synthesis in seeds of the Euphorbiaceae species Euphorbia lagascae. To clarify the biosynthetic origin of vernolic acid in E. lagascae seed, an expressed sequence tag analysis was conducted. Among 1,006 randomly sequenced cDNAs from developingE. lagascae seeds, two identical expressed sequence tags were identified that encode a cytochrome P450 enzyme classified as CYP726A1. Consistent with the seed-specific occurrence of vernolic acid in E. lagascae, mRNA corresponding to theCYP726A1 gene was abundant in developing seeds, but was not detected in leaves. In addition, expression of the E. lagascae CYP726A1 cDNA in Saccharomyces cerevisiae was accompanied by production of vernolic acid in cultures supplied with linoleic acid and an epoxy fatty acid tentatively identified as 12-epoxyoctadeca-9,15-dienoic acid (12-epoxy-18:2Δ9,15) in cultures supplied with α-linolenic acid. Consistent with this, expression of CYP726A1 in transgenic tobacco (Nicotiana tabacum) callus or somatic soybean (Glycine max) embryos resulted in the accumulation of vernolic acid and 12-epoxy-18:2Δ9,15. Overall, these results conclusively demonstrate that Asteraceae species and the Euphorbiaceae E. lagascae have evolved structurally unrelated enzymes to generate the Δ12-epoxy group of vernolic acid.

Metabolic Engineering of Isoflavone Biosynthesis

Isoflavones are phenolic secondary metabolites found mostly in legumes. These compounds play key roles in many plant–microbe interactions and are associated with the health benefits of soy consumption. Because of their biological activities, metabolic engineering of isoflavonoid biosynthesis in legume and nonlegume crops have significant agronomic and nutritional impact by enhancing plant disease resistance and providing dietary isoflavones for the improvement of human health. This review first outlines the current understanding of isoflavone biosynthetic pathways, with focus on key structural enzymes and transcription factors that directly relate to the pathways. Then it summarizes recent progress on metabolic engineering of isoflavone biosynthesis in both legume and nonlegume plants. The major limitations of these approaches, as well as the “metabolic channeling” theory, which is proposed to explain some of the results from the engineering works, are also discussed.

Chapter eight Metabolic engineering of soybean for improved flavor and health benefits

Summary and Future Directions The food choices that consumers make are informed by a variety of criteria including cost, safety, environmental impact, and especially perceived health benefits and taste. Over the past several years these criteria have caused consumers to include more soyfood products in their diets. Two things are necessary for the continuation of this trend. First, the scientific community must more rigorously prove the health benefits associated with eating soybean; specifically which components of soybean cause the health benefits and the physiological mechanisms under which these benefits are obtained. These are difficult studies to carry out. In the past, some of these studies have been conducted using soyfoods that have been treated in such a way as to remove a given compound, although this typically results in the removal of many classes of compounds. Metabolic engineering of soybeans will allow the creation of beans with specific compounds (or lack there of) that can then be used to test the role these compounds play in human health. Second, soyfoods must be developed that individual consumers consider atasty part of their diet. Only foods that appeal to an individual are likely to continue to remain a part of that individuals diet, no matter how many other good properties are associated with that food. Progress in formulation of soyfoods has created foods with significantly greater acceptance. However, it may be that specific changes resulting from metabolic engineering will be required to produce new generations of tasty and healthy soyfoods.