Bottlenecks for metabolic engineering of isoflavone glycoconjugates inArabidopsis

Abstract

In view of their perceived chemopreventive activities against hormone-dependent cancers, cardiovascular disease, and postmenopausal ailments, there is considerable interest in engineering plants to contain isoflavone phytoestrogens. However, attempts to date have only resulted in low levels of isoflavone accumulation in non-legumes. Introducing soybean isoflavone synthase (IFS) into Arabidopsis thaliana leads to accumulation of low levels of genistein glycosides. Leaves of wild-type A. thaliana contain high levels of similar conjugates of the flavonols quercetin and kaempferol, which could be increased by threefold on introduction of an alfalfa chalcone isomerase transgene. Levels of genistein were not increased by expressing both IFS and alfalfa chalcone isomerase, but levels of flavonol conjugates were reduced to a greater extent than could be accounted for by flux into isoflavone. Introduction of IFS into the tt6͞tt3 double mutant blocked in flavonol, and anthocyanin synthesis resulted in high levels of genistein. The bottleneck for constitutive isoflavone production in Arabidopsis is, therefore, competition for flavanone between IFS and endogenous flavonol synthesis, and the flavonol pathway is reciprocally but disproportionately affected by IFS.flavonol ͉ phytoestrogen ͉ antioxidant ͉ metabolic engineering ͉ transgenic plants

Combinatorial Biosynthesis of Non-bacterial and Unnatural Flavonoids, Stilbenoids and Curcuminoids by Microorganisms

“…2), which is a common precursor of the biosynthesis of isoflavonoids, has attracted much attention because of its phytoestrogen activity [54]. Several groups have tried to produce genistein in nonlegume plants but the yields were very low probably because of competitive use of naringenin (2g) between isoflavone synthase (IFS) and the endogenous flavonoid pathway [44,55,56]. On the other hand, microbial production of isoflavonoids has been hampered because IFS, a key enzyme catalyzing the aryl migration of (S)-naringenin (2g) to yield isoflavonoid (2m) [57ϳ59], is a membrane-bound cytochrome P450 enzyme that requires a specific electron transfer system.…”

Regulation of Isoflavonoid Biosynthesis in Soybean Seeds

“…However, attempts in these lines have produced very low concentration of isoflavonoids . Since IFS is the entry point enzyme into isoflavonoid biosynthesis and is not present in nonlegume plants, these earlier efforts for metabolic engineering of isoflavonoids were focussed on introducing soybean IFS into corn, tobacco and Arabidopsis thaliana , Liu et al 2002. Introduction of soybean IFS gene into Arabidopsis could yield low levels of genistein glycosides (Liu et al 2002).…”

“…Since IFS is the entry point enzyme into isoflavonoid biosynthesis and is not present in nonlegume plants, these earlier efforts for metabolic engineering of isoflavonoids were focussed on introducing soybean IFS into corn, tobacco and Arabidopsis thaliana , Liu et al 2002. Introduction of soybean IFS gene into Arabidopsis could yield low levels of genistein glycosides (Liu et al 2002). However, this constitutive production of isoflavonoids in Arabidopsis resulted into competition for flavanone between endogenous flavonol synthesis and IFS, affecting the flavonol pathway disproportionally.…”

Cloning and transformation analysis of isoflavone synthase gene into Minshan Trifolium pratense

“…Isoflavone synthase (ISF) is a key enzyme in the biosynthesis pathway of isoflavones as it catalyzes flavanones in the pathway (Overkamp et al, 2000;Liu et al, 2002). To obtain new T. pratense lines of high-yielding isoflavones, we cloned and introduced the IFS gene into T. pratense calluses.…”