Purification of flavan-3-ol biosynthesis for co-localization onto nanocarriers and a multi-enzyme assay

Flavonoids are polyphenolic secondary metabolites that serve a variety of purposes, from assisting pollination in plants to providing anti-cancerous activity in humans. It is the wide range of functions that give these molecules the highly revered status they have today. Range in function can be linked with the range in structure, and with over 10,000 different flavonoids known, they make up one of the largest groups of natural products. One common sub-class of flavonoids is the flavan-3-ols, which are known for their health benefits and tendency to condense to form proanthocyanidins, or condensed tannins. Both of these classes of flavonoids represent the valuable end products of a highly investigated, yet still not fully understood, section of flavonoid biosynthesis. Furthermore, some of the intermediates are highly unstable and have never been isolated in planta. The enzymes that comprise this section are highly interconnected and regulated, suggesting that a possible multi-enzyme complex exists. With this evidence, we propose to create a synthetic complex using a polymer nanocarrier using anthocyanidin synthase and reductase (ANS and ANR, respectively), the final two enzymes in flavan-3-ol biosynthesis. Along with the preceding enzyme, dihydroflavonol 4-reductase (DFR), they have been separately and recombinantly expressed in E. coli. Both ANR and ANS are fused to a specific tag for binding to the nanocarrier, ANR – 6xHis and ANS – (monomeric) streptavidin. Specifying binding sites will allow adjustable enzyme concentration and distribution on the nanocarrier. DFR, which has a stable substrate, allows for generation of the highly unstable ANS substrate. All three enzymes have been designed and purified for co-localization on the nanocarrier and all have exhibited activity in an assay designed for the multi-enzyme reaction.