Abstract
Only very little is known about the resin composition of natural rubber from the dandelion species Taraxacum koksaghyz, thus its full characterization could provide new insights into how the isoprenoid end-products influence the physical properties of natural rubber, and this resin might be a good source of highly diverse triterpenoids. Here, we present a comprehensive analysis of the triterpenoid composition in an acetone extract and identified 13 triterpenes and triterpenoids also including the so far unknown pentacyclic compounds lup-19(21)-en-3-ol (1) and its ketone lup-19(21)-en-3-one (2). We purified single triterpenes from the acetone extract by developing a two-step HPLC system that is adapted to the structural differences of the described triterpenoids. Furthermore, we isolated six different oxidosqualene cyclases (OSCs) and two P450 enzymes, and we functionally characterized TkOSC1 and CYP716A263 in Nicotiana benthamiana and Saccharomyces cerevisiae in detail. TkOSC1 is a multifunctional OSC that was capable of synthesizing at least four of the latex-predominant pentacyclic triterpenes (taraxasterol, α-, β-amyrin and lup-19(21)-en-3-ol) while CYP716A263 oxidized pentacyclic triterpenes at the C-3 position. The identified enzymes responsible for biosynthesis and modification of pentacyclic triterpenes in T. koksaghyz latex may represent excellent tools for bioengineering approaches to produce pentacyclic triterpenes heterologously.
Highlights
Over 20,000 angiosperm species produce latex, a highly specialized cytoplasm, in tubular cells called laticifers[1]
To gain a detailed overview about the single triterpenes that play a role in natural rubber characteristics, a lipid fraction was extracted from T. koksaghyz NR using acetone as a solvent
We detected different pentacyclic triterpenes that are described to be highly abundant in the roots of Taraxacum koksaghyz[17] in four of the seven C18 fractions; these pentacyclic triterpenes included taraxasterol and β-amyrin in fraction F4 (Fig. 1c), α-amyrin in F6, lupeol in F1 and taraxerol in F3 (Supplementary Fig. S1)
Summary
Over 20,000 angiosperm species produce latex, a highly specialized cytoplasm, in tubular cells called laticifers[1]. The latex of Taraxacum species contains substantial amounts of pentacyclic triterpenes[7] These pentacyclic triterpenes exhibit extraordinary biological activities against fungi and bacteria, making them highly attractive for agricultural and pharmaceutical applications[8]. Cyclization of the chair-boat-chair conformation of 2,3-oxidosqualene yields the tetracyclic protosteryl cation intermediate that generates sterols, whereas cyclization of the chair-chair-chair conformation of 2,3-oxidosqualene produces the tetracyclic dammarenyl cation that can undergo subsequent ring expansion to form pentacyclic triterpenes. These can further be modified by cytochrome P450 monooxygenases (P450s) or glycosyltransferases, yielding triterpenoids and saponins, respectively. Regarding P450s, studies have predominantly identified the CYP716 family members as responsible for oxidizing pentacyclic triterpenes, most commonly at the C-28 position[16]
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