The enzymes OSC1 and CYP716A263 produce a high variety of triterpenoids in the latex of Taraxacum koksaghyz

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.

Uncovering mechanisms of rubber biosynthesis in Taraxacum koksaghyz – role of cis‐prenyltransferase‐like 1 protein

“…Analyzing triterpenes in roots of TkCPTL1 ‐RNAi and TkCPTL2 ‐RNAi plants as well as corresponding controls, we noticed equivalent results (Figure d and Table ; detailed overview in Table S1a,b). Whereas, for TkCPTL1 knockdown lines (24.18 mg/g root DW) substantially elevated levels (compared with the wild‐types; 8.43 mg/g root DW) of pentacyclic triterpenes such as taraxasterol as well as α‐ and β‐amyrin, which are supposed to be highly abundant in latex (Pütter et al ., ), were detected, triterpenes in TkCPTL2 knockdown plants (10.33 mg/g root DW) remained on the same levels as in control plants (10.49 mg/g root DW). Sterols were unaffected in the TkCPTL1 ‐RNAi lines.…”

“…2,3‐oxidosqualene functions as precursor to either pentacyclic triterpenes or sterols. Other proteins that were less abundant in TkCPTL1 knockdowns included two oxidosqualene cyclases (OSCs) responsible for converting of 2,3‐oxidosqualene to several pentacyclic triterpene products (Unland et al ., ; Pütter et al ., ). Interestingly, REF and SRPPs exhibited very high protein levels in the wild‐type and remained unaffected in the RNAi lines, as it was indicated in the SRRP‐specific western blot analysis (Figure f).…”

Oxidosqualene Cyclase Knock-Down in Latex of Taraxacum koksaghyz Reduces Triterpenes in Roots and Separated Natural Rubber

“…As shown in Figure 2b the expression of TkOSC1 was significantly reduced in the latex in all transgenic lines, with only 15% for line sL2 and 8% for line gL2 of the wild-type expression level. Selected plants ( n = 2–4) from all transgenic lines were then analysed regarding their expression of other known OSC genes ( TkOSC2-6 and TkLUP [17]) in the latex (Figure 2c). In TkOSC -RNAi lines (gL), the expression of another predominant latex OSC gene (TkOSC2 , [17]) was significantly reduced.…”

“…Selected plants ( n = 2–4) from all transgenic lines were then analysed regarding their expression of other known OSC genes ( TkOSC2-6 and TkLUP [17]) in the latex (Figure 2c). In TkOSC -RNAi lines (gL), the expression of another predominant latex OSC gene (TkOSC2 , [17]) was significantly reduced. TkOSC2 expression was also slightly reduced in sL2 and sL4 plants, which results obviously from the partial sequence similarity with TkOSC1 .…”

“…Recently, we provided a comprehensive analysis of the triterpene composition in NR extracts from T. koksaghyz and identified 13 triterpenes and triterpenoids, also including the so far unknown pentacyclic compound lup-19(21)-en-3-ol [17]. Furthermore, we isolated and functionally characterized seven different T. koksaghyz OSCs ( TkOSC1-6 and TkLUP) and could demonstrate that TkOSC1 is highly expressed in laticifers and represents a multifunctional OSC capable of synthesizing at least four of the latex-predominant pentacyclic triterpenes (taraxasterol, α-, β-amyrin and lup-19(21)-en-3-ol) [17,18] (Figure 1).…”