A BAHD acyltransferase catalyzing 19‐O‐acetylation of tabersonine derivatives in roots of Catharanthus roseus enables combinatorial synthesis of monoterpene indole alkaloids

Abstract

While the characterization of the biosynthetic pathway of monoterpene indole alkaloids (MIAs) in leaves of Catharanthus roseus is now reaching completion, only two enzymes from the root counterpart dedicated to tabersonine metabolism have been identified to date, namely tabersonine 19-hydroxylase (T19H) and minovincine 19-O-acetyltransferase (MAT). Albeit the recombinant MAT catalyzes MIA acetylation at low efficiency in vitro, we demonstrated that MAT was inactive when expressed in yeast and in planta, suggesting an alternative function for this enzyme. Therefore, through transcriptomic analysis of periwinkle adventitious roots, several other BAHD acyltransferase candidates were identified based on the correlation of their expression profile with T19H and found to localize in small genomic clusters. Only one, named tabersonine derivative 19-O-acetyltransferase (TAT) was able to acetylate the 19-hydroxytabersonine derivatives from roots, such as minovincinine and hörhammericine, following expression in yeast. Kinetic studies also showed that the recombinant TAT was specific for root MIAs and displayed an up to 200-fold higher catalytic efficiency than MAT. In addition, gene expression analysis, protein subcellular localization and heterologous expression in Nicotiana benthamiana were in agreement with the prominent role of TAT in acetylation of root-specific MIAs, thereby redefining the molecular determinants of the root MIA biosynthetic pathway. Finally, identification of TAT provided a convenient tool for metabolic engineering of MIAs in yeast enabling efficiently mixing different biosynthetic modules spatially separated in the whole plant. This combinatorial synthesis associating several enzymes from Catharanthus roseus resulted in the conversion of tabersonine in tailor-made MIAs bearing both leaf and root-type decorations.

The assembly of (+)‐vincadifformine‐ and (−)‐tabersonine‐derived monoterpenoid indole alkaloids in Catharanthus roseus involves separate branch pathways

“…In marked contrast, both (+)‐minovincinine (Figure , inset lane 8) and its O ‐acetylated derivative (+)‐echitovenine (Figure , inset lane 9) accumulated, but not (+)‐vincadifformine (Figure , inset lane 7). While enantiomeric forms of the MIAs were not specified, hairy roots, adventitious roots and roots from whole C. roseus plants (Carqueijeiro et al ., ,b) were reported to accumulate tabersonine and lochnericine together with lower levels of hörhammericine, vincadifformine, minovincinine and echitovenine, while only traces of 19‐hydroxytabersonine, 19‐ O ‐acetyltabersonine or 19‐ O ‐acetylhörhammericine were detected.…”

“…The results obtained with the T3O homolog showed that these two separate enzymes, sharing 27% amino acid identity, are specific for enantiomer‐specific hydroxylation of (+)‐ and (−)‐aspidosperma skeletons, respectively (Figure ). The results suggest that while 19‐ O ‐acetylhörhammericine is derived from (−)‐tabersonine (Figure ) (Carqueijeiro et al ., ,b), formation of (+)‐echitovenine involved a separate two‐step pathway from (+)‐vincadifformine (Figure ). The T3O homolog that catalyzes this regiospecific and stereospecific hydroxylation was named (+)‐vincadifformine 19‐hydroxylase (V19H).…”

“…It has been suggested (Carqueijeiro et al ., ,b) that TAT rather than MAT (Laflamme et al ., ) catalyzes the terminal reactions for the biosynthesis of 19‐ O ‐acetylhörhammericine and echitovenine (Figure ). When (+)‐minovincinine (Figure j) was incubated with recombinant purified MAT and acetyl‐CoA, (+)‐echitovenine (Figure k) was formed, while TAT was not able to accept this (+)‐enantiomer as a substrate (Figure l).…”

“…The enantiomeric specificity of T19H, V19H, MAT and TAT (Figure ) clearly highlight the existence of separate pathways for the formation of (+)‐echitovenine from (+)‐vincadifformine, of 19‐ O ‐acetylhörhammericine from (−)‐tabersonine, and of (−)‐echitovenine from (−)‐vincadifformine (Figure ). The presence of hörhammericine and the trace levels of 19‐ O ‐acetylhörhammericine in 3‐week‐old roots (Figure ), in adventitious roots and in hairy roots (Carqueijeiro et al ., ,b) clearly suggests that TAT is poorly active in these organs. The accumulation of (+)‐echitovenine in these roots (Figure ) combined with the substrate specificity of MAT for (+)‐minovincinine (Figure n) show that MAT is responsible for its formation rather than TAT (Figure ).…”

“…While less is known about MIA biosynthesis in C. roseus roots, the pathway for assembly of 19‐ O ‐acetylhörhammericine from tabersonine involves two cytochrome P450‐mediated reactions for 6,7‐epoxide (Rodriguez et al ., ; Carqueijeiro et al ., 2018a) and 19‐hydroxyl (Giddings et al ., ) formation followed by 19‐ O ‐acetylation catalyzed by minovincinine 19‐hydroxy‐ O ‐acetyltransferase (MAT) (Laflamme et al ., ) (Figure 2). However, the recent discovery of another root‐specific 19‐hydroxytabersonine, 19‐ O ‐acetyltransferase (TAT) (Carqueijeiro et al ., 2018b), capable of acetylating both minovincinine and hörhammericine with 200‐fold higher catalytic efficiency than MAT, led to the suggestion that MAT may only play a minor role in O ‐acetylation of these tabersonine and vincadifformine derivatives in C. roseus roots.…”