21-Hydroxpregnane 21-O-malonyltransferases: Heterologuous expression in Escherichia coli and Saccharomyces cerevisiae, characterization and significance in cardenolide biosynthesis
Final Report Abstract
The main aim of the project was to identify and isolate genes coding for malonyltransferases being capable of accepting 21-hydroxypregnanes as substrates. We isolated cDNAs from seven different candidate genes (Atpmat1, Atpmat2, Ecmat1, Dlmat1, Dlmat2, Dlmat3, Dlmat4) all encoding enzymes of the BAHD-type family. The gene sequences were isolated from three different plant species, two cardenolide-producing plants (Digitalis lanata and Erysimum crepidifolium), and the cardenolide-free model plant Arabidopsis thaliana. Five of them were successfully expressed in E. coli and all of the enzymes produced accepted various 21-hydroxypregnanes as substrates. AtPMaT1 had remarkably higher specific activities as compared to the others, 14β-hydroxylated 21-O-pregnanes, such as 3-O- acetylketol and 3-O-digitoxosylketol were preferred over others. AtPMaT1 was regarded a suitable candidate to be introduced into a yeast biotechnological platform, which already contained four genes that provide an enzymatic system able to transform pregnenolone to compounds occurring in early steps in cardenolide biosynthesis. Feeding of pregnenolone to the newly built construct did not result in a malonylated product since the system still lacks a 14β-hydroxylase. On the other hand, when only AtPMaT1 was expressed in yeast and fed with 14β-hydroxylated 21-O-pregnanes, conversion of up to 23 % of the substrate was observed. Hence Atpmat1 is a potential candidate to expand the already established biotechnological platform, which now has to be completed by the 14β- hydroxylation step.
Publications
- BAHD-like malonyltransferase genes from Digitalis lanata and Arabidopsis thaliana and their putative role in cardenolide biosynthesis. 67th International Congress and Annual Meeting of the Society for Medicinal Plant and Natural Product Research (GA), Innsbruck, September 2019
M Tropper, S Höhn, J Munkert, W Kreis
(See online at https://doi.org/10.1055/s-0039-3399788) - 21-Hydroxypregnane 21-O-malonylation, a crucial step in cardenolide biosynthesis, can be achieved by substrate-promiscuous BAHD-type phenolic glucoside malonyltransferases from Arabidopsis thaliana and homolog proteins from Digitalis lanata. Phytochemistry 2021
Tropper M, Höhn S, Wolf L-S, Fritsch J, Kastner-Detter N, Rieck C, Munkert J, Meitinger N, Lanig H, Kreis W
(See online at https://doi.org/10.1016/j.phytochem.2021.112710) - In silico, in vitro, and structural investigations on BAHD-enzymes from different species able to malonylate 21-hydroxypregnanes. 69th International Congress and Annual Meeting of the Society for Medicinal Plant and Natural Product Research (GA), Bonn, September 2021
M Tropper, L-S Wolf, H Lanig, W Kreis
(See online at https://doi.org/10.1055/s-0041-1736873)
