21-Hydroxpregnane 21-O-malonyltransferases: Heterologuous expression in Escherichia coli and Saccharomyces cerevisiae, characterization and significance in cardenolide biosynthesis

强心内酯，如地高辛，仍然是通过从毛地黄的干燥叶中提取而产生的。所有强心内酯的一个结构特征是与类固醇支架的C-17连接的γ-内酯环（“丁烯二酸环”）。据推测，丁烯二酰环的形成是通过形成21-O-丙二酰基-5 α-癸烷-3 β，14 β-二醇-20-酮而引发的。该步骤由丙二酰辅酶A：21-羟基胆甾烷-21-0-丙二酰转移酶（21 MaT）催化，其将丙二酰部分从丙二酰辅酶A转移至适当胆甾烷前体的21-羟基。在没有酶参与的情况下，通过14β-羟基化21-O-丙二酰孕烷的脱羧和随后的脱水，证明了丁烯二酰环的形成在体外自发发生。从产腰果酚的植物中纯化21 MaTs的尝试导致了几种酶的（部分）纯化。到目前为止，纯化的21 MaTs的测序失败。在拟南芥AtPMaT 1，我们确定了第一个候选基因，可能编码的酶能够丙二酰化21-羟基孕烷。AtPMaT 1的重组形式（称为rAtPMaT 1）确实被证明编码一种酶，该酶催化5 α-胆甾烷-14 β，21-二醇-20-酮的21-O-丙二酰化，产生21-O-丙二酰-5 α-胆甾烷-14 β-醇-20-酮，洋地黄毒苷的直接前体。D.也已经鉴定、克隆了羊毛状植物，并在大肠杆菌中进行了功能性表达。已经鉴定了更多的候选基因。这些基因将在大肠杆菌中表达，表征和比较。在酿酒酵母中表达21 MaT时，将进一步扩展用于强心内酯的现有生物制造平台。 我们推测，异氟烷-21-O-丙二酰化是Cardenolide生物合成中的中心步骤，提供-像某些外源性物质的代谢-液泡膜转运的代谢物，其可以进入液泡，Cardenolides的储存位点。21 MaT酶和基因的分离和表征将使我们能够研究21-O-丙二酰化孕烷的代谢及其与Cardenoprotein生物合成的相关性，以最终实现Cardenoprotein途径在酵母中。

Cardenolides, such as digoxin, are still produced by extraction from dried leaves of Digitalis lanata. One structural feature of all cardenolides is the gamma-lactone ring ("butenolide ring") attached to C-17 of a steroid scaffold. It is assumed that butenolide ring formation is initiated by the formation of 21-O-malonyl-5ß-pregnane-3ß,14ß-diol-20-one. This step is catalyzed by a malonyl coenzyme A:21-hydroxypregnane-21-O-malonyltransferase (21MaT) which transfers a malonyl moiety from malonyl-CoA to the 21-hydroxy group of an appropriate pregnane precursor. Butenolide ring formation was demonstrated to occur spontaneously in vitro by decarboxylation and subsequent dehydration of 14β-hydroxylated 21-O-malonylpregnanes without participation of an enzyme. The attempts to purify 21MaTs from cardenolide-producing plants resulted in the (partial) purification of several enzymes. Sequencing of purified 21MaTs so far failed. In Arabidopsis thaliana AtPMaT1 we identified a first candidate gene that might encode an enzyme able to malonylate 21-hydroxpregnanes. The recombinant form of AtPMaT1 (termed rAtPMaT1) was indeed demonstrated to encode an enzyme that catalyzes the 21-O-malonylation of 5ß-pregnane-14ß,21-diol-20-one, yielding 21-O-malonyl-5ß-pregnane-14ß-ol-20-one, the direct precursor of digitoxigenin. A homologuous gene in D. lanata has also been identified, cloned and functionally expressed in E.coli. Further candidate genes have been identified. These genes will be expressed in E.coli , characterized and compared. In expressing 21MaT also in Saccharomyces cerevisiae an existing biomanufacturing platform for cardenolides will further be extended. We presume that pregnane-21-O-malonylation is a central step in cardenolide biosynthesis providing - like in the metabolism of certain xenobiotics - a tonoplast-transportable metabolite, that can enter the vacuole, a storage site for cardenolides. The isolation and characterization of 21MaT enzymes and genes will enable us to study the metabolism of 21-O-malonylated pregnanes and their relevance for cardenolide biosynthesis, in order to finally realize a cardenolide pathway in yeast.