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

像地高辛这样的硬核内酯仍然是通过从毛地黄的干叶子中提取而产生的。所有核桃内酯的一个结构特征是-内酯环（“丁烯内酯环”）附着在类固醇支架的C-17上。假设丁烯内酯环的形成是由21- o -丙二醇-5ß-孕烷-3ß，14ß-二醇-20- 1的形成引发的。这一步骤由丙二醇辅酶a催化：21-羟基孕烯-21- o -丙二醇转移酶（21MaT）将丙二醇基部分从丙二醇辅酶a转移到合适的孕烷前体的21-羟基上。丁烯内酯环的形成是在体外自发发生的，在没有酶参与的情况下，14β-羟基化21- o -丙二烯孕烷的脱羧和随后的脱水。从香芋内酯产生植物中纯化21MaTs的尝试导致了几种酶的（部分）纯化。迄今为止，纯化的21MaTs测序失败。在拟南芥AtPMaT1中，我们发现了第一个候选基因，该基因可能编码一种能够丙二酸21-羟基孕烷的酶。AtPMaT1的重组形式（称为rAtPMaT1）确实被证明编码一种酶，该酶催化5ß-孕烷-14ß，21-二醇-20- 1的21- o丙二醛化，产生21- o -丙二酰-5ß-孕烷-14ß-醇-20- 1，这是地地黄黄苷的直接前体。一种同源基因也已在大肠杆菌中被鉴定、克隆和功能表达。进一步的候选基因已被确定。这些基因将在大肠杆菌中表达、表征和比较。在酿酒酵母菌中表达21MaT，将进一步扩展现有的木心脂生物制造平台。我们推测孕烯-21- o -丙二醛酰化是核桃内酯生物合成的核心步骤，提供了一种可运输的代谢物，这种代谢物可以进入液泡，这是核桃内酯的储存场所。通过对21MaT酶和基因的分离和鉴定，我们可以研究21- o丙二酰化孕激素的代谢及其与香薯内酯生物合成的相关性，最终实现酵母中香薯内酯的途径。

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.