Biosynthetic approach to combine the first steps of cardenolide formation in Saccharomyces cerevisiae

A yeast expression plasmid was constructed containing a cardenolide biosynthetic module, referred to as CARD II, using the AssemblX toolkit, which enables the assembly of large DNA constructs. The genes cloned into the vector were (a) a Delta(5)-3 beta-hydroxysteroid dehydrogenase gene from Digitalis lanata, (b) a steroid Delta(5)-isomerase gene from Comamonas testosteronii, (c) a mutated steroid-5 beta-reductase gene from Arabidopsis thaliana, and (d) a steroid 21-hydroxylase gene from Mus musculus. A second plasmid bearing an ADR/ADX fusion gene from Bos taurus was also constructed. A Saccharomyces cerevisiae strain bearing these two plasmids was generated. This strain, termed CARD II yeast, was capable of producing 5 beta-pregnane-3 beta,21-diol-20-one, a central intermediate in 5 beta-cardenolide biosynthesis, starting from pregnenolone which was added to the culture medium. Using this approach, five consecutive steps in cardenolide biosynthesis were realized in baker's yeast.