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

We describe the construction of a novel yeast strain capable to reconstitute an important part of the cardenolide pathway in Digitalis plants. The intermediates are synthesized from a simple steroid in correct stereospecific manner with the help of five heterologously expressed genes from different bacteria, plants, and mammals. AbstractA 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 Δ5‐3β‐hydroxysteroid dehydrogenase gene fromDigitalis lanata, (b) a steroid Δ5‐isomerase gene fromComamonas testosteronii, (c) a mutated steroid ‐5β‐reductase gene fromArabidopsis thaliana, and (d) a steroid 21 ‐hydroxylase gene fromMus musculus. A second plasmid bearing an ADR/ADX fusion gene fromBos taurus was also constructed. ASaccharomyces cerevisiae strain bearing these two plasmids was generated. This strain, termed “CARD II yeast”, was capable of producing 5β‐pregnane‐3β,21‐diol‐20‐one, a central intermediate in 5β‐cardenolide biosynthesis, starting from pregnenolone which was added to the culture medium. Using this approach, five consecutive steps in cardenolide biosynthesis were realized i n baker's yeast.

Source: MicrobiologyOpen - August 21, 2019 Category: Microbiology Authors: Christoph Rieck, Daniel Geiger, Jennifer Munkert, Katrin Messerschmidt, Jan Petersen, Juliane Strasser, Nadine Meitinger, Wolfgang Kreis Tags: ORIGINAL ARTICLE Source Type: research