Open AccessBiosynthetic approach to combine the first steps of cardenolide formation in Saccharomyces cerevisiae
Author(s) -
Rieck Christoph,
Geiger Daniel,
Munkert Jennifer,
Messerschmidt Katrin,
Petersen Jan,
Strasser Juliane,
Meitinger Nadine,
Kreis Wolfgang
Publication year - 2019
Publication title -
microbiologyopen
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.881
H-Index - 36
ISSN - 2045-8827
DOI - 10.1002/mbo3.925
Subject(s) - cardenolide , saccharomyces cerevisiae , pregnenolone , biology , plasmid , gene , biochemistry , yeast , steroid , botany , glycoside , hormone
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 Δ 5 ‐3β‐hydroxysteroid dehydrogenase gene from Digitalis lanata , (b) a steroid Δ 5 ‐isomerase gene from Comamonas testosteronii , (c) a mutated steroid‐5β‐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 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 in baker's yeast.