Towards a biomanufacturing platform for cardenolides

 

Cardenolides are drugs used to treat congestive heart failure. More recently their antiproliferative action was brought into focus [1]. Cardenolides are still extracted from plants grown in the field. Though attempts have been tried in the past to produce cardenolides by plant cell tissue culture, farming of foxglove Digitalis sp. still remains the sole source of cardenolides. We are about to engineer yeast cells to produce cardenolides starting from a simple sugar source. Basically, we intend to follow an adapt the strategy reported for hydrocortisone biosynthesis in yeast [2], however, we are focusing on plant genes in several places where mammalian genes have been used. We combine enzyme discovery, enzyme engineering, as well as pathway optimization to realize this. Cardenolide aglycone formation from a sterol precursor requires the following steps: 1 sterol side chain cleavage, 2 pregnenolone 3-O-dehydrogenation, 3 isoprogesterone isomerisation, 4 progesterone 5β-reduction, 5 pregnane-3-one reduction, 6 pregnane 21-hydroxylation, 7 pregnane 14-hydroxylation, 8 malonyl 21-O-hemiester formation and 9 butenolide ring formation. Presently, we are mainly working on steps (1) – (5) using plant genes (steps 2, 4, 5), a bacterial gene (step 3) and mammalian genes (steps 1 and 6). No candidate gene has as yet been identified for steps (7) and (8). The proof of principle has been demonstrated in modules converting pregnenolone to 21-hydroxyprogesterone in yeast and pregnenolone to 5β-pregnanes in E. coli. These modules will be developed further. Studies to shape step (4) for improved co-substrate usage are in progress. Candidate genes for step (8) are about to be identified using two different approaches, namely enzyme isolation and homology search. The modular strategy followed here also takes into consideration that more than one organism (co-cultivation) can be used in a synthetic biology approach towards the production of cardenolides.

Keywords: Pathway engineering, synthetic biology, cardenolides

References:

[1] Nolte E, Sobel A, Wach S, Hertlein H, Ebert N, Müller-Uri F, Slany R, Taubert H, Wullich B, Kreis W. The new semisynthetic cardenolide analog 3β-[2-(1-amantadine)-1-on-ethylamine]-digitoxigenin (AMANTADIG) efficiently suppresses cell growth in human leukemia and urologic tumor cell lines. Anticancer Res 2015; 35: 5271 – 5276

[2] Szczebara FM, Chandelier C, Villeret C, Masurel A, Bourot S, Duport C, Blanchard S, Groisillier A, Testet E, Costaglioli P, Cauet G, Degryse E, Balbuena D, Winter J, Achstetter T, Spagnoli R, Pompon D, Dumas B. Total biosynthesis of hydrocortisone from a simple carbon source in yeast. Nat Biotechnol 2003; 21: 143 – 149