Suppressing the side reactions of pregnenolone acetylation in transgenic yeast using competitive inhibition and optimal vector‒host system

Abstract

The rational design of the vector‒host system and the conditions for its use is the key to its most effective use as a biocatalyst for obtaining valuable products or for testing potential bioregulators. In this work, we will consider examples of such solutions for some yeasts from the literature over the past 5 years and our experience in optimizing the reactions of 17α-hydroxylation of pregnanes with recombinant strains of the yeast S. cerevisiae and Y. lipolytica. Thus, it was known that pregnenolone and its product pregnenolone and the products of its transformation by cytochrome P450 17α-hydroxylase/17,20-lyase (P450c17) ‒ 17 hydroxypregnenolone, dehydroepiandrosterone ‒ undergo 3-O-acetylation by the Atf2p enzyme of the yeast Saccharomyces cerevisiae. We have found that adding isoamyl or amyl alcohol to the cultivation medium significantly reduced the formation of 3-O-acetylated products by both uninduced and induced cells of transgenic yeast S. cerevisiae GRF18 YEp5117α expressing P450c17 under the control of the GAL10 promoter. In this case, the Atf2p substrate competition model was applied. A more effective solution was to use a different host microorganism and an expression promoter. The analysis of the genome of the yeast Y. lipolytica using the BLAST program showed the absence of potential analogues of Atf2p, which was confirmed by the absence of pregnenolone acetylation products. Selecting the optimal host organism is an alternative to using a strain with a deleted gene.