Mammalian Cells Engineered To Produce New Steroids

Abstract

Steroids can be difficult to modify through traditional organic synthesis methods, but many enzymes regio- and stereoselectively process a wide variety of steroid substrates. We tested whether steroid-modifying enzymes could make novel steroids from non-native substrates. Numerous genes encoding steroid-modifying enzymes, including some bacterial enzymes, were expressed in mammalian cells by transient transfection and found to be active. We made three unusual steroids by stable expression, in HEK293 cells, of the 7α-hydroxylase CYP7B1, which was selected because of its high native product yield. These cells made 7α,17α-dihydroxypregnenolone and 7β,17α-dihydroxypregnenolone from 17α-hydroxypregnenolone and produced 11α,16α-dihydroxyprogesterone from 16α-hydroxyprogesterone. The last two products were the result of CYP7B1-catalyzed hydroxylation at previously unobserved sites. A Rosetta docking model of CYP7B1 suggested that these substrates' D-ring hydroxy groups might prevent them from binding in the same way as the native substrates, bringing different carbon atoms close to the active ferryl oxygen atom. This new approach could potentially use other enzymes and substrates to produce many novel steroids for drug candidate testing.

Keywords: biosynthesis; cytochromes; hydroxylation; steroids; synthetic biology.

Figure 1.

Overview of our method for novel steroid biosynthesis. Steroid-modifying enzymes are expressed heterologously in mammalian cells and exposed to a non-native substrate, resulting in novel steroid product(s).

Figure 2.

Extracted ion mass chromatograms comparing products of cells supplied with 3 and 7. CYP7B1 cells stably expressed that enzyme, while the negative control cells stably expressed green fluorescent protein. CYP7B1 cell samples were diluted tenfold. A) Only CYP7B1 cells exposed to 3 produced peaks corresponding to the [M+H-H₂O]⁺, [M+H-2H₂O]⁺, and [M+H-3H₂O]⁺ ions of 4 and 5. B) Only CYP7B1 cells exposed to 7 produced a single peak corresponding to the [M+H]₊ ion of 8.

Figure 3.

Predicted structures of the CYP7B1 active site with steroid substrates 1 (blue), 3 (orange), and 7 (green). A) Cartoon representation of holo-CYP7B1 with 1. B-E) Structural models of CYP7B1 permitting 7α-hydroxylation of 1 (B) and 3 (C), 7β-hydroxylation of 3 (D),and 11α-hydroxylation of 7 (E). The red dashed line highlights the positions of the reactive ferryl oxygen and the observed hydroxylation site. F) Side view of native substrate 1 in active site. Yellow dashed lines indicate the proximity of steroid carbons C-16 and C-17 to the edge of the binding pocket. Distances are in Angstroms.

Scheme 1.

Steroid-modifying reactions observed from CYP7B1. A) CYP7B1 hydroxylates native substrate pregnenolone (1) to form 7α-hydroxypregnenolone (2). Yield is in μg of steroid per mL of media extracted. B) CYP7B1 hydroxylates non-native substrate 17α-hydroxypregnenolone (3) to form novel product isomers 7α,17α-dihydroxypregnenolone (4) and 7β,17α-dihydroxypregnenolone (5). C) CYP7B1 hydroxylates non-native substrate 16α-hydroxyprogesterone (7) to form 11α,16α-dihydroxyprogesterone (8). The anticipated product 7α,16α-dihydroxyprogesterone (6) was not observed.