Biosynthesis and Industrial Production of Androsteroids

Abstract

Steroids are a group of organic compounds that include sex hormones, adrenal cortical hormones, sterols, and phytosterols. In mammals, steroid biosynthesis starts from cholesterol via multiple steps to the final steroid and occurs in the gonads, adrenal glands, and placenta. This highly regulated pathway involves several cytochrome P450, as well as different dehydrogenases and reductases. Steroids in mammals have also been associated with drug production. Steroid pharmaceuticals such as testosterone and progesterone represent the second largest category of marketed medical products. There heterologous production through microbial transformation of phytosterols has gained interest in the last couple of decades. Phytosterols being the plants sterols serve as inexpensive substrates for the production of steroid derivatives. Various genes and biochemical pathways involved in phytosterol degradation have been identified in many Rhodococcus and Mycobacterium species. Apart from an early investigation in mammals, presence of steroids such as androsteroids and progesterone has also been demonstrated in plants. Their main role is linked with growth, development, and reproduction. Even though plants share some chemical features with mammals, the biosynthesis is different, with the first C22 hydroxylation as an example. This is performed by CYP11A1 in mammals and CYP90B1 in plants. Moreover, the entire plant steroid biosynthesis is not fully elucidated. Knowing this pathway could provide new processes for the industrial biotechnological production of steroid hormones in plants.

Keywords: androsteroids; cholesterol biosynthesis; phytosterols; pregnenolone; progesterone; steroid hormones; testosterone.

Figure 1

Overview of the biosynthesis of steroids in mammals. This overview combines the classic pathway (in blue) and the backdoor pathway (in red). In the classic pathway, cholesterol is converted into pregnenolone through cleavage of the side chain, followed by multiple steps towards testosterone synthesis. 3β-hydroxysteroid dehydrogenase type 2 (HSD3B2) converts Δ5 steroids to Δ4 steroids. In humans, production of testosterone is favored through 17OH-pregnenolone, as the catalytic efficiency of CYP17A1 is largely higher with this derivative than with 17OH-progesterone. The backdoor pathway bypasses production of dehydroepiandrosterone and androstenedione. Actions of 5α-reductase of type 1 (SRD5A1) and 3α-hydroxysteroid dehydrogenase (AKR1C4) lead to production of androsterone, the major androsteroid of this pathway.

Figure 2

Production of steroid compounds such as testosterone (TS) via a biotransformation process. Phytosterols (P—in green) from agricultural waste, represented here by β-Sitosterol, are used as relevant and cost-effective substrates. (A) They are degraded by microbial cell factories (in yellow), such as mycobacteria, through a multiple-step process where a chemical synthesis (in blue) of TS follows the biotransformation of androsterone (AD), (B) or through a one-step process where microbial strains are engineered to directly convert phytosterols to TS. 9OH-AD, 9α-hydroxy-4-androstene-3,17-dione; 9OH-ADD, 9 alpha-hydroxyandrosta-1,4-diene-3,17-dione; KstD, 3-ketosteroid Δ1-dehydrogenase; Ksh, 3-cetosteroide-9α hidroxilasa; 17β-HSD, 17-β hydroxy steroid dehydrogenase; Cyp125-3, steroid C27-monooxygenase.

Figure 3

Steroid biosynthesis pathway in plants. This overview shows the interpretation of the classic steroid biosynthesis pathway in plants. The enzyme is responsible for the conversion of cholesterol to pregnenolone and pregnenolone to progesterone, having been characterized in different plant species. However, the full elucidation of the remaining steps is unknown. Δ⁵-3β-HSD, Δ⁵-3β-Hydroxysteroid dehydrogenase; Δ⁵-Δ⁴-KSI, Δ⁵-Δ⁴-ketosteroid isomerase.

Figure 4

Conversion of phytosterols in pregnenolone. The figure shows the steroid core (A) and the possible hydroxylation reactions for phytosterols: cholesterol (B), campesterol (C) and beta-sitosterol (D). In vertebrates the conversion is carried out by CYP11A1 (in red), but it also functions with plants sterols. In plants, the enzymes involved are shown in blue. It is still unknown how the side-chain cleavage between C20 and C22 (represented by dotted blue line) occurs in the plant kingdom.