Unlocking Testosterone Production by Biotransformation: Engineering a Fungal Model of Aspergillus nidulans Strain Deficient in Steroid 11α-Hydroxylase Activity and Expressing 17β-Hydroxysteroid Dehydrogenase Enzyme as Proof of Concept

Abstract

Testosterone holds significant medical and economic importance, with the global market for testosterone replacement therapies valued at approximately USD 1.9 billion in 2023. This hormone is essential for the development and maintenance of male sexual characteristics as well as bone and muscle health. It plays a key role in conditions such as hypogonadism, muscle disorders, and andropause. However, the industrial production of testosterone often involves complex chemical processes that result in low yields, high costs, and environmental damage. Microbial biotransformation of steroids presents an eco-friendly alternative to traditional chemical synthesis. A knockout strain of Aspergillus nidulans deficient in steroid 11α-hydroxylase activity was developed, rendering it incapable of hydroxylating androstenedione, progesterone, and testosterone. In these strains, two newly identified CYP450 enzymes, CYP68L1 from A. nidulans and CYP68L8 from Aspergillus ochraceus, were expressed to confirm their roles as steroid 11α-hydroxylases of androstenedione, progesterone, and testosterone. The availability of these 11α-hydroxylases represents significant progress toward achieving efficient single-step steroid fermentation. Furthermore, the A. nidulans knockout strain serves as an effective model for studying the conversion of androstenedione to testosterone upon the expression of the enzyme 17β-hydroxysteroid dehydrogenase, due to its inability to hydroxylate testosterone.

Keywords: 17β-hydroxysteroid dehydrogenase (17β-HSD) EC 1.1.1.51; Aspergillus nidulans; Aspergillus ochraceus; androstenedione; progesterone; steroid 11α-hydroxylase; testosterone.

Figure 1

A schematic representation of reactions catalyzed by different strains of Aspergillus nidulans. Reactions with AD and TS are represented as mediated by the wild-type strain of A. nidulans, the steroid 11α-hydroxylase (CYP68L1) knockout strain of A. nidulans KO strain, (ΔhydAN), and the A. nidulans KO strain (ΔhydAN) expressing the 17β-hydroxysteroid dehydrogenase enzyme from C. lunatus. In the wild-type strain of A. nidulans, conversion occurs, whereas no conversion of AD or TS is observed in the A. nidulans knockout strain (ΔhydAN). The expression of the 17β-hydroxysteroid dehydrogenase enzyme from C. lunatus in the A. nidulans KO strain (ΔhydAN) could facilitate the conversion of AD to TS, as no hydroxylation of either AD or TS is produced.

Figure 2

Appearance of 11α-hydroxylated androstenedione from AD in A. nidulans cultures previously grown with AD (blue) and without AD (red), and then transferred to media with AD. The number of biological replicates was n = 3. HPLC measurements were taken at a wavelength of 240 nm.

Figure 3

Alignment of genomic DNA, cDNA obtained from database, and cDNA sequence obtained by us from AN8530 (CYP68L1) from A. nidulans in this study. The portion of the exon expansion is shown in this figure.

Figure 4

Chromatograms obtained from the analysis of fermentation media of strains (72 h): (A) KOhydAN + hydAN at 32 °C, (B) KOhydAN + hydJ5AO at 32 °C and (C) at 37 °C, (D) KOhydAN +hydAO at 32 °C, and (E) KOhydAN + hydAO at 37 °C. The chromatograms of the culture media containing AD (blue), PG (green), and the KOhydAN expressing the CYP68L8 and CYP68J5 proteins (from A. ochraceus) were analyzed. The construction of the KOhydAN expressing CYP68L1 protein was analyzed in media with AD to verify the recovery of activity. New peaks were identified in the chromatogram results, including the 11α-hydroxylation of AD (11-OHAD) in light blue and the 11α-hydroxylation of PG (11-OHPG) in light green. Temperatures of 32 °C and 37 °C were tested to determine the optimal enzymatic activity of A. ochraceus proteins expressed in A. nidulans, reflecting the different growth temperatures of these Aspergillus species.

Figure 4

Chromatograms obtained from the analysis of fermentation media of strains (72 h): (A) KOhydAN + hydAN at 32 °C, (B) KOhydAN + hydJ5AO at 32 °C and (C) at 37 °C, (D) KOhydAN +hydAO at 32 °C, and (E) KOhydAN + hydAO at 37 °C. The chromatograms of the culture media containing AD (blue), PG (green), and the KOhydAN expressing the CYP68L8 and CYP68J5 proteins (from A. ochraceus) were analyzed. The construction of the KOhydAN expressing CYP68L1 protein was analyzed in media with AD to verify the recovery of activity. New peaks were identified in the chromatogram results, including the 11α-hydroxylation of AD (11-OHAD) in light blue and the 11α-hydroxylation of PG (11-OHPG) in light green. Temperatures of 32 °C and 37 °C were tested to determine the optimal enzymatic activity of A. ochraceus proteins expressed in A. nidulans, reflecting the different growth temperatures of these Aspergillus species.

Figure 5

Alignment of proteins: CYP68L1 from A. nidulans with CYP68J5 (CYP68AQ1) and CYP68L8 from A. ochraceus. The identity of CYP68L1 with CYP68J5 was 36%. CYP68L1’s identity with CYP68L8 was 62%.

Figure 6

Chromatograms obtained from the analysis of fermentation media (72 h) of (A) WTAn + hsdAN; (B) KOhydAN + hsdAN; (C) WTAn + hsdCL; and (D) KOhydAN + hsdCL supplemented with AD (blue) and added TS (red).

Figure 7

Chromatograms obtained from the analysis of fermentation media of KOhydAN + hsdCL. The transformation of AD to TS was measured at 0 h (green), 72 h (purple), and 120 h (orange). AD (blue) and TS (red) standards are shown. Different initial concentrations of AD added to the media were added: (A) 1 g/L of AD, (C) 8 g/L of AD, and (E) 15 g/L of AD. The TS production in g/L was calculated for each initial AD concentration: (B) 1 g/L of AD, (D) 8 g/L of AD, and (F) 15 g/L of AD.