**1. Introduction**

Dehydroepiandrosterone (DHEA) is a major C19 steroid hormone produced by the adrenal cortex. Meanwhile, it is also produced in small quantities in the gonads and brain [1]. Due to its long half-life in plasma, most DHEA would become dehydroepiandrosterone sulfate ester (DHEAS), reserved, and converted into specific hormones when needed [2]. As a kind of important pharmaceutical steroid, hydroxylation at different positions would exhibit diversified biological activities. For example, hydroxylation at position 9α/16α is crucial for the bioactivities of glucocorticoids (dexamethasone, triamcinolone, etc.) [3,4]. Hydroxylation at position 11α is essential for anti-inflammatory activities (hydrocortisone, prednisolone) [5,6]. Hydroxylation at position 14α is vital for the production of the 21-acetoxy analog of proligestone, which is a prodrug of Promegestone [7]. Hydroxylation at position 15α is a key intermediate for the production of progesterone [8].

Previous biotransformation investigation towards DHEA-analog steroids had demonstrated a varieties of metabolites spectrum. Huang et.al reported that 15α-hydroxy-17 a-oxa-D-homo-androst-4-ene-3,17-dione and androst-4-en-3,17-dione, were produced by *Penicillium griseopurpureum* [9]. Kołek et al. used androstenediol as a substrate and produced di- and trihydroxylation products such as 3β,17β-Dihydroxyandrost-5-en-7-one, 3β,7α,17β-trihydroxyandrost-5-ene, 3β,7β,17β-Trihydroxyandrost-5-ene [10,11].

When 7-oxo-DHEA was used as substrate, 3β,16β-dihydroxy-androst-5-en-7,17-dione, 3β-hydroxy-17α-oxa-D-homo-androst-5-en-7,17-dione, and 3β-acetoxy-androst-5-en-7,17 dione could be produced by *Laetiporus sulphureus* AM498, *Fusicoccum amygdali* AM258 and *Spicaria divaricata* AM423 [12]. Incubation of DHEA with *Ulocladium chartarum* MRC 72584 produced seven DHEA derivatives, such as 3β-hydroxyandrost-5-en-7,17-dione, 3β,7β-dihydroxyandrost-5-en-17-one, 3β,7α-dihydroxyandrost-5-en-17-one, etc. [13]. A

**Citation:** Song, M.; Fu, R.; Cai, S.; Jiang, X.; Wang, F.; Xu, W.; Xu, W. 7*α* and 7*β* Hydroxylation of Dehydroepiandrosterone by *Gibberella sp.* and *Absidia Coerulea* Biotransformation. *Catalysts* **2023**, *13*, 272. https://doi.org/10.3390/ catal13020272

Academic Editors: Zhilong Wang and Tao Pan

Received: 13 December 2022 Revised: 19 January 2023 Accepted: 20 January 2023 Published: 25 January 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

7α,15α-dihydroxyl-DHEA product was reported by Li et al. using *Colletotrichum lini* [14]. Microbial transformation by using *Mortierella isabellina* AM212 produced 7-Oxo-DHEA, 7α-Hydroxy-DHEA, 7β-Hydroxy-DHEA [10], and *Backusella lamprospora* VKM F- 944 could transform DHEA into 7α-hydroxy-DHEA [15]. These single, double, and triple hydroxylation reactions greatly enriched the DHEA metabolite ingredients. [14]. Microbial transformation by using *Mortierella isabellina* AM212 produced 7-Oxo-DHEA, 7α-Hydroxy-DHEA, 7β-Hydroxy-DHEA [10], and *Backusella lamprospora* VKM F-944 could transform DHEA into 7α-hydroxy-DHEA [15]. These single, double, and triple hydroxylation reactions greatly enriched the DHEA metabolite ingredients. In this research, two out of twelve filamentous strains of fungi demonstrated their

en-7,17-dione could be produced by *Laetiporus sulphureus* AM498, *Fusicoccum amygdali* AM258 and *Spicaria divaricata* AM423 [12]. Incubation of DHEA with *Ulocladium chartarum* MRC 72584 produced seven DHEA derivatives, such as 3β-hydroxyandrost-5-en-7,17-dione, 3β,7β-dihydroxyandrost-5-en-17-one, 3β,7α-dihydroxyandrost-5-en-17-one, etc. [13]. A 7α,15α-dihydroxyl-DHEA product was reported by Li et al. using *Colletotrichum lini*

In this research, two out of twelve filamentous strains of fungi demonstrated their metabolic abilities for DHEA (Table 1, Figure S1). After the culture, extraction, and isolation, it could be identified that 7α-Hydroxy-DHEA was produced by *Gibberella sp.* CICC 2498 and 7β-Hydroxy-DHEA was produced by *Absidia coerulea* CICC 41050 (Figure 1). Previous literature had reported several optimizations for the production of 7α-hydroxy-DHEA. As far as we know, it is the first time that the 7β-Hydroxy-DHEA is obtained by *Absidia coerulea* CICC 41050 biotransformation. Thus, we focused on the optimization of the 7β-Hydroxy-DHEA in this study, and the optimized transformation rate of 7β-Hydroxy-DHEA is 69.61%. metabolic abilities for DHEA (Table 1, Figure S1). After the culture, extraction, and isolation, it could be identified that 7α-Hydroxy-DHEA was produced by *Gibberella sp*. CICC 2498 and 7β-Hydroxy-DHEA was produced by *Absidia coerulea* CICC 41050 (Figure 1). Previous literature had reported several optimizations for the production of 7α-hydroxy-DHEA. As far as we know, it is the first time that the 7β-Hydroxy-DHEA is obtained by *Absidia coerulea* CICC 41050 biotransformation. Thus, we focused on the optimization of the 7β-Hydroxy-DHEA in this study, and the optimized transformation rate of 7β-Hydroxy-DHEA is 69.61%.

**Table 1.** Ability of DHEA biotransformation by the tested microorganisms. **Table 1.** Ability of DHEA biotransformation by the tested microorganisms. **Microorganism Ability\* Microorganism Ability\***

*Catalysts* **2022**, *12*, x FOR PEER REVIEW 2 of 13


\* Ability of DHEA biotransformation: (+) able, (−) not able. \*Ability of DHEA biotransformation: (+) able, (-) not able.

**Figure 1***.* 7α- and 7β-hydroxylated DHEA obtained by *Gibberella* sp. CICC 2498 and *Absidia coerulea* CICC 41050. **Figure 1.** 7α- and 7β-hydroxylated DHEA obtained by *Gibberella sp.* CICC 2498 and *Absidia coerulea* CICC 41050.

#### **2. Results and Discussion 2. Results and Discussion**

#### *2.1. Whole-cell Biotransformation Results of DHEA 2.1. Whole-Cell Biotransformation Results of DHEA*

Thin layer chromatography (TLC) was used to identify whether *Gibberella sp*. CICC 2498 and *Absidia coerulea* CICC 41050 could transform dehydroepiandrosterone (DHEA). Figure 2 showed that the substrate control group (Group1, DHEA) has an ocher band, and the transformation groups (Group 4 and 6) have blue bands of different shades with good separation between bands, and almost no ocher substrate bands can be seen. The blue bands (products) are below the red band (DHEA), indicating that the product polarity is greater than DHEA. In addition to the major metabolite, some other products were also generated but could not be further identified due to their lower concentration. Thin layer chromatography (TLC) was used to identify whether *Gibberella sp.* CICC 2498 and *Absidia coerulea* CICC 41050 could transform dehydroepiandrosterone (DHEA). Figure 2 showed that the substrate control group (Group1, DHEA) has an ocher band, and the transformation groups (Group 4 and 6) have blue bands of different shades with good separation between bands, and almost no ocher substrate bands can be seen. The blue bands (products) are below the red band (DHEA), indicating that the product polarity is greater than DHEA. In addition to the major metabolite, some other products were also generated but could not be further identified due to their lower concentration.

## *2.2. HPLC Analysis of DHEA Transformed by Gibberella sp. CICC 2498 and Absidia Coerulea CICC 41050*

Figure 3A showed the transformation results of DHEA by *Absidia coerulea* CICC 41050. It can be seen from the comparison between the transformation group 4, and the substrate control group 3 that the substrate (DHEA) peak in the transformation group was significantly reduced. This indicated that DHEA was transformed by *Absidia coerulea* CICC 41050. By comparing the results of transformation group 4, strain control group 1, and cosolvent control group 3, the increased peak in transformation group 4 was most likely the metabolite of DHEA transformed by *Absidia coerulea* CICC 41050, rather than the substance produced by microbial growth and metabolism. The types of metabolites of

DHEA transformed by *Absidia coerulea* CICC 41050 were few, and the content of metabolite I was significant, which was conducive to the later separation and purification. The retention time of metabolite I was 8.588 min (Figure 3A). In brief, incubation of *Absidia coerulea* CICC 41050 with DHEA (1 g/L) resulted in selective accumulation of the metabolite I. *Catalysts* **2022**, *12*, x FOR PEER REVIEW 3 of 13

*Catalysts* **2022**, *12*, x FOR PEER REVIEW 3 of 13

**Figure 2.** TLC analysis of microbial transformation of DHEA. Group 1, substrate (DHEA); Group 2, cosolvent (acetone); Group 3, *Absidia coerulea* CICC 41050; Group 4, *Absidia coerulea* CICC 41050 + DHEA; Group 5, *Gibberella sp*. CICC 2498; Group 6, *Gibberella sp*. CICC 2498+ DHEA. Colored with a 10% sulfuric acid-ethanol. **Figure 2.** TLC analysis of microbial transformation of DHEA. Group 1, substrate (DHEA); Group 2, cosolvent (acetone); Group 3, *Absidia coerulea* CICC 41050; Group 4, *Absidia coerulea* CICC 41050 + DHEA; Group 5, *Gibberella sp.* CICC 2498; Group 6, *Gibberella sp.* CICC 2498 + DHEA. Colored with a 10% sulfuric acid-ethanol. 41050. It can be seen from the comparison between the transformation group 4, and the substrate control group 3 that the substrate (DHEA) peak in the transformation group was significantly reduced. This indicated that DHEA was transformed by *Absidia coerulea* CICC 41050. By comparing the results of transformation group 4, strain control group 1, and cosolvent control group 3, the increased peak in transformation group 4 was most

*2.2. HPLC Analysis of DHEA Transformed by Gibberella sp. CICC 2498 and Absidia Coerulea CICC 41050* Figure 3 A showed the transformation results of DHEA by *Absidia coerulea* CICC 41050. It can be seen from the comparison between the transformation group 4, and the Figure 3B showed that the transformation of DHEA by *Gibberella sp.* CICC 2498. The separation of the metabolites of DHEA transformed by *Gibberella sp.* CICC 2498 was good, which was conducive to the later separation and purification. The main metabolite is named metabolite II, and the retention time of metabolite II is 11.211 min (Figure 3B). likely the metabolite of DHEA transformed by *Absidia coerulea* CICC 41050, rather than the substance produced by microbial growth and metabolism. The types of metabolites of DHEA transformed by *Absidia coerulea* CICC 41050 were few, and the content of metabolite I was significant, which was conducive to the later separation and purification. The retention time of metabolite I was 8.588 min (Figure 3 A). In brief, incubation of *Absidia* 

#### substrate control group 3 that the substrate (DHEA) peak in the transformation group was significantly reduced. This indicated that DHEA was transformed by *Absidia coerulea 2.3. Isolation, Purification and Structural Identification of Metabolites coerulea* CICC 41050 with DHEA (1 g/L) resulted in selective accumulation of the metabo-

CICC 41050. By comparing the results of transformation group 4, strain control group 1, and cosolvent control group 3, the increased peak in transformation group 4 was most likely the metabolite of DHEA transformed by *Absidia coerulea* CICC 41050, rather than the substance produced by microbial growth and metabolism. The types of metabolites of DHEA transformed by *Absidia coerulea* CICC 41050 were few, and the content of metabo-The fermentation broth was further purified by semi-preparative HPLC. Figure 4showed that the retention times of metabolites I and II were 15.198 min and 17.698 min, respectively. Metabolite I and II obtained by semi-preparative HPLC were confirmed by HPLC (retention times were 8.588 min and 11.211 min, respectively). Finally, metabolites I and II can be isolated from fermentation broth with purities of 94.0% and 96.0%, respectively. lite I. Figure 3 B showed that the transformation of DHEA by *Gibberella sp* CICC 2498*.* The separation of the metabolites of DHEA transformed by *Gibberella sp* CICC 2498 was good, which was conducive to the later separation and purification. The main metabolite is named metabolite II, and the retention time of metabolite II is 11.211 min (Figure 3 B).

**Figure 3.** *Cont*.

*Catalysts* **2022**, *12*, x FOR PEER REVIEW 4 of 13

**Figure 3.** HPLC analysis of biotransformation of DHEA by *Absidia coerulea* CICC 41050 (**A**), and *Gibberella sp*. CICC 2498 (**B**). Group 1, strain; Group 2, cosolvent (acetone); Group 3, substrate (DHEA); Group 4, strain + substrate (DHEA). **Figure 3.** HPLC analysis of biotransformation of DHEA by *Absidia coerulea* CICC 41050 (**A**), and *Gibberella sp.* CICC 2498 (**B**). Group 1, strain; Group 2, cosolvent (acetone); Group 3, substrate (DHEA); Group 4, strain + substrate (DHEA). HPLC (retention times were 8.588 min and 11.211 min, respectively). Finally, metabolites I and II can be isolated from fermentation broth with purities of 94.0% and 96.0%, respectively.

respectively. Metabolite I and II obtained by semi-preparative HPLC were confirmed by

**Figure 4.** Semi-preparative HPLC separation diagram. (**A**), the semi-preparative separation of metabolite I from fermentation broth after incubation of *Absidia coerulea* CICC 41050 with DHEA (5 days, 1 g/L), metabolite I was identified by HPLC. (**B**), the semi-preparative separation of metabolite II from fermentation broth after incubation of *Gibberella sp.* CICC 2498 with DHEA (5 days, 1 g/L), metabolite II was identified by HPLC.

According to MS (ESI) m/z [M+H]<sup>+</sup> 305.1 and [M−H]<sup>+</sup> 303.2, the relative molecular weight of metabolite I is calculated to be 304. Compared with the relative molecular weight of 288 of the substrate DHEA (C<sup>19</sup> H<sup>28</sup> O2), an oxygen atom is added, and the molecular formula is C<sup>19</sup> H<sup>28</sup> O<sup>3</sup> (Figures S2 and S3).

According to MS (ESI) m/z [M+H]<sup>+</sup> 305.1 and [M−H]<sup>+</sup> 303.2, the relative molecular weight of metabolite II is calculated to be 304. Compared with the relative molecular weight of 288 of the substrate DHEA (C<sup>19</sup> H<sup>28</sup> O2), an oxygen atom is added, and the molecular formula is C<sup>19</sup> H<sup>28</sup> O<sup>3</sup> (Figures S4 and S5).

The position of the introduced hydroxyl group is analyzed according to the <sup>13</sup> C NMR and <sup>1</sup> H NMR spectral data (Figures S6–S9).

Metabolite I: 7β-Hydroxy-DHEA: <sup>1</sup> H-NMR (600 MHz, CDCl3) δH: 0.90 (3 H, s, 18-Me); 1.08 (3 H, s, 19-Me); 1.23–1.28 (1 H, m), 1.31 (1 H, d, *J* = 6.7 Hz, 6-H), 1.42–1.62 (7 H, m), 1.67–1.72 (1 H, m), 1.82–1.89 (4 H, m), 2.08–2.14 (1 H, m), 2.22–2.29 (2 H, m), 2.33–2.37 (1 H, m), 2.45–2.49 (1 H, m), 3.52–3.59 (1 H, m, 3α-H); 3.96 (1 H, dt, *J*<sup>1</sup> = 5.3 Hz, *J*<sup>2</sup> = 14.8 Hz, 7α-H); 5.31 (1 H, t, *J* = 1.8 Hz, 6-H). <sup>13</sup> C-NMR (151 MHz, CDCl3) δC: 13.5 (18-C), 19.1 (19-C), 20.3 (11-C), 24.1 (15-C), 31.2 (2-C), 31.4 (12-C), 35.9 (16-C), 36.6 (10-C), 36.8 (1-C), 40.4 (8-C), 41.6 (4-C), 47.7 (13-C), 48.2 (9-C), 51.1 (14-C), 71.2 (3-C), 72.8 (7-C), 125.4 (6-C), 143.7 (5-C), 221.1 (17-C).

Metabolite II: 7α-Hydroxy-DHEA: <sup>1</sup> H-NMR (600 MHz, CDCl3) δH: 0.89 (3 H, s, 18- Me), 1.02 (3 H, s, 19-Me), 1.10–1.15 (1 H, m), 1.26–1.31 (2 H, m), 1.50–1.61 (3 H, m), 1.66–1.73 (4 H, m), 1.78–1.91 (4 H, m), 2.09–2.19 (2 H, m), 2.28–2.33 (1 H, m), 2.35–2.39 (1 H, m), 2.45–2.50 (1 H, m), 3.55–3.61 (1 H, m, 3α-H), 3.98 (1 H, t, *J* = 4.7 Hz, 7β-H), 5.65 (1 H, d, *J* = 7.4 Hz, 6-H). <sup>13</sup> C-NMR (151 MHz, CDCl3) δC: 13.4 (18-C), 18.4 (19-C), 20.2 (11-C), 22.1 (15-C), 31.2 (2-C), 31.4 (12-C), 35.9 (16-C), 37.1 (10-C), 37.3 (1-C), 37.7 (8-C), 42.1 (4-C), 42.8 (13-C), 45.1 (9-C), 47.2 (14-C), 64.4 (7-C), 71.3 (3-C), 123.7 (6-C), 146.7 (5-C), 221.3 (17-C).The <sup>1</sup> H and <sup>13</sup> C NMR data of metabolites I and II are in agreement with those reported in the literature [10], which indicated that both 7 α/β hydroxylation DHEA were obtained (Figure 1) [10].
