3.8.3. Biotransformation Conditions Were Studied by Single-Factor Experiment and Orthogonal Experiment

On the basis of the best cosolvent and the best culture medium, the single-factor experiment of transformation conditions was conducted. Set inoculum at 4%, 6%, 8%, 10%, 12%, 14%, and 16% (*v*/*v*, 50 mL). Fill a 250 mL Erlenmeyer flask with 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, and 80 mL of transformation medium, respectively. The biotransformation times are 6 h, 12 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, and 168 h. Add 25 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, and 400 mg of DHEA to 50 mL of transformation medium, respectively. Inoculate volume, medium volume, biotransformation time, and concentration of substrate (DHEA) were investigated to explore the effect on the transformation rate.

The orthogonal experiment with four factors and three levels L<sup>9</sup> (3<sup>4</sup> ) was designed according to the inoculate volume, medium volume, biotransformation time, and concentration of substrate (DHEA) determined by the single-factor experiment.

#### *3.9. Statistical Analysis*

All the experiments were carried out in triplicate, and each presented value was the average of three independent experiments. Standard deviations (SD) were estimated using the following equation and shown as errors on the graphs:

$$\text{SD} = \sqrt{\frac{\sum \left| \mathbf{x} - \boldsymbol{\mu} \right|^2}{\mathbf{N}}}$$

where ∑ means "sum of", x is a value in the data set, µ is the mean of the data set, and N is the number of data points in the population. SPSS 20.0 software was used to conduct a *t*-test on the data to determine the statistical difference; *p* < 0.05 was significant (\*), *p* < 0.01 was extremely significant (\*\*).

#### **4. Conclusions**

The aim of this study was to evaluate 7α and 7β hydroxylation of dehydroepiandrosterone (DHEA) by *Gibberella sp.* CICC 2498 and *Absidia coerulea* CICC 41050 biotransformation. The biotransformation products were analyzed by HPLC. The retention time of the main product of DHEA transformation by *Absidia coerulea* CICC 41050 was 8.588 min. The retention time of the main product of DHEA transformation by *Gibberella sp.* CICC 2498 was 11.211 min. A semi-preparative HPLC method was successfully established to separate the biotransformation products of DHEA. The purity of the two metabolites was 94% and 96%, respectively. The isolated products were identified by NMR and MS, and the product of DHEA transformation by *Absidia coerulea* CICC 41050 was 7 β- OH-DHEA, and the product of DHEA transformation by *Gibberella sp.* CICC 2498 was 7α-OH-DHEA. Determined by single-factor experiment and subsequent orthogonal experiment, the optimized media composition to produce 7 β-OH-DHEA by *Absidia coerulea* CICC 41050 was consisted of 30 g/L sucrose, 20 g/L peptone, 10 g/L corn steep liquor, 2 g/L K<sup>2</sup> HPO4, 1.6 g/L KH<sup>2</sup> PO4, 0.5 g/L MgSO4, and 0.05 g/L FeSO<sup>4</sup> with pH 6.5. The transformation rate of 7β-OH-DHEA reached 50.48%. The optimal biotransformation conditions (DHEA 1 g/L, medium volume 60 mL, biotransformation time 48 h, and inoculum 10%) provided maximum production (69.61%) of 7β-OH-DHEA by *Absidia coerulea* CICC 41050. The transformation of the DHEA substrate by *Absidia coerulea* CICC 41050 was described for the first time. Meanwhile, the conversion period was shortened to 48 h.

Previous literature had reported that *Absidia griseolla* var. igachii could provide C6β, C7α/β and C14α hydroxylation on androst-4-ene-3,17-dione (4-AD) [4], and *Absidia coerulea* AM93 could generate C7α/β hydroxylation on androstenediol [11]. This work extended our knowledge of DHEA hydroxylation on C7 position to the *Absidia coerulea* CICC 41050. It seems that the *Absidia* species possesses the C7 hydroxylation potential. Further genomic and proteomic data mining are worthy fo exploration for the rest of the story.

Steroids are lipophilic compounds with a gonane skeleton and play an important role in higher organisms. Due to different hydroxylations of steroid molecules, they vary greatly in their mode of action [16]. Hydroxylation of dehydroepiandrosterone (DHEA) to positions 3,7, and15 is an essential step in the synthesis of many steroidal drugs [17]. However, low hydroxylation of DHEA production is a difficult issue that must be solved urgently in industry. At present, DHEA and other steroid substrates could be hydroxylated by cytochromes P450 [18]. However, cytochrome P450 is a membrane bound protein, that is not very easy to get in a purified form for extensive research. To overcome at least some of these drawbacks, whole-cell systems are the method of choice to accomplish hydroxylation of the DHEA. Thus, this strategy would provide a possible way to enhance the 7β-OH-DHEA yield in the pharmaceutical industry.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/catal13020272/s1, Figure S1. Morphology of *Absidia coerulea* and *Gibberella sp.* Figure S2. (ESI) m/z [M+H]<sup>+</sup> diagrams for the metabolite I. Figure S3. (ESI) m/z [M+H]<sup>−</sup> diagrams for the metabolite I. Figure S4. (ESI) m/z [M+H]<sup>+</sup> diagram for the metabolite II. Figure S5. (ESI) m/z [M+H]<sup>−</sup> diagram for the metabolite II. Figure S6. <sup>1</sup> H NMR diagram for the 7α-OH-DHEA. Figure S7. <sup>13</sup> C NMR diagram for the 7α-OH-DHEA. Figure S8. <sup>1</sup> H NMR diagram for the 7β-OH-DHEA. Figure S9. <sup>13</sup> C NMR diagram for the 7β-OH-DHEA. Figure S10. Standard curve of 7β-OH-DHEA by HPLC.

**Author Contributions:** M.S.: Data Curation, Investigation; R.F.: Investigation, Methodology; S.C. and X.J.: Data Curation; F.W.: Resource; W.X. (Weizhuo Xu) and W.X. (Wei Xu): Resources, Supervision, Writing-Review and Editing. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Data Availability Statement:** Data are available upon reasonable request.

**Conflicts of Interest:** The authors declare no conflict of interest.
