Production of 14α-Hydroxy Progesterone Using a Steroidal Hydroxylase from Cochliobolus lunatus Expressed in Escherichia coli

Abstract

Steroids with hydroxylation at C14 are drawing increased attention because of their diverse biological activities and applications. P-450_lun from Cochliobolus lunatus is the first fungal cytochrome P450 reported to have 14α-hydroxylase activity. Studies have shown that P-450_lun catalyzes the hydroxylation of progesterone (PROG) at C14α with low regiospecificity and activity. To improve its regiospecificity and activity for PROG, truncated forms of P-450_lun and its cognate redox partner CPR_lun were functionally co-expressed in Escherichia coli. Then, a semi-rational protein engineering approach was applied to P-450_lun, resulting in a double-site mutant E109A/F297W with enhanced 14α-position selectivity for PROG compared with the wild-type P-450_lun (97% vs. 28%). Protein structure analysis revealed that the F297W substitution can hinder the binding pose for 11β-hydroxylation product formation. Finally, whole-cell catalysis was optimized, and the final titer of 14α-OH-PROG reached 16.0 mg/L. This is the first report where a fungal 14α-hydroxylase was functionally expressed in Escherichia coli. The steroid hydroxylation system obtained in this study can serve as a basis for the synthesis of 14α-hydroxylated PROG and the rapid evolution of eukaryotic cytochrome P-450_lun.

1. Introduction

Steroids, pervasive terpene lipids in nature, have long served as therapeutic agents for a range of clinical diseases, including rheumatologic, autoimmune, and inflammatory disorders [1,2]. The diverse physiological and pharmacological activities of steroids result from the strategic introduction of various functional groups onto the rigid gonane ring [3,4]. A pivotal modification is the hydroxylation of the steroid backbone, a process that heightens the polarity of hydrophobic steroid molecules, thereby influencing their toxicity, cell membrane penetration ability, and the biological effects of steroid drugs ultimately [5]. Of particular interest are 14-position hydroxylated steroids. 14α-OH steroids exhibit specific biological activities, demonstrating potential antigonadotrophic and anticancer properties [6,7,8]. In contrast, 14β-OH substituent is normally found in cardiotonic steroids, which are commonly used in treating congestive heart failure because of their cardiac effects, particularly the positive transducer effect [9,10,11].

Two approaches are currently used to incorporate 14-OH groups into steroids: chemical synthesis and biocatalysis. Nevertheless, the chemical synthesis route for steroid hydroxylation is beset by its complexity, low yields, and environmental unfriendliness [12,13]. In contrast, biocatalysis is gaining prominence because of its high selectivity and atom utilization efficiency [14]. Diverse microorganisms, including prokaryotes and fungi, can efficiently catalyze the production of steroids containing 14α-OH [15,16]. Conversely, only certain plants and amphibians can yield 14β-hydroxylated sterols in small quantities [17]. Fortunately, 14β-OH steroids can be obtained using the facile conversion of 14α-OH counterparts through chemical methods, enhancing the significance of investigating the biocatalytic synthesis of 14α-OH steroids.

Although many species can produce steroids with 14α-OH, research on the identification and characterization of these hydroxylation genes has been few reported. To our knowledge, 14α-hydroxylases have been obtained from Cochliobolus lunatus (anamorph Curvularia lunatus) [15,18,19], Bipolaris sp. [20], Thamnidum elegans [21], Crocus sativus, and Bufo toadstool [17]. In contrast, the 14β-hydroxylases remain elusive. Although Crocus sativus and Bufo toadstool can produce 14β-hydroxylated steroids, the results of transcriptome data analysis and P450 gene screening experiments only proved the existence of 14α hydroxylation genes [17]. This fact supports the hypothesis that nature exhibits a preference for generating 14α-OH, subsequently undergoing conformational conversion to 14β-OH through processes such as dehydration and hydration.

Two 14α-hydroxylases have been obtained from Cochliobolus lunatus: P-450_lun (from C. lunatus ATCCTM12017, equivalent to CYP103168 from C. lunatus CECT 2130) [15,19] and CYP14A (from C. lunatus CGMCC 3.3589 and C. lunatus JTU 2.406) [18]. The corresponding electron transport protein, CPR_lun, was identified using transcriptome analysis and genetic screening. P-450_lun and CYP14A, with approximately 82% sequence identity, all have low C14-hydroxylation specificity for C17-substituted steroids, such as progesterone (PROG), when co-expressed with CPR_lun in Saccharomyces cerevisiae [15,18]. Specifically, P-450_lun converts PROG into a mixture of products with 14α-OH and 11β-OH at a 1:1 ratio [18]. PROG is the fundamental steroidal core for synthesizing C21 steroid derivatives of pharmaceutical significance. 14-OH-PROG is a crucial intermediate in the chemical and biological synthesis of active steroids, such as cardenolides [10,22]. Therefore, we performed engineering of P-450_lun to enhance its C14 regioselective hydroxylation of PROG in this study.

Yeasts are the primary choice for the heterologous expression of membrane-bound eukaryotic cytochrome P450 (CYP) because the composition and structure of the endoplasmic reticulum membrane of yeast cells allow the anchoring of membrane-bound proteins [23,24]. The reported 14α-hydroxylases such as CYP11411, CYP44476, P-450_lun and CYP14A all employed S. cerevisiae as the host for investigating the transformation of steroid substrates [17]. However, this expression system also comes with some drawbacks, including the unexpected formation of by-products, altered protein glycosylation, challenges in predicting plasma membrane permeability for specific compounds, and unpredictable targeting of recombinant proteins [25]. Moreover, the burdensome operation and slow growth rate of yeasts limit the size and screening speed of mutant libraries for the directed evolution of enzymes with low activity and selectivity [23,26]. Here, we propose using E. coli as an expression system because of its successful use in the heterologous expression of mammalian or fungal CYPs and operational convenience during enzyme directed evolution.

In this study, we report the functionally heterologous co-expression of N-terminally modified P-450_lun and CPR_lun in E. coli. We also engineered the truncated P-450_lun, resulting in a double-site mutant (E81A/F269W) with enhanced regioselectivity and activity for the C14α hydroxylation of PROG. Finally, the whole-cell catalysis was optimized, and the production of 14α-OH-PROG was further increased. This is the first report of producing 14α-OH-PROG in E. coli with a fungal 14α-hydroxylase.

2. Results and Discussion

2.1. Identification of a Functional Truncated Form of P-450_lun (ΔP-450_lun)

The P-450_lun and CPR_lun were chemically synthesized and codon-optimized for expression in E. coli. As a redox partner, Flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) containing CPR_lun mediates electron transfer and is essential for P-450_lun catalytic activity [14,27,28]. Class II P450 P-450_lun and its partners CPR_lun can be typically co-expressed in a vector system using one or two “promoter-ORF(s)-terminator“ expression cassettes [29]. With the single cassette approach, P-450_lun and CPR_lun can be spaced apart by a sequence containing the ribosome entry/binding site, and two distinct proteins will be expressed. The expression of the P-450_lun and CPR_lun will be heterogeneous, with the upstream gene displaying higher expression [30,31]. In addition, P-450_lun and CPR_lun can also be linked by a nucleotide sequence (can be translated into a flexible or rigid linker) and expressed as a fusion protein. However, the nucleotide length and amino acid composition can severely affect the activity of the fusion protein [32,33,34]. To avoid variability in expression efficiency and the selection of complex linkers, plasmid pRSFDuet with two expression cassettes was applied to express P-450_lun and CPR_lun.

As membrane-bound proteins, eukaryotic P-450_lun and its cognate partner CPR_lun possess N-terminal hydrophobic domains (residues 2–29 and 2–31, respectively) that anchor to the plasma membrane. However, E. coli lacks membrane-bound organelles, potentially leading to inclusion body formation or low protein expression due to its poor recognition ability of unique signaling sequences when expressing membrane-bound proteins [26,35]. Fortunately, these limitations can be addressed by N-terminal modifications, including amino acid substitutions and truncations of hydrophilic sequences [26,36,37].

Firstly, we produced a recombinant E. coli BL21 (DE3) strain containing a modified P-450_lun (by replacing the second codon with alanine or substituting the N-terminal region with the hydrophilic sequences MAKKTSS or MALLLAVFL) [38,39,40] and ΔCPR_lun (containing a truncated transmembrane region), while no detectable by-products of PROG were produced during whole-cell biotransformation. Then, transmembrane helices truncated P-450_lun and CPR_lun (ΔP-450_lun and ΔCPR_lun, respectively) were introduced into E. coli BL21 (DE3), and detected products were generated (Figure 1A). High-performance liquid chromatography (HPLC) analyses revealed that PROG was converted into two major compounds and several uncharacterized hydroxylated by-products. Isolation, purification, and structural analysis using nuclear magnetic resonance (NMR) of the main compounds showed that they were 14α-OH-PROG and 11β-OH-PROG, respectively (with ratio of ~2:1) (Figure 1B; see NMR Data and spectrums in the Supporting Information). While the molar ratio of 14α-OH-PROG/11β-OH-PROG was different from that obtained from 1:1 in S. Cerevisiae, this could be the result of different culture conditions such as the pH, incubation time, and substrate dose [18]. Therefore, the N-terminally modified P-450_lun and CPR_lun from C. lunatus were functionally expressed in E. coli. However, the activity and regioselectivity of ΔP-450_lun for C14α position were still low (approximately 21% yield at a concentration of 3.5 mg/L).

2.2. Engineering ΔP-450_lun to Improve Its Regiospecificity and Catalytic Activity

In pursuit of refining the regiospecificity of ΔP-450_lun at C-14α of PROG, a hybrid approach involving a semi-rational design and directed evolution was employed. Given that the crystal structure of P-450_lun is not available, a three-dimensional model was constructed using SWISS-MODEL [41] based on the crystal structure of CYP3A4 (PDB ID 5VCD, identity: 28%; similarity: 46%) [42]. Although the sequence identity is not high, they possess a comparable overall fold and a conserved heme-binding core structure [43]. To enhance model accuracy, GalaxyRefine [44,45] and Gromacs [46] were employed for side-chain repacking and global structural relaxation, resulting in an optimized model with an initial Z-score [47] of −8.25 (Figure S1) and more than 91.0% of residues located in the most favored regions of the Ramachandran plot [48,49] (Figure S2).

Molecular docking of PROG into the presumed structure of ΔP-450_lun was conducted to identify residues potentially influencing selectivity. The analysis of docking poses with the lowest binding free energy revealed two distinct binding modes (pose I and II), corresponding to 14α- and 11β-hydroxylation, respectively (Figure 2A). The lower free energy associated with mode II suggests a preference for 11β-OH. In this mode, the four-ring structure of PROG assumed a slightly oblique orientation to the heme ring, and its β-side faced the heme group. The C17 acetyl group of PROG was close to the I-helix and formed hydrogen bonds with His121 and Ser294. While in pose I, the ligand rotated its plane by approximately 180°, so the α-side faced the heme group. This binding mode was stabilized by a hydrogen bond between the C20-keto group of PROG and Thr369 and the hydrophobic interaction of the gonane ring structure with residues Phe297, Ala298, and Met365 (Figure 2B).

Molecular docking also showed the presence of eight residues (P108, E109, L122, S294. F297, T364, S367, and F368) within 5 Å of the ligands in both binding modes (Figure 2C). P108, E109, and L122 were located in substrate recognition site 1 (SRS-1). Residues S294 and F297 were located in the I-helix near the conserved sequence motif AGXXT (325–329), which was involved in selectivity. Residues T364, S367, and F368 were located at positions +5, +8, and +9 in the highly conserved EXXR motif in SRS-5. Given their proximity to the heme group, these three residues had a high potential for substrate binding and control of regioselectivity [50]. We hypothesized that these eight residues contributed to the selectivity or activity of ΔP-450_lun and that replacing these amino acids could improve the activity or selectivity of hydroxylation at C14α.

Alanine scanning was performed across the eight residues to identify the key residues with a remarkable substantial effect on region-selectivity or activity. The substitutions E109A and F297A significantly improved the selectivity from 28% in the wild type to 57% and 73%, respectively. E109A also increased the PROG conversion from 75% to 83%. Although F297A decreased the conversion to 8%, only a trace amount of by-products was detected (Figure 2D). Motivated by these findings, E109 and F297 were selected for further analyses. We performed site saturation mutagenesis in these two residues using NNK codons in 24-well plates. To achieve a 95% coverage of all possible library variants, approximately 60 variants were screened in each single-site library. HPLC results showed that all screened E109 substitutions displayed similar or decreased PROG conversion and selectivity compared to E109A. Conversely, F297W substitution had the highest PROG conversion (41%) and selectivity (93%). To further enhance the regioselectivity and activity of ΔP-450_lun, we combined these two substitutions to obtain a double mutant E109A/F297W, which had higher selectivity (approximately 97%) and catalytic activity (Figure 3A,B;

Table 1. Conversion rate of PROG hydroxylation and product distribution of P-450_lun and mutants from the bioconversion assay.

Mutants	Conversion (%) 1	Selectivity (%)
		14α	11β	Others
WT	75	28	52	20
E109A	84	57	27	16
F297A	9	69	26	6
F297W	41	93	6	1
E109A/F297W	71	97	2	1

¹ Reaction conditions: recombinant E. coli cells were suspended in phosphate buffer (pH 7.4, 50 mM) containing 50 μM PROG; reactions were conducted at 28 °C, 250 rpm for 8 h.

).

2.3. Computational Analysis of ΔP-450_lun and Its Variants

Structural and computational analyses were performed to understand the molecular basis for the selectivity and activity difference between ΔP-450_lun and its mutants for PROG. The binding modes of PROG in ΔP-450_lun were identical to PROG-bound CYP260A1, which can catalyze hydroxylation of PROG at C1α or C17α [51]. Molecular docking revealed that the introduction of the larger indole group in F297W reduced the binding pocket volume of ΔP-450_lun by 168.5 ų and brought in a significant steric hindrance against the substrate in pose I (Figure S7 and Figure 4C). The shift in selectivity might arise from the destabilization of the alternative binding mode due to the steric hindrance and hydrogen bond disruption caused by F297W.

Access tunnels facilitate the movement of ligands between the active site and solvent environment, particularly in enzymes with a buried active site [52,53]. Using the Caver3.0 server [54], we identified two distinctive access tunnels in ΔP-450_lun with features shared by cytochrome P450s [55] (Figure 4A). Tunnel 1, located between the F’ helix, B–C loop, and β1 sheet, behaved as a 2a-like channel. In contrast, Tunnel 2, regressed through the B–C loop, could be merged and behaved as a 2e-like channel. Both channels were located in the P450 region involved in substrate specificity.

Detailed analysis revealed that the trajectories of these two channels converged at a node near the heme moiety. Residue E109, situated at the intersection of these two channels, might act as a gate controlling the spatial size of the node. E109 also could form an ionic bond with K489, serving as a critical bottleneck in access Tunnel 1. Therefore, substituting E109 with alanine widened both channels (bottleneck radius increased from 1.36 Å to 1.94 Å for Tunnel 1, from 1.23 Å to 1.50 Å for Tunnel 2) (Figure 4A,B). This modification might enhance the channel’s suitability for substrate and water transport, consequently elevating the overall activity of ΔP-450_lun. Moreover, changes in the size, physicochemical properties, or dynamics of the access tunnels of an enzyme can affect its regioselectivity and catalytic activity. Cheng et al. found that a single residue mutation in the access tunnel of PpNHase or CtNHase could invert its regioselectivity toward aliphatic α,ω-dinitriles [56]. Meng et al. replaced the bottleneck residue F79 in the access tunnel of P450_BsβHI with relatively small amino acid alanine resulting the change of hydroxylation products’ distribution toward myristic acid and pentadecanoic acid [57]. So, substituting residue E109, which was located at the end of the access tunnel and the entrance to the binding pocket, with Alanine might contribute to the difference in the size and shape of the substrate access tunnel, and thus resulted in the diversity of reaction regioselectivity [56].

2.4. Optimizing the Conditions of Whole-Cell Biocatalyst

To establish the optimal conditions for whole-cell biocatalysis, various parameters of the biotransformation process, including temperature, pH, and the additives, were systematically evaluated. A preliminary analysis showed the highest yield of 14α-OH-PROG obtained by ΔP-450_lunE109A/F297W. Therefore, we used the E. coli strain containing pRSFDuet_ΔP-450_lunE109A/F297W_ΔCPR_lun to analyze the effects of different reaction conditions on 14α-OH-PROG yield. Results showed that the optimal reaction temperature and pH were 30 °C and 7.4 (50 mM phosphate buffer), respectively (Figure 5A,B). Subsequent reaction variables were all evaluated under these conditions.

Since P450s are heme-containing enzymes, enhancing the intracellular concentrations of heme can potentially enhance the activity of whole-cell biocatalysts. We investigated the impact of adding different heme precursors, such as hemin, 5-ALA, and FeSO₄·7H₂O during the expression of P-450_lun [58]. Given the limited import of heme into the BL21 (DE3) strain [59], adding heme did not significantly increase 14α-OH-PROG yield. However, supplementation with 5-ALA demonstrated an improvement, achieving the highest yield of 12.7 mg/L when 0.5 mM ALA was added (Figure 5C). In contrast, only trace amount of products were observed in the absence of 5-ALA during the induction of P-450_lun, indicating that E. coli relied mainly on exogenous 5-ALA for the biosynthesis of heme. Furthermore, the inclusion of iron might also contribute to the biosynthesis of heme. Adding 0.5 mM ALA to the medium and varying final concentrations of FeSO₄·7H₂O (5, 10, 20, 30, and 40 mg/L) were introduced, while no noticeable change in 14α-OH-PROG yield was observed.

As a member of class II P450s, P-450_lun requires two electrons from NADPH delivered by CPR_lun to increase its catalytic activity [14,27]. Thus, the nicotinamide cofactor may also be a limitation in current whole-cell systems. Various concentrations of NADPH (1, 1.5, or 2 equivalents) were introduced to the biotransformation system, and the production of 14α-OH-PROG was assessed at different time points (Figure 5D). The results were compared with the control reaction (without supplemented NADPH). The findings revealed that additional NADPH increased the initial rates and conversions, suggesting that the regeneration of the cofactor in cells was insufficient to sustain the catalytic reaction at the achieved rates and with the current coupling efficiency. Growth was interrupted when 1.5 equivalents of NADPH were added to the reaction system, possibly due to the low protein expression levels of P-450_lun or CPR_lun. These results indicated that introducing the enzymatic regeneration systems (such as GDH/glucose) in the host as a substitute for nicotinamide cofactors may represent an economical approach to produce 14α-OH-PROG in this system. Following the analysis and optimization of reaction conditions, the highest conversion of PROG was achieved at 99%, corresponding to a 14α-OH-PROG yield of 16.0 mg/L.

Thereafter, the effect of substrate concentration (ranging from 25 to 200 μM) was investigated using those optimized conditions mentioned above. The yield of 14α-OH-PROG was estimated after 8 h of transformation in the Erlenmeyer flasks. Results suggested that the maximum titer of 14α-OH-PROG was found at 50 μM substrate (Figure 6). At higher substrate concentrations (>50 μM), the accumulation of 14α-OH-PROG stopped or even showed a downward trend, which may result from the limited protein expression levels, poor substrates/products transport capacity of E. coli or substrates/products inhibition.

3. Materials and Methods

3.1. Strains and Reagents

E. coli DH5α was used for DNA cloning and plasmid construction. E. coli BL21 (DE3) served as hosts for whole-cell biotransformation. Yeast extract and tryptone were purchased from Qxoid (Basingstoke, UK). Q5 Hot Start High-Fidelity DNA polymerase and restriction endonucleases were obtained from Thermo Scientific (Waltham, MA, USA). The plasmid purification kit was sourced from Omega Laboratories (Cleveland, OH, USA). Oligonucleotide synthesis and sequence analysis were performed by Novogene (Tianjin, China). PROG, 5-ALA and isopropy1 β-D-1-thiogalactopyranoside (IPTG) were purchased from Energy Chemical (Shanghai, China). Other chemicals were obtained from Genview (Beijing, China) with the highest available commercial grade.

3.2. Plasmid Construction and Protein Expression

The wild-type gene P-450_lun (GenBank accession numbers: MN061487) and CPR_lun (GenBank accession numbers: MN061485) were chemically synthesized and optimized considering the codon preferences of E. coli by Novogene (Tianjin, China). The expression construct for the normal or modified P-450_lun and CPR_lun were designed in pRSFDuet, respectively, using primers presented in Table S1. Sequences confirmation was performed by sequencing in Novegene (Tianjin, China). The recombinant cells transformed with expression vectors (pRSFDuet) harboring the normal or modified P-450_lun gene and CPR_lun gene were cultivated at 37 °C in Luria–Bertani (LB) medium with 50 μg/mL kanamycin. In total, 0.5 mM IPTG and 0.5 mM 5-ALA were added When cells’ OD₆₀₀ reached 0.6, and they continued to cultivate for 16 h at 28 °C. Then, cells were collected using centrifugation (10,000× g, 10 min, 4 °C) and resuspended (in 50 mM pH 7.4 potassium phosphate buffer) for the whole-cell biotransformation.

3.3. Whole-Cell Conversion of Progesterone Using E. coli in the Erlenmeyer flask

In total, 10 mL potassium phosphate buffer (50 mM, pH 7.4) with 50 g cell wet weight/L (cww/L) recombinant E. coli and 50 μM PROG was shaken at 250 rpm and 30 °C for 8 h. In total, 2 mL samples were taken and then extracted with equal volume ethyl acetate, dried with air-blowing, re-dissolved in 200 μL chromatography methanol, and filtered through a 0.2 μm filter. Product formation was analyzed using reverse-phase HPLC to calculate the final conversions. The reaction mixture was separated by a Thermo Hypersil Gold C18 Column (4.6 × 250 mm, 5 μm) using solvent A (ddH₂O with 0.1% v/v formic acid) and solvent B (acetonitrile). The flow rate was 1 mL/min. The gradient was set as 0–15 min 30% B—95% B, 15–20 min 95% B, 20–21 min 95% B—30% B, 25 min 30% B.

3.4. Engineering of P-450_lun for Improved Regioselectivity and Activity

Single-site mutation was introduced in P-450_lun with PCR using designed primers (Table S1) and Q5 hot-start high-fidelity DNA polymerase. The PCR reaction mixture (25 μL) was prepared, which included primers (1.25 μL, 10 μM), template plasmid (1 μL, 50 ng/μL), Q5 hot-start high-fidelity DNA polymerase (12.5 μL), and dd H₂O (9 μL). The PCR product was digested by restriction enzyme Nhel/BspTI for 1.5 h at 37 °C and then connected into corresponding restriction sites of pRSFDuet_ΔCPR_lun using T4 DNA ligase (Thermo Scientific, Waltham, MA, USA). The final ligated products were transformed into E. coli BL21 (DE3) via electroporation and plated on LB agar plates containing 50 μg/mL kanamycin. Mutant colonies were inoculated in separate wells of 24-deep-well plates (0.2 mL, LB medium with 50 μg/mL kanamycin per well) and incubated at 300 rpm and 37 °C for 8 h. Then, 1.0 mL TB medium containing 50 μg/mL kanamycin, 0.5 mM IPTG, and 0.5 M 5-ALA was added to each well, and the plates continued to be incubated at 300 rpm and 28 °C for another 16 h. The cells were harvested by centrifuging the plates at 4000× g for 20 min, washed with 0.2 mL potassium phosphate buffer (50 mM, pH 7.4), and centrifuged again. Cells in each well were then resuspended in 0.5 mL of 50 mM pH 7.4 potassium phosphate buffer containing 50 μM PROG. The plates were shaken at 300 rpm and 30 °C for 12 h. Each sample was extracted with 500 μL ethyl acetate, dried with air-blowing, re-dissolved in 100 μL chromatography methanol, and product formation was analyzed using reverse-phase HPLC (described above). Desired mutations were confirmed using DNA sequencing.

3.5. Structural Modeling Analysis, Molecular Docking and Tunnel Analysis

Swiss-Model server (https://swissmodel.expasy.org/, accessed on 10 February 2023) was used to build a 3D structure model of ΔP-450_lun and its variants. To enhance the geometry and energy of the overall predicted model, energy minimization was conducted using the steepest gradient minimization algorithm, employing specific GROMACS routines. This involved performing 1500 minimization steps in a vacuum environment, with a maximum force convergence threshold set at 1.0 kJ/mol/nm. Additionally, a cut-off range of 1.4 nm was applied for both van der Waals and Coulomb interactions. Then, the optimized protein structure was refined using the GalaxyWeb server (https://galaxy.seoklab.org/index.html, accessed on 11 February 2023). The quality of the final generated model was assessed using a Ramachandran plot (SAVES server, https://saves.mbi.ucla.edu/, accessed on 12 February 2023) and Z-score (ProSA web, https://prosa.services.came.sbg.ac.at/prosa.php, accessed on 12 February 2023). Structural images were produced using PyMOL and Discovery studio visualizer 2019.

The molecular structure of the ligand PROG was constructed and optimized using ChemDraw. A grid box with dimensions of 5 Å surrounding the active site was generated, centered on the heme iron of P-450_lun or its variants. Molecular docking simulations were conducted using Autodock 4.2 and subsequently visualized using PyMOL. Each variant underwent 20 docking runs, and the resulting poses were clustered based on a Root Mean Square Deviation (RMSD) cutoff of 5 Å, employing default parameters.

Tunnel analysis was performed using Caver Analyst 2.0 (https://www.caver.cz/index.php?sid=121, accessed on 17 May 2023), with the following parameters: a probe radius of 1.4 Å, shell depth of 4 Å, shell radius of 3 Å, clustering threshold of 3.5, and the starting point set at the Fe atom of the heme co-factor. Following the tunnel calculations, the bottleneck radius and tunnel-lining residues for each tunnel were extracted from the tunnel statistics and residue graph functions.

3.6. Optimization of the Whole-Cell Biocatalytic Process

Optimization of bio-transformations was carried out with varying different parameters of the reactions (pH, temperature, and the additives). The whole-cell conversion process of PROG using E. coli containing pRSFDuet_ΔP-450_lunE109A/F297W_ΔCPR_lun in the Erlenmeyer flask is similar to that mentioned above. The pH or reaction temperature varied according to different optimization purposes. Additives heme, 5-ALA or FeSO₄·7H₂O were added along with IPTG at the start of protein induction. Additive NADPH was added at the beginning of whole-cell conversion. Product formation was analyzed using reverse-phase HPLC to calculate the final yield. After all parameters were determined, the cell was induced with 0.5 M IPTG along with 0.5 mM 5-ALA. The final biocatalytic reaction was performed in 10 mL 50 mM pH 7.4 potassium phosphate buffer containing 50 g cww/L recombinant E. coli, 50 μM PROG and 75 μM NADPH at 30 °C for 16 h.

4. Conclusions

This study described the successful expression and development of the C. lunatus derived steroid 14α-hydroxylase P-450_lun in the E. coli expression system. The N-terminal hydrophobic region truncated strategy was applied to express the eukaryotic membrane protein P-450_lun and its cognate partner CPR_lun in E. coli. Using a semi-rational design and directed evolution, a ΔP-450_lun variant E109A/F297W with ~97% regioselectivity to the C14α position of PROG was obtained. Then, whole-cell catalyzed reaction conditions were optimized, and the final yield of 14α-OH-PROG was approximately 16.0 mg/L. The E. coli system obtained in this study can serve as a basis to produce 14α-OH-PROG and a rapid evolution platform to eukaryotic cytochrome P-450_lun. Nonetheless, the yield and productivity of this system are still low. Thus, research is underway to construct a NADPH regeneration system in host cells, promote the transportation of hydrophobic substrates and products, and further increase the catalytic activity of P-450_lun.