Divergent evolution of fungal P450 monooxygenase unlocks simultaneous access to C12β and C15α oxyfunctionalization of steroids

Domain: physics
Published: 2025-11-28
Editorial outcome: major_revision

Abstract
Steroidal C12β/15α-hydroxylation are pivotal in synthesizing steroid drugs but remain challenging via chemical and biological methods. To address this, structure-guided divergent evolution is applied to the fungal P450 monooxygenase CYP68J5_fg. Two optimized variants, W12M5 (F107S/Q112R/N295T/V299T/R368K) and W15M4 (Q112C/D126V/V299L/A362M) are created, achieving high selectivity (97.7% for C12β- and 99.6% for C15α-hydroxylation of progesterone) alongside enhanced catalytic efficiency, effectively overcoming the classic activity-selectivity trade-off. Molecular dynamics simulations reveal that key mutations reorient the substrate by reshaping the binding pocket’s polarity and hydrogen-bonding network, enabling hydroxylation at distinct positions. High-density fermentation with engineered Pichia pastoris yields titers of 4.6 g/L 12β-OH progesterone, 10.9 g/L 15α-OH progesterone and 14.1 g/L 15α-OH androstenedione. These products serve as key intermediates for streamlined synthesis of C12-/C15-functionalized steroids such as drospirenone and C-nor-D-homo derivatives. Collectively, this study demonstrates the successful divergent evolution of a fungal P450, a strategy which has so far not been reported in the literature, highlights its broad applicability for the scalable synthesis of complex bioactive molecules.

Introduction
Steroids represent a therapeutically essential class of molecules, accounting for over 17% of FDA-approved drugs, with an annual production exceeding 1 million tons1–3. Selective C-H oxidation is pivotal for synthesizing bioactive derivatives, particularly at the C12 and C15 positions, which are critical for a number of pharmaceuticals4,5. For instance, C12α-hydroxylated deoxycholic acid is FDA-approved for fat reduction6, while 12β-hydroxy steroids serve as precursors for bioactive C-nor-D-homo steroids like cyclopamine and veratramine7,8. Similarly, C15α-hydroxylated steroids are as key intermediates for contraceptives (e.g., estetrol/drospirenone)9,10 and anti-inflammatory drugs11–13 (Fig. 1a, b). Despite their pharmaceutical value, these hydroxylated steroids are naturally scarce, necessitating synthetic access. Moreover, their near-identical C(sp3)-H bonds pose significant regio- and stereoselective challenges1. Existing chemical methods for C12β and C15α hydroxylation often require pre-functionalization, harsh conditions, and multi-step routes, leading to low efficiency and yield (Fig. 1c, d)14–17. This highlights the urgent need for direct and selective C12/C15-hydroxylation strategies to streamline steroid functionalization.

On the other hand, the biocatalytic strategies employing cytochrome P450s (CYPs) for direct steroid hydroxylation in a highly selective fashion, has gained more and more attention due to its high catalytic efficiency and atom economy4,18–20. For example, microbial P450-catalyzed C11α-hydroxylation of progesterone (1, PG) streamlined cortisone production by eliminating 10–12 steps from the conventional chemical synthesis route, while reducing the cost from $200 to $1 per gram21. However, the direct C12β/C15α hydroxylation of steroids by microbial (bacterial/fungal) P450s remains underexplored. For bacterial P450s, CYP107D1 (OleP)22 from Streptomyces antibioticus exhibited only 5–10% conversion in the C12β-hydroxylation of 100 μM testosterone (5, TS). The engineered P450 BM323 from Bacillus megaterium showed 82% conversion for 2 mM 5, but with C15α-selectivity of only 73%. In contrast, fungal P450 enzymes demonstrate superior suitability for industrial-scale production, as exemplified in C11 steroid hydroxylation with fungi whole cell as catalyst21,24. Nevertheless, in terms of C12β/C15α hydroxylation, the reported C15 hydroxylases CYP5150A P2 from Thanatephorus cucumeris expressed in Mycolicibacterium neoaurum and CYP-N2 from Nigrospora sphaerica expressed in Pichia pastoris (P. pastoris) exhibited only 70% and 58.8% selectivity toward 200 mg/L and 500 mg/L substrate 1, respectively25,26. Our prior work identified an unique fungal P450 CYP68J5_fg from Fusarium graminearum with both C12β and C15α selectivity, but it exhibited poor selectivity, yielding a product ratio of ~3:5:2 (C12β, C15α- mono-hydroxylated and C15α-/C12β- dihydroxylated)6.

Enzyme directed evolution is an effective approach to enhance their activity and/or selectivity20,27–32. But current fungal CYP engineering efforts remain sparse since most fungal cytochrome P450s are typically endoplasmic reticulum (ER) localized with limited mechanistic insights29,33–36, which hinder their heterologous expression as well as further investigations on improving their activity, selectivity and practical applications37. Although semi-rational design improved activity of Absidia orchidis CYP5311B2 (11β-hydroxylase), yielding a 3-fold increase in hydrocortisone productivity over the wild type (WT), while maintaining unchanged stereoselectivity36. The rational engineering of Cochliobolus lunatus CYP14A improved C-14 specificity and activity towards various steroids (90–95% site selectivity, 72–78% conversion rate), but mechanistic insights and broader regioselectivity modulation require exploration29. Additionally, no studies demonstrate the divergent evolution of a fungal P450 to achieve two variants with distinct selectivity. Therefore, more research work need to be done in this aspect, especially for P450-catalyzed direct steroid C12β/C15α hydroxylation.

Herein, our previously identified CYP68J5_fg was selected as the template, the combined structure-guided site-saturation mutagenesis (SSM) and focused rational iterative site-specific mutagenesis (FRISM) were employed to achieve the divergent evolution of CYP68J5_fg for simultaneous access to C12β/15α-hydroxylated steroids in highly selective manner (Fig. 1e). Computational analysis was performed to reveal the key residues that are responsible for the improved activity and selectivity. The substrate scope was explored for both C12β and 15α-selective variants to document their generality. High-density fermentation of recombinant P. pastoris containing the best variants was employed to produce the C12β/15α-hydroxylated steroids to demonstrate their practical applications. Finally, the chemoenzymatic process was proposed based on the isolated C12-/C15-functionalized steroids for streamlined synthesis of steroid drugs such as drospirenone and C-nor-D-homo derivatives, demonstrating a tailored fungal P450 engineering strategy broadly applicable to complex bioactive molecule synthesis.

Results
Mutational scanning on the key residues of CYP68J5_fg
CYP68J5_fg from F. graminearum was heterologously expressed in S. cerevisiae, but unfortunately resulted in poor selectivity and catalytic activity for the hydroxylation of 1, yielding a product ratio of ~3:5:2 (C12β, C15α- and C15α-/C12β- mono and dihydroxylated) (Fig. 2a)6. Due to CYP68J5_fg’s membrane-bound nature and the absence of high-identity structural templates in the Protein Data Bank (PDB), its tertiary structure was predicted using AlphaFold2 and subsequently docked with substrate 1. Substrate binding analysis revealed 15 key residues (F107, A111, Q112, S115, F125, D126, A127, L131, F212, N295, V299, A362, L363, R368, I488) located within 4 Å of the two distinct binding cavities in the C12- or C15-docked conformations of 1 (Fig. 2b). These residues, which encapsulate the 1 molecule, are likely involved in stabilizing its orientation for hydroxylation at the heme iron center. The investigation of their sequence-function relationships can guide the further engineering process. Thus, mutational scanning at all selected 15 residues was conducted by performing site saturation mutagenesis (SSM) using the 22c-trick strategy38. Variants were screened in 96-deep-well plates via high-performance liquid chromatography (HPLC) to assess hydroxylation selectivity and activity38,39.

After screening the library (~1395 variants), the results revealed that most variants exhibited reduced activity or selectivity. However, seven residual sites (F107, Q112, D126, N295, V299, A362, R368) emerged as exceptions, showing enhanced activity or selectivity (Fig. 2b). Notably, variants with improved activity with moderate-to-high selectivity for 1 at each site were presented in Fig. 2c. Strikingly, selectivity diverged between two pathways: C12β selectivity primarily correlated with five residues (F107, Q112, N295, V299, R368), while C15α selectivity involved four residues (Q112, D126, V299, A362). Mutations at Q112 and V299 were pivotal for switching regioselectivity in CYP68J5_fg, toggling between C12β and C15α hydroxylation of 1 when Q112 alternated long-chain (R) and short-chain (A/C/S/T/M) residues or V299 switched between polar (T) and nonpolar (I/L) amino acids (Fig. 2c). However, these results suggest that single mutations at key residues can modulate activity or selectivity but are insufficient to fully invert selectivity. Thus, we hypothesized that combinatorial mutations at multiple sites might synergistically enhance pathway-specific selectivity with the aid of revealed sequence-function relationships. To test this, we implemented focused rational iterative site-specific mutagenesis (FRISM)40–42 since it avoided laborious mutant library screening with minimal screening effort for selectivity and activity evaluation (Fig. 3a). Two distinct mutagenesis routes were designed to optimize selectivity for either C12β or C15α, as detailed in the subsequent study.

FRISM applied on CYP68J5_fg for improved selectivity and activity
Next, based on the revealed sequence-function relationships by mutational scanning, FRISM was carried out by combining the beneficial mutations from single variants that showed positive effects on activity (conversion rate of substrate 1) and/or selectivity. Given that most single mutations in CYP68J5_fg related to C12β selectivity of 1 are accompanied by reduced activity, the variant N295T (W12M1), which exhibited the highest activity, was chosen as the starting template for further mutagenesis. Next, four double variants were developed by combining N295T with mutations at four additional sites (F107S, Q112R, V299T, R368K). After screening the resulting variants, the optimal double variant Q112R/N295T (W12M2) was obtained, which demonstrated a 20% increase in activity and a 46% enhancement in selectivity (relative to WT). Subsequently, based on W12M2, three more triple mutants were generated by individually introducing mutations F107S, V299T and R368K. The best-performing triple mutant, Q112R/N295T/V299T (W12M3) boosted the selectivity to 97% with 51% conversion. Finally, to further enhance the activity, quadruple (W12M4 (F107S/Q112R/N295T/V299T)) and quintuple (W12M5 (F107S/Q112R/N295T/V299T/R368K)) mutants were correspondingly constructed by integrating mutations F107S and R368K. Consequently, variant W12M5 showed the best catalytic performance, achieving 82.3% conversion towards 1 while maintaining its selectivity at 97.7% (Fig. 3b, d).

Similarly, to enhance both selectivity and activity of CYP68J5_fg-catalyzed C15α-hydroxylation of 1, the identified single variant V299L (W15M1), which demonstrated a 39% increase in selectivity over the WT, was selected as the initial template. Then, based on W15M1, double variants were created by individually introducing mutations at sites Q112, D126, and A362 (Q112A/M/T/S/C, D126T/V, A362M). Among them, the double variant D126V/V299L (W15M2) demonstrated the most significant improvement, achieving 17% increased activity and 8% enhanced C15α selectivity relative to W15M1. Subsequently, based on W15M2, tertiary and quaternary variants were generated by introducing mutations of Q112A/M/T/S/C and A362M, respectively. As a result, the quadruple mutant Q112C/D126V/V299L/A362M (W15M4) emerged as the optimal variant, exhibiting 99.6% C15α selectivity with 72.7% substrate conversion (Fig. 3c, e).

It is noteworthy that, while engineering P450 enzymes often results in a “trade-off” between selectivity and catalytic activity, where high selectivity typically diminishes at elevated substrate conversions43. However, our combined SSM and FRISM strategies for P450 engineering successfully overcame this classical limitation by just screening 29 variants. Consequently, divergent evolution of CYP68J5_fg was achieved, leading to two distinct CYP68J5_fg variants W12M5 and W15M4 for highly active and selective C12β and C15α-hydroxylation, respectively. Variant W12M5 boosted the C12β selectivity from 37.1% (for WT) to 97.7% with 82.3% substrate conversion (only 28.1% for WT). Parallel to this achievement, variant W15M4 enhanced the C15α-selectivity to 99.6% from 49.3% with 72.7% substrate conversion.

Molecular mechanism of CYP68J5_fg variants for selective steroid hydroxylation
To elucidate the mechanisms underlying the enhanced catalytic efficiency and selectivity of the CYP68J5_fg variants W12M5 (F107S/Q112R/N295T/V299T/R368K) and W15M4 (Q112C/D126V/V299L/A362M), comprehensive structural and computational analyses were performed by using molecular dynamics (MD) simulations.

The W12M5 variant achieved high selectivity for the C12β-hydroxylation of 1. MD simulation results showed that the F107S mutation created a larger hydrophilic space, which forces the nonpolar methyl groups of 1 to relocate from the polar environment toward more compatible hydrophobic regions. Meanwhile, the Q112R mutation introduced a positively charged guanidinium group, while the N295T substitution reduced local hydrophilicity by removing a polar side chain, collectively altering the electrostatic landscape around the C3 keto group and driving it toward the BC loop through polarity-mediated spatial rearrangement (cyan arrows, Fig. 4a). In addition, the R368K and V299T mutations, which reduce side-chain polarity and enhance hydrophilicity respectively, flank the C20 carbonyl group of 1 and generate asymmetric polarity gradients. These, together with a weak hydrogen bond between H302 and the substrate ((H302) N···O(1)=3.86±0.74 Å, Supplementary Fig. 5a), collectively guide the C20 carbonyl group of 1 towards the I helix (magenta arrows, Fig. 4a). These concerted modifications establish an optimized catalytic microenvironment favoring C12-specific oxidation, with a mean 12C···O=Fe(heme) distance of 3.56±0.22 Å and a mean angle of 134±13° formed by O(Fe=O)-(1)-H(12C)-(1)-C(12) (Fig. 4b, c). The close agreement between these structural parameters and the QM-optimized transition state (TS) for C12β-H abstraction (4TS H,C12, Supplementary Fig. 4) is consistent with the geometric requirements essential for efficient TS formation and reaction feasibility.

In contrast to W12M5-1 in pose 12 (positioning C12 proximal to the heme for β-hydroxylation), the V299L mutation in W15M4 introduces a steric hindrance effect through the elongated hydrophobic side chain of leucine, thereby driving a reverse orientation of the substrate’s nonpolar methyl groups to minimize hydrophobic packing conflicts. The Q112C and D126V mutations collectively reduce local polarity, while the N295 residue forms a strong hydrogen bond with the substrate ((N295) N···O(1)=2.84±0.12 Å, Supplementary Fig. 5b), orienting the C3 keto group of 1 toward the I-helix (cyan arrows, Fig. 4d). The C20 carbonyl group of 1 is sandwiched between the hydrophobic side chain of M364 and the polar residue R368, forming a polarity-asymmetric cleft that drives the functional group to migrate toward the polar side. Moreover, R368 further anchors the C20 carbonyl oxygen via hydrogen bonding ((R368) N···O(1)=3.12±0.39 Å, Supplementary Fig. 5c), guiding the carbonyl group toward the β2 sheet (magenta arrows, Fig. 4d). Consequently, pose 15 (positioning C15 proximal to the heme for α-hydroxylation) emerges as the dominant active conformation in the W15M4-1 complex, characterized by a mean C15···O=Fe(heme) distance of 3.92±0.33 Å and a mean angle formed by O(Fe=O)-(1)-H(15C)-(1)-C(15) of 144±9° (Fig. 4e, f), both of which are consistent with QM-predicted values for C15α-H abstraction (4TS H,C15, see Supplementary Fig. 4). Moreover, MD simulations of CYP68J5_fg with substrate 1 in poses 12 and 15 (Supplementary Fig. 6a–d), variant W12M5 with 1 in pose 15 (Supplementary Fig. 6e, f), and variant W15M4 with 1 in pose 12 (Supplementary Fig. 6g, h) revealed that the majority of substrate-binding conformations sampled during the trajectory proved to be non-productive, as expected. Interestingly, in variant W12M5, substrate 1 flipped ~90° at 20 ns and ~180° by 25 ns relatiive to its initial pose, ultimately adopting a C12-binding mode (Supplementary Fig. 7) This spontaneous rearrangement further confirms the instability of C15-binding and strongly supports the C12-hydroxylation specificity of W12M5.

In summary, our MD simulation results offer valuable molecular insights into the binding of 1 in CYP68J5_fg variants. The introduced mutations reshaped the polar distribution of substrate binding pocket, altering the key residue network involved in hydrogen bonding with the polar functional groups (C3 keto group and C20 carbonyl group) of 1. These findings elucidate how polarity remodeling at the binding pocket governs regioselective hydroxylation at C12β or C15α.

Exploring substrate scope of variants of CYP68J5_fg
The catalytic capacity of CYP68J5_fg variants to catalyze regio- and stereoselective hydroxylations at the C15α and C12β of steroids was evaluated. We tested the activity and selectivity of different variants of CYP68J5_fg on a range of steroids: canrenone (2), bisnoralcohol (3), 17α-hydroxyprogesterone (4), testosterone (5), androstenedione (6), followed by scaling up of top-performing mutants (Supplementary Fig. 8). Variants favoring C15α hydroxylation on substrate 1 also selectively hydroxylated substrates 2, 3, 5, and 6, with substrate conversion ranging from 46.7% to >99.0% and selectivity ranging from 93.8% to >99.0% (Table 1). Due to the endogenous C17 reductase in S. cerevisiae44, 6 can be reduced to 5, the selectivity of variant W15M4 towards 6 was confounded by its conversion to 5. The capability of this reductase for converting 6 to 5 was further confirmed by its heterologous expression in E. coli (Supplementary Fig. 9). Future work on optimization of the yeast chassis (e.g., reductase gene knock out) could potentially eliminate this reduction activity. However, substrate 4 was not accepted by this variant. MD simulations revealed that the C17 hydroxyl group of 4 forms a stable hydrogen bond with the heme Fe=O center (d4, Supplementary Fig. 10), enforcing a near-planar conformation and resulting in a suboptimal O(Fe=O)-(4)-H(15C)-(4)-C(15) attack angle (θ=120±13° vs. ideal 170°), which sterically hinders hydrogen abstraction, leading to the loss of the activity. On the other hand, the variants that prefer C12β hydroxylation at position 1 also selectively act on substrates 2, 3, and 4, exhibiting a preference for C12β hydroxylation. These mutants, exhibiting 72.6% to >99.0% substrate conversions with the selectivity all being above 94.0% (Table 1). Compared to the acetyl side chain of 1, the computational analysis showed that the enhanced polarity at the C17 position in 5 and 6 promotes strong hydrogen bonding with residue K368 (d5 and d6, Supplementary Fig. 10) in W12M5, inducing a conformational shift that displaces C12 from the Fe=O center and blocks oxidation. Significantly, structural characterization revealed three previously unreported steroidal hydroxylated products: the C12β-hydroxylated derivatives of 2 and 3, as well as the 15α-hydroxylated derivative of 3. Notably, differences in substrate chemical properties influence hydrogen bonding and conformational changes, which in turn affect the catalytic performance of variants and lead to substrate-specific inactivation. Thus, leveraging sequence-function relationship, these variants can be further optimized to enhance their selectivity or/and activity towards these steroidal substrates. Given the special synthetic value of C15 and C12 positional functionalization of steroids, as exemplified in the production of glucocorticoids or deoxycholic acid4,6. Consequently, the discovery of CYP68J5_fg and its variants further diversifies the enzyme toolkit and provides excellent biocatalysts for industrial steroid drug production.

Scale-up reactions with generated variants W12M5 and W15M4
Compared to S. cerevisiae, which shows limited recombinant protein production despite robust fermentation, P. pastoris achieves higher yields due to its Crabtree-negative metabolism that prevents carbon loss to ethanol under respiration, increasing biomass and protein production45,46. We finally evaluated the performance of the variants W12M5 and W15M4 for hydroxylated steroid production under fed-batch fermentation by using a P. pastoris strain GS115 (Supplementary Fig. 11). For the recombinant strain GS115-W12M5, methanol induction began after 49 h of glycerol growth. After 24 h of induction, 10 g of 1 and cyclodextrin was added at a molar ratio of 1:1. As in Fig. 5a, after 144 h of fermentation, the yield of product 1a reached 4.6 g/L, with an isolation yield of 45.1% (purity >95%). This non-linear reaction profile suggested product inhibition, a hypothesis we validated by adding varying concentrations of 1a to a fixed concentration of substrate 1 (Supplementary Fig. 12). Worth noting is that, the conversion of 1 to 1a catalyzed by GS115-W12M5 represents the highest yield of 12β-hydroxylation of 1a achieved by any fungal-derived P450 mutant in yeast to date. Moreover, the rearrangement of 12β-hydroxysteroids could yield highly bioactive C-nor-D-homo-steroids featuring an unusual 6-6-5-6 ring system, significantly simplifying synthetic routes to natural products containing this scaffold. As exemplified by the previous study7, it had been demonstrated that 1c undergoes rapid skeletal rearrangement within 20 min. Therefore, 1a could potentially undergo skeletal rearrangement via this approach to afford corresponding C-nor-D-homo-steroids (Fig. 5a).

For the recombinant strain GS115-W15M4, we also tested the strain’s ability to transform compounds 1 and 6. As shown in Supplementary Fig. 13, for the transformation of 1, cyclodextrin-solubilized 1 was added in batches (10 g and 8 g) after 24 h of induction, and after 148 h of fermentation, the production of 1b finally increased to 10.9 g/L, with an isolation yield of 71.6% (purity >95%). Furthermore, as the 15α-hydroxylated derivative of 6 (15α-OH-AD) is a key intermediate in the synthesis of the contraceptive drug drospirenone47,48. Using the engineered strain GS115-W15M4, we achieved a 15α-OH-AD (6a) titer of 14.1 g/L in fed-batch fermentation with 20 g/L AD loading, which represent the highest reported yield for steroid hydroxylation by a fungal-derived P450 in yeast (Fig. 5b). Conventional fungi (e.g., Fusarium culmorum47, Penicillium griseopurpureum49, or Fusarium equiseti48) catalyze AD 15α-hydroxylation, but generally yield off-target byproducts (6β-, 7α-, or 11α-hydroxylated derivatives). In contrast, W15M4 exhibits exceptional regioselectivity for C15 hydroxylation. Coupled with the high-density fermentation capacity of P. pastoris, this system offers a superior alternative route for efficient drospirenone production47,48.

These results indicate that P. pastoris serves as a suitable host organism for engineering a promising cell factory for high-yield steroid production, advancing the feasibility of industrial-scale steroidal drug manufacturing. Notably, as shown in Fig. 1a, b, C12-hydroxylated and C15-hydroxylated sterols possess critical pharmacological activities. By achieving high-density fermentation of CYP68J5_fg and its mutants in P. pastoris, optimization of the synthetic pathway of these steroidal drugs offers significant synthetic possibilities.

Methods
Functional expression of fungal P450 in S. cerevisiae
S. cerevisiae strain INVSc1 cells were prepared for transformation with pYES2 expression plasmid harboring CYP68J5_fg or mutants through a standard protocol for lithium acetate transformations51. Transformed cells were plated on uracil dropout plates containing 2% (w/v) glucose. After 2–3 d of growth at 30 °C, colonies were inoculated in 2 mL uracil dropout minimal medium containing 2% (w/v) glucose and grown for 24 h at 30 °C with shaking at 220 rpm. The seed solution was then transferred to 20 mL uracil dropout minimal medium containing 2% (w/v) glucose for 2 days. Cultures were induced for expression by resuspension (2:1) in uracil dropout minimal medium containing 4% (w/v) galactose. Expression cultures were grown at 30 °C with shaking at 220 rpm. For biotransformations, substrates were dissolved in DMF or 2-hydroxypropyl-β-cyclodextrin solution and spiked into the expression culture at the point of induction. After reaction, ethyl acetate was added with an equal volume for product extraction, the organic phase was then obtained by centrifugation, followed by evaporation to dryness before resuspended in methanol for HPLC analysis.

Site saturation mutagenesis (SSM)
Site saturation mutagenesis (SSM) was performed by two-step PCR approach with non-overlapping primers. Additionally, degenerate 22-codon trick primers were designed for each of the 15 active site residues required for SSM (gene sequences and associated primers are provided in the Supplementary Data 1)38. In the first step, the primers were to be mixed in a molar ratio of 12 (NDT): 9 (VHG): 1 (TGG) before mixing with a non-mutagenic (silent) primer in a 1:1 ratio. The mixture was then subjected to first PCR for 28 cycles to obtain short DNA fragments containing mutagenic sites with KOD -Plus- Neo Polymerase. The recovered DNA fragments then serve as a megaprimer to perform the second PCR with KOD-Plus-Neo Polymerase for whole plasmid amplification with 28 cycles, and the resulting PCR products were digested with DpnI (NEB) overnight at 37 °C to eliminate the parental template. And the PCR products were transformed into E. coli DH5α by electroporation. Subsequently, cells were recovered for 45 min at 37 °C in LB medium, aliquots were plated on LB agar plates supplemented with 100 μg/mL ampicillin (LB-Amp plates), and plates were incubated at 37 °C overnight. Resulting colonies were grown up and miniprepped to give a plasmid DNA library. S. cerevisiae strain INVSc1 cells were prepared for transformation with the plasmid DNA library through a standard protocol for lithium acetate transformations. Transformed cells were plated on uracil dropout plates containing 2% (w/v) glucose and incubated at 25 °C for 3 d. Resulting colonies were used directly in library screening.

Site-directed mutagenesis (SDM)
Site-directed mutagenesis (SDM) was performed by one-step PCR approach with non-overlapping primers like SSM. For SDM, mutagenic primer was added in a 1:1 ratio with non-mutagenic (silent) primer and then 28 cycles of PCR were performed using PrimerSTAR Max DNA Polymerase. Finally, the clones developed on agar plate were picked and subjected to DNA sequencing for confirmation.

High throughput biotransformations
Uracil dropout minimal media containing 2% (w/v) glucose was added to sterile 96-well culture plates to a volume of 500 μL39. S. cerevisiae library colonies were inoculated into each well of the plate except for row A1, A2 and A3, which were reserved for S. cerevisiae control colony, CYP68J5_fg wild type and no colony control. Cultures were grown for 36 h at 30 °C with shaking at 800 rpm. Typically, an aliquot of 100 μL was transferred to a glycerol stock plate and stored at −80 °C for culture collection. Culture plates were centrifuged at 2820 × g for 10 min to pellet cells and the growth media was removed. Cells were resuspended with 200 μL of uracil dropout minimal media containing 4% (w/v) galactose and 300 μM progesterone (PG, 1). The biotransformations were incubated at 30 °C with shaking at 800 rpm for 2–3 days. Reactions were quenched with the addition of 3 equivalents of ethyl acetate and centrifugation at 2820 × g for 10 min. The organic phase was transferred into a 96-wells shallow plate and placed in the fume cupboard until the solvent complete evaporation. The dried samples were resuspended in 200 μL methanol and passed through a PTSF 96-well plate filter to remove solid particles (Pall, VWR, Germany) into a new 96-wells shallow plate. The plates were closed using silicon lids and subjected to HPLC analysis.

Hit validation and sequencing
Functional validation of selected hit variants was performed by culturing from glycerol stocks and conducting triplicate biotransformation assays followed by HPLC analysis. Variants demonstrating enhanced activity relative to the parental template were selected for sequencing to identify mutations in active sites. For this purpose, plasmid DNA was extracted from S. cerevisiae using the TIANprep Yeast Plasmid DNA Kit, transformed into DH5α E. coli competent cells, and subsequently purified using a Plasmid Miniprep Kit. The purified plasmids were submitted to Sangon Biotech (Shanghai, China) for Sanger sequencing.

Screening of CYP68J5_fg mutants for hydroxylation of diverse steroids
Sterile 96-well plates were filled with 500 μL of uracil dropout minimal medium containing 2% (w/v) glucose. Each well was inoculated with a distinct CYP68J5_fg mutant colony, with each mutant replicated three times per substrate. The plates were incubated at 30 °C for 36 h with shaking at 800 rpm. Subsequently, the cultures were centrifuged at 2820 × g for 10 min to pellet the cells, and the supernatant was carefully removed. Cells were resuspended in 200 μL of uracil dropout minimal medium supplemented with 4% (w/v) galactose and 300 μM of the respective substrate: canrenone (2), bisnoralcohol (3), 17α-hydroxyprogesterone (4), testosterone (5), or androstenedione (6). The biotransformations were carried out at 30 °C with shaking at 800 rpm for 48–72 h. Reactions were terminated by adding three volumes of ethyl acetate relative to the reaction mixture, followed by centrifugation at 2820 × g for 10 min. The organic extracts were transferred to a shallow 96-well plate and evaporated to dryness in a fume hood. The residues were reconstituted in 200 μL methanol and filtered through a PTSF 96-well filter plate (Pall, VWR, Germany) into a new shallow 96-well plate. Plates were sealed with silicon lids prior to HPLC analysis.

Scale-up reaction for product preparation with mutants of CYP68J5_fg
For large-scale product preparation, a preculture of S. cerevisiae INVSc1-CYP68J5_fg mutant was used as the inoculum. A single colony was inoculated into 100 mL of SC-Ura medium (2% (w/v) glucose) in a 1 L shake flask and cultivated at 30 °C with shaking at 250 rpm for 48 h. Cells were harvested by centrifugation and resuspended in 50 mL of fresh SC-Ura medium (4% (w/v) galactose). To the culture, we added a steroid substrate dissolved in 2-hydroxypropyl-β-cyclodextrin to a final concentration of 1–2 mM, along with 5-aminolevulinic acid hydrochloride (5-ALA) at a final concentration of 0.5 mM. The biotransformation was continued for 3 days under the same temperature and shaking conditions. This process can be readily repeated and scaled as required.

Functional expression in P. pastoris
The expression plasmids harboring CYP68J5_fg mutants was linearized with PmeⅠ restriction enzyme (NEB), and 1-2 μg of linearized DNA was transferred into GS115 electrocompetent cells by electroporation. Electroporated cells were grown on YPD plates containing 100 μg/mL zeocin at 30 °C for 2–3 days. For screening transformants, colonies were inoculated in BMGY medium and grown for 2 days at 30 °C with shaking at 220 rpm, cultures were induced for expression by resuspension (2:1) in BMMY medium at 25 °C with shaking at 220 rpm and supplemented daily with methanol to maintain 1% (v/v) methanol in the cultures. For biotransformations, substrate was spiked into the expression culture at the point of induction.

Fed-batch fermentation in P. pastoris
Fermentation control was adapted from the Pichia Fermentation Process Guidelines (PFPG, Invitrogen) and the fermentation process included three phases. First, the batch phase (phase Ⅰ), the pH of BSM medium was maintained at 5.5 by adding aqueous ammonia, and the temperature was maintained at 28 °C, and the aeration rate of 2–3 vvm. A single colony was first inoculated into a 1 L Erlenmeyer flask containing 150 mL of YPD medium at 30 °C with shaking at 220 rpm for ~24 h. Then, 10% (v/v) of the seed culture was inoculated into 1.5 L BSM medium with 2 g/L histidine in a 5 L fermenter for fed-batch fermentation. After glycerol was exhausted (~24–28 h) (phase Ⅱ), the fed-batch phase was initiated by feeding 50% (w/v) glycerol with 2 g/L histidine and PTM1 at a speed of 4–5 mL/min to biomass increasing, and the dissolved oxygen (DO) was maintained at ~30%. After the OD reached ~150 (~24–30 h), glycerol feeding was stopped to allow cells to deplete the glycerol and shifted to methanol feeding (phase Ⅲ), the methanol feeding rate at 4–7 mL/min until the end of fermentation, the temperature was set to 25 °C and the DO was maintained at ~20%. The substrate 1 and cyclodextrin are added in a molar ratio of 1:1.

Quantum Mechanical (QM) calculations
The P450 enzyme, coordinated with an iron-bound porphyrin complex, features an Fe=O double bond denoted Compound I (Cpd I). To simplify the model of Cpd I, the porphyrin macrocycle was represented with methyl groups for all substituents, and the axial Cys449 ligand was modeled as ethylthiolate anion. To preserve the protein scaffolding, geometry optimizations were performed with the atoms at the truncation sites constrained to their crystallographic positions. All calculations were carried out using unrestricted density functional theory (UDFT) with the hybrid functional B3LYP, as implemented in the Gaussian 16 program package52. Geometry optimizations were performed with the LANL2DZ basis set for the Fe atom, the 6–31G+(d,p) basis set for all other atoms. Based on the optimized geometries, more accurate single-point energies were obtained using larger basis sets, namely LANL08+ for Fe and cc-pVTZ(-f)+ for non-metallic elements. To incorporate the solvation effects of the protein environment, single-point calculations at the same level of theory were conducted based on the conductor-like polarizable continuum model (CPCM)53,54. The dielectric constant (ε) is set to 4, which is a conventional value for modeling protein environments55,56. Frequency calculations were performed at the same level of theory as the geometry optimizations to characterize the stationary points and provide zero-point energy (ZPE) corrections. Following geometry optimization of the enzyme-substrate complex in various electronic states, the quartet state was identified as the ground state (Supplementary Tables 1 and 2). This finding is consistent with previous research57, which establish that the quartet C-H abstraction transition state (TS) consistently exhibits lower energy. Thus, all subsequent calculations were conducted exclusively in the quartet state. P450-mediated hydroxylation involves the hydrogen atom abstraction from the C12/C15 position of PG by Cpd I, followed by a hydroxyl radical rebound. With computational results confirming hydrogen abstraction as the rate-determining step (Supplementary Fig. 3), its TS structure was used as a key reference to evaluate the prereactive conformations sampled in MD simulations.

Molecular docking
Molecular docking was conducted employing LeDock software58, recognized for its high performance and accuracy. Initial ligand preparation involved converting the PG substrate structure (retrieved in SDF format from PubChem) to mol2 format. Concurrently, the receptor structures, including the predicted CYP68J5_fg and its variants, were prepared in PDB format. Docking poses were clustered based on a Root Mean Square Deviation (RMSD) cutoff of 1.0 Å. A docking box was defined as a 5 Å-radius sphere centered on the heme using PyMOL’s GetBox plugin59, delineating the expected three-dimensional ligand-binding region. The simulation was set to produce 20 distinct binding poses, which were ranked by their predicted binding energy scores. From these energy-ranked poses, catalytically competent conformations were identified by evaluating both the distance and orientation of the target C12-H or C15-H bonds relative to the ferryl oxygen of Compound I. Thus, the final selected conformation represents the pose that is both thermodynamically stable (most negative binding energy) and mechanistically viable for C12/C15 hydroxylation.

Molecular Dynamics (MD) simulations
Molecular dynamics (MD) simulations were performed using AMBER 2260 based on the CYP68J5_fg (or its variants)/PG complex obtained by molecular docking. Parameters for the substrate (PG) were generated with the Antechamber module in AmberTools 23, employing the Generalized AMBER Force Field (GAFF)61. Atomic partial charges were obtained by fitting the electrostatic potential at the HF/6-31G(d) level using the restrained electrostatic potential (RESP) approach. Parameters for heme compound I and axial cysteine residue were taken from established computational protocols for heme-protein modeling62. The remaining protein residues were described by the Amber ff14SB force field63, and the protonation states of titratable residues were predicted using the PROPKA3 server64. The complex structures were solvated in a cubic TIP3P65 water box, maintaining a minimum distance of 16 Å between the protein surface and the box edges. Each system was neutralized with Na+ counterions.

The complex system underwent initial energy minimization through 5000 steps of steepest descent followed by 5000 steps of conjugate gradient optimization, with positional restraints applied to the protein using a force constant of 500 kcal·mol−1Å−2. An additional 5000 steps of steepest descent and 5000 steps of conjugate gradient minimization were then performed to fully optimize the system without restraints. Subsequently, the system was heated from 0 to 300 K over 500 ps under the NVT ensemble, with backbone atoms constrained by a force constant of 25 kcal·mol−1Å−2. Next, density equilibration was conducted for 1.0 ns using the NPT ensemble to achieve uniform density.

The Langevin thermostat66 was used to control the temperature at 300 K with a collision frequency of 1 ps−1, while the pressure was kept at 1 bar with a relaxation time of 2 ps. Covalent bonds involving hydrogen atoms were constrained with the SHAKE method67, employing a 2 fs integration time step. A cutoff radius of 12 Å was set for nonbonded interactions. Following 10 ns of equilibration under the NPT ensemble, a 100-ns productive MD simulation was performed in the NPT ensemble. Trajectory analysis was conducted using the AmberTools 23 CPPTRAJ module.

HPLC analysis
The conversion analysis was performed via reversed phase HPLC technique using a Shimadzu LC2030C system with an Ultimate® XB-C18 column (4.6 × 250 mm, 5 μm, Welch Materials (Shanghai) Co., Ltd.). For steroids (1, 3 and 4), the mobile phase consisted of acetonitrile (CH3CN) and water with a gradient program of CH3CN: H2O=30:70–80:20 for 0–12 min, 80:20–70:30 for 12–15 min at 1.5 mL/min flow rate and 40 °C oven temperature. The injection volume was 10 μL and UV detection was accomplished at 254 nm. For steroids (2, 5 and 6), the mobile phase consisted of CH3CN and water with a gradient program of CH3CN: H2O=10:90 for 0–2 min, 10:90-40:60 for 2–10 min, 40:60–70:30 for 10–15 min, 70:30–90:10 for 15–16 min, 90:10 for 16–17 min at 1.5 mL/min flow rate and 40 °C oven temperature. The injection volume was 10 μL and UV detection was accomplished at 254 nm expect for 2, for which 310 nm was used.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Wang et al. presents a significant advancement in fungal P450 engineering for regioselective steroid\nhydroxylation. It is well known that engineering fungal P450 enzymes poses considerable challenges due to the\nabsence of crystal structures, their membrane-bound nature, and the difficulty in achieving efficient heterologous expression. To address\nthese issues, the authors employed a combined SSM and FRISM mutagenesis approach to facilitate the divergent evolution\nof the fungal P450 CYP68J5_fg. This strategy successfully yielded two variants capable of catalyzing C12β- and C15α-\nhydroxylation with high selectivity and activity, providing an important example of overcoming the classical trade-off between\nselectivity and activity in enzyme engineering.\nMolecular dynamics simulations revealed that key mutations alter the polarity landscape of the enzyme’s active site and\nreconfigure hydrogen-bonding networks, thereby directing hydroxylation to distinct positions. Additionally, high-density\nfermentation achieved unprecedented product titers exceeding 10 g/L, which represent the highest yields reported to date for\nyeast-based steroid hydroxylation. Importantly, these products are valuable intermediates for the synthesis of C12- and\nC15-functionalized steroid drugs, such as drospirenone, as well as C-nor D-homo derivatives.\nOverall, this study provides valuable insights into the precise control of regio- and stereoselectivity through the evolution of\nCYP68J5_fg mutants. It also highlights the utility of computational simulations in engineering non-structure-dependent P450\nenzymes derived from fungi. In addition, the experimental work appears thorough and convincing, and the manuscript is\ngenerally well written. I recommend its publication after addressing the following points to enhance clarity, scientific rigor,\nand impact:\n1. Justification of why SSM+FRISM was chosen over established methods like CASTing. Compare library sizes/screening\nefforts (e.g., Line 153: \"small collections\" is vague; quantify variant numbers screened).\n2. In Table 1, substrate 6 (androstenedione) shows reduced conversion to testosterone, likely due to endogenous\nreductases. Is it possible to identify which reductase(s) are responsible for this reduction? Additional discussion or\nexperiments addressing this point would strengthen the results.\n3. Isolation yields for 1a (45.1%) and 1b (71.6%) differ significantly (Lines 328, 342). Please discuss potential reasons for\nthis discrepancy, such as cytotoxic effects or losses during purification.Some errors and typos:\n4. There are some typographical errors and inconsistent terminology:\nLine 175: \"catalzyed\" should be corrected to \"catalyzed.\"\nLine 363: \"chemocatalytic\" should be corrected to \"chemoenzymatic.\" Please use \"chemoenzymatic\" consistently\nthroughout the manuscript.\nFigures 3b and 3c: Please explicitly define \"activity\"—for example, specify whether it refers to conversion rate, turnover\nnumber, or another metric for clarity.\n5. The abstract could be slightly shortened to improve conciseness.", "reviewer_2": "In the manuscript entitled “Divergent Evolution of Fungal P450 Monooxygenase Unlocks Simultaneous Access to C12β and\nC15α oxyfunctionalization of Steroids”, Wang et al. performed divergent evolution with combined structure-guided site-\nsaturation mutagenesis and focused rational iterative site-specific metagenesis, achieving rather good selectivity for\nC12beta and C15alpha while simultaneously enhancing catalytic efficiency. the N-oxidation selective potential of P450BM3\nfor N-heterocyclic compounds. The reason behind the mutations was further uncovered by MD simulations. Importantly, this\nwork represents a first example of the divergent evolution of a fungal P450. The manu. is well-written and the results seem\ninteresting. I suggest a major revision before its publication due to following concerns.\n1. The labels of the substrates (at least 1) may be supplied.\n2. Is the regioselectivity coupled with the stereoselectivity?\n3. I am curious about the performance of W15M4 and W12M4 for substrate 3, 4 5 and 6. Only some have been tested. Why\nother mutants were used in Table 1?\n4. it is quite confusing to use “pose 12” and “pose 15”.\n5. The binding conformations shown in Figure 4 are not clear. Additionally, the polar H atoms may be shown in all related\nfigures to make them clear.\n6. Optimized geometries (it seems key information is missing in Figure S4) and energy profiles obtained with QM are\nmissing.\n7. It is confusing to say “To obtain the optimal geometry for the transition state (TS), the calculations were performed using\nunrestricted density functional theory (UDFT) with the hybrid functional B3LYP as implemented in the Gaussian 16 program\npackage.”\n8. Instead of 6-31G(d,p)+, it is more common to use 6-31+G(d,p).\n9. More details may be supplied for how the binding conformations are obtained. For example, whether the chosen\nconformation is the most favorable one?\n10. What is the reference value (distQM) in Figure 4, the distance between Cpd I and hydrogen atoms or carbon atoms? Why\nuse values of the transition state?\n11. The energy difference between doublet and quartet states is 3.2 kcal/mol in supplementary Table 3. However, it is only\n0.1 kcal/mol in supplementary Table 4. What is the reason?\n12. According to Figure S5b, it is hard to explain the C15 reactivity of WT enzyme.\n13. It would be interesting to see what happened at 20 ns in Figure S5C.", "reviewer_3": "The C12β- or15α-hydroxylated steroids are leading compounds in developing steroid pharmaceuticals and medicines.\nHowever, it is a challenging role for regioselective and stereoselective hydroxylation of steroid compounds either by\nchemical or biological approaches. Cytochrome P450 enzymes are known as an important biological hydroxylase for\nbiosynthesis of steroid hormones in vivo. This manuscript presents a groundbreaking work for engineering fungal P450\nCYP68J5_fg to achieve highly selective C12β- and C15α-hydroxylation of steroids. The combination of structure-guided\nSSM and FRISM mutagenesis successfully overcomes the classic activity-selectivity trade-off, yielding two variants (W12M5\nand W15M4) with exceptional regioselectivity (>97%). MD simulations revealed that the key mutations reshape the\nsubstrate-binding conformation, thereby directing hydroxylation to distinct positions (C12β or C15α). The industrial relevance\nis supported by unprecedented titers (up to 14.1 g/L) in P. pastoris fermentation. The manuscript is suitable for publishing in\nNature Communications after a further revision.\n1) From the perspective of directed evolution, this is a surprisingly smooth job! The first step of directed evolution screening\nbased on SSM strategy is a routine operation, but I am a little concerned about calling the second stage of directed evolution\nFRISM. From the description in the text, it appears that this is somewhat different from the classic FRISM. The author doesn't\neven need to perform a few limited tests at the target site, essentially just cross combining the single mutations obtained\nthrough the SSM strategy in the first round. Although the results may seem perfect, a more accurate description of this\nprocess should still be considered.\n2) In line 63, the authors describe that “fungal P450 enzymes demonstrate superior suitability for industrial-scale production”,\nplease provide more evidences or references.\n3) As shown in Figure 2a, the dehydroxylated product 1c can be formed in this reaction. The direct dihydroxylation of\nsteroids is also very important and interesting (see recent example, Wang et al. Catal. Sci. Technol., 2025, 4170-4178.). It is\nobserved that there is an unknown product by W12M1 in Figure 3d. Is this compound a dihydroxylation product? I am\nwondered if the author has detected and identified the generation of dihydroxy products during directed evolution. If so, it\nmaybe your next interesting paper.\n4) In Table 1, can the selectivity and conversion rate be accurately measured to two decimal places?\n5) In line 329, the authors describe that “the low yield of 1a is potentially due to the cytotoxicity of 1a”, please provide the\nexperiment details or references.\n6) In line 382 and 383, the authors describe that “the integrated approach of SSM coupled with FRISM strategy effectively\nmodulates selectivity and enhances activity in these fungal-derived P450 enzymes”, to some extent, the approach may just\neffectively suitable for CYP68J5_fg, it would be better to state more precisely.\n7) Except for substrate 6, the other four structurally similar substrates all showed excellent selectivity and conversion rates.\nDid the author also examine for other substrates? If so, it is best to report the results, even if it is only included in the\nsupporting data. Because natural steroid hydroxylation P450 enzymes often have very strict substrate limitations. Similar\nresults are of great significance for the development of substrate-promiscuity P450 isoenzymes through protein\nengineerings.\n8) English language and style are required to fine spell check, such as “in line 175, catalzyed should be catalyzed; in line\n324, W1M4 should be W15M4.”"}, "author_response": "Point-by-point response to the reviewers’ comments\nReviewer #1: The manuscript by Wang et al. presents a significant advancement in\nfungal P450 engineering for regioselective steroid hydroxylation. It is well known\nthat engineering fungal P450 enzymes poses considerable challenges due to the\nabsence of crystal structures, their membrane-bound nature, and the difficulty in\nachieving efficient heterologous expression. To address these issues, the authors\nemployed a combined SSM and FRISM mutagenesis approach to facilitate the\ndivergent evolution of the fungal P450 CYP68J5_fg. This strategy successfully\nyielded two variants capable of catalyzing C12β- and C15α-hydroxylation with high\nselectivity and activity, providing an important example of overcoming the classical\ntrade-off between selectivity and activity in enzyme engineering.\nMolecular dynamics simulations revealed that key mutations alter the polarity\nlandscape of the enzyme’s active site and reconfigure hydrogen-bonding networks,\nthereby directing hydroxylation to distinct positions. Additionally, high-density\nfermentation achieved unprecedented product titers exceeding 10 g/L, which\nrepresent the highest yields reported to date for yeast-based steroid hydroxylation.\nImportantly, these products are valuable intermediates for the synthesis of C12- and\nC15-functionalized steroid drugs, such as drospirenone, as well as C-nor-D-homo\nderivatives.\nOverall, this study provides valuable insights into the precise control of regio-\nand stereoselectivity through the evolution of CYP68J5_fg mutants. It also highlights\nthe utility of computational simulations in engineering non-structure-dependent P450\nenzymes derived from fungi. In addition, the experimental work appears thorough\nand convincing, and the manuscript is generally well written. I recommend its\npublication after addressing the following points to enhance clarity, scientific rigor,\nand impact:\n1. Justification of why SSM+FRISM was chosen over established methods like\nCASTing. Compare library sizes/screening efforts (e.g., Line 153: “small collections”\nis vague; quantify variant numbers screened).\nAnswer: Thank you very much for your feedback and meticulous review of our\nmanuscript.\nRegarding the rationale for selecting SSM+FRISM over CASTing: the\nSSM+FRISM is a well-established methodology (J. Am. Chem. Soc. 2019, 141, 19,\n7934-7945; Mol. Catal. 2024, 553, 113755) that allows for the generation of a\nfocused library with significantly reduced screening effort. While CASTing\n(Combinatorial active-site saturation test) is effective, it requires screening a large\nnumber of variants. In contrast, our approach enabled us to identify the best variants\nby screening only 29 total mutants for both the C12β and C15α hydroxylation of\nsteroids.\nRegarding the library sizes and screening efforts for the two methods: In our\nstudy, FRISM enabled efficient screening of just 29 mutants for both C12β and C15α\noxyfunctionalizations of steroids. In contrast, the CASTing approach requires\nscreening of 96 mutants (2×2×2×2×2×3=96, 3 times oversampling to ensure≥95%\nlibrary coverage) for C12β-hydroxylation and 324 mutants (6×3×3×2×3=324, 3\ntimes oversampling to ensure≥95% library coverage) for C15α-hydroxylation,\ntotaling 420 mutants.\nRegarding the term “small collections” in the current line 148-149 (previously\nline 153), we have added the specific number of screened mutants (“by just screening\n29 variants”) on line 188 to ensure logical coherence of the manuscript. Furthermore,\nthe term “small collections” of this statement on line 148 has been adjusted.\n2. In Table 1, substrate 6 (androstenedione) shows reduced conversion to testosterone,\nlikely due to endogenous reductases. Is it possible to identify which reductase(s) are\nresponsible for this reduction? Additional discussion or experiments addressing this\npoint would strengthen the results.\nAnswer: We thank the reviewer for this insightful comment.\nExtensive studies have characterized yeast-mediated specific 17β-reduction of\nsteroids. As demonstrated in the article “Dehydroepiandrosterone (DHEA)\nmetabolism in Saccharomyces cerevisiae expressing mammalian steroid hydroxylase\nCYP7B: Ayr1p and Fox2p display 17beta-hydroxysteroid dehydrogenase activity\n(Yeast, 2002, 19(10): 873-86.)”, the reductase AYR1 (1-acyl dihydroxyacetone\nphosphate reductase) can reduce the 17-carbonyl group of DHEA to a hydroxyl group.\nOur experimental validation confirmed that AYR1 indeed converts androstenedione\n(AD) to testosterone (TS) (Supplementary Fig. 9). Our future work will be focusing\non optimizing the yeast chassis (such as to knock out the AYR1 gene) to minimize\nC17-reduction of steroids. All relevant experiments and statements have been added\nin both the main text and supplementary information.\nSupplementary Figure 9. Bioconversion of 1 mM AD by AYR1 from P. pastoris\nin E. coli. Reaction conditions: 3 mL E. coli cell lysate containing 1 mM AD, 3%\n(w/v) glucose, 1 U/mL glucose dehydrogenase (GDH), and 0.2 mM NAD(P)+\ncofactor.\n3. Isolation yields for 1a (45.1%) and 1b (71.6%) differ significantly (Lines 328, 342).\nPlease discuss potential reasons for this discrepancy, such as cytotoxic effects or\nlosses during purification.\nAnswer: Thank you very much for the comments.\nThe variance in the isolated yields of 1a and 1b can be attributed to the following:\n(1) Different catalytic efficiency: the biocatalytic conversion of 1 to 1a reached\napproximately 75%, which is lower than its conversion of 1 to 1b (approximately\n90%).\n(2) Different isolation approaches: 1a was purified by column chromatography\nbecause of the large amount of remaining substrate. This approach resulted in\nsubstantial loss due to the co-elution of 1 and 1a, a consequence of their similar\npolarities. Conversely, the higher conversion rate for 1b allowed for its efficient\nisolation through a high-yielding recrystallization.\n4. Some errors and typos:\nThere are some typographical errors and inconsistent terminology:\nLine 175: “catalzyed” should be corrected to “catalyzed.”\nLine 363: “chemocatalytic” should be corrected to “chemoenzymatic.” Please use\n“chemoenzymatic” consistently throughout the manuscript.\nFigures 3b and 3c: Please explicitly define “activity”—for example, specify whether\nit refers to conversion rate, turnover number, or another metric for clarity.\nAnswer: Thank you for your valuable feedback. We have addressed all the points\nraised:\n1) On the current line 172 (previously Line 175), “catalzyed” has been corrected to\n“catalyzed”.\n2) On the current line 370 (previously Line 363), “chemocatalytic” has been\nreplaced with “chemoenzymatic”, and this term was used consistently throughout\nthe manuscript.\n3) The term “activity” now clearly refers to the conversion rate of steroid substrates,\nand this clarification has been added in line 155 to the revised manuscript.\n5. The abstract could be slightly shortened to improve conciseness.\nAnswer: Thank you very much for your feedback.\nWe have streamlined the abstract to keep it concise, and a revised version of the\nabstract is presented below:\n“Steroidal C12β/15α-hydroxylation are pivotal in synthesizing steroid drugs but\nremain challenging via chemical and biological methods. To address this, the fungal\nP450 monooxygenase CYP68J5_fg underwent structure-guided divergent evolution.\nTwo optimized variants, W12M5 (F107S/Q112R/N295T/V299T/R368K) and W15M4\n(Q112C/D126V/V299L/A362M) were created, achieving exceptional selectivity (97.7%\nfor C12β- and 99.6% for C15α-hydroxylation of progesterone) alongside enhanced\ncatalytic efficiency, effectively overcoming the classic activity-selectivity trade-off.\nMolecular dynamics simulations revealed that key mutations reorient the substrate by\nreshaping the binding pocket’s polarity and hydrogen-bonding network, enabling\nhydroxylation at distinct positions. High-density fermentation with engineered Pichia\npastoris yielded unprecedented titers of 4.6 g/L 12β-OH progesterone, 10.9 g/L 15α-\nOH progesterone and 14.1 g/L 15α-OH androstenedione. These products serve as key\nintermediates for streamlined synthesis of C12-/C15-functionalized steroids such as\ndrospirenone and C-nor-D-homo derivatives. Collectively, this study demonstrates the\nfirst successful divergent evolution of a fungal P450 and highlights broad applicability\nfor the scalable synthesis of complex bioactive molecules.” (150 words)\nReviewer #2: In the manuscript entitled “Divergent Evolution of Fungal P450\nMonooxygenase Unlocks Simultaneous Access to C12β and C15α\noxyfunctionalization of Steroids”, Wang et al. performed divergent evolution with\ncombined structure-guided site-saturation mutagenesis and focused rational iterative\nsite-specific metagenesis, achieving rather good selectivity for C12beta and\nC15alpha while simultaneously enhancing catalytic efficiency. The reason behind the\nmutations was further uncovered by MD simulations. Importantly, this work\nrepresents a first example of the divergent evolution of a fungal P450. The manu. is\nwell-written and the results seem interesting. I suggest a major revision before its\npublication due to following concerns.\n1. The labels of the substrates (at least 1) may be supplied.\nAnswer: Thank you very much for your feedback and meticulous review of our\nmanuscript.\nIn Fig. 1e, we have labeled the carbon atom numbering for the model substrate\n1 (progesterone, PG). And the specific positions targeted for modifications (C12 and\nC15) are highlighted in distinct colors in Figs. 4a and 4d.\n2. Is the regioselectivity coupled with the stereoselectivity?\nAnswer: Thank you very much for the comments.\nIn our study, CYP68J5_fg and its mutants consistently produced either C12β-\nand/or C15α-hydroxylated products from progesterone and other steroid substrates\n(canrenone, testosterone, androstenedione, etc.). However, hydroxylated products at\neither the C12α or C15β position were not observed. Thus, the stereoselectivity\ninversion or switch was not involved.\n3. I am curious about the performance of W15M4 and W12M4 for substrate 3, 4\n5 and 6. Only some have been tested. Why other mutants were used in Table 1?\nAnswer: Thank you for your insightful feedback.\nDuring our initial evaluation of the activities of W15M4 and W12M5 against\nsubstrates 2-6, we observed insufficient activity of these variants toward certain\nsubstrates. Considering the sequence-function relationships between the substrates\nand the enzyme, we ultimately decided to test the activity of all variants (W12M1,\nW12M2, W12M3, W12M4 and W12M5; W15M1, W15M2, W15M3 and W15M4)\nagainst substrates 2-6 (Supplementary Figure 8). Only the top-performing variants\nfor each of substrates 2-6 are presented in Table 1.\n4. it is quite confusing to use “pose 12” and “pose 15”.\nAnswer: Thank you for your comments.\nWe agree that the original terms “pose 12/15” lacked functional context. “pose\n12” designates the substrate-binding conformation that positions C12 of the steroids\nwithin catalytic proximity of the heme iron for β-hydroxylation, while “pose 15”\nrefers to the conformation enabling C15α-hydroxylation. To clarify, the revised\nmanuscript now defines these terms parenthetically at first occurrence:\npose 12 (positioning C12 proximal to the heme for β-hydroxylation)\npose 15 (positioning C15 proximal to the heme for α-hydroxylation)\n5. The binding conformations shown in Figure 4 are not clear. Additionally, the\npolar H atoms may be shown in all related figures to make them clear.\nAnswer: Thank you for these constructive suggestions.\nWe have revised Fig. 4 accordingly by rotating the view to better expose the\nsubstrate-binding pocket. In addition, polar hydrogen atoms have been shown both in\nFig. 4 and in all related figures.\nFig. 4 Mechanism analysis of 1 hydroxylation by the CYP68J5_fg variants. a, d,\nRepresentative snapshots of the MD simulation revealing the substrate-enzyme-substrate binding\nmode in the active sites of W12M5 (a) and W15M4 (d). Mutated residues are shown in\nred. b, e, Plots of the C12, C15(1)···O=Fe distances (y primary axis) and the O(Fe=O)-\n(1)-H(12C, 15C)-(1)-C(12, 15) angles (y secondary axis) along the simulation time (x\naxis) for one of the replicas of W12M5 (b) and W15M4 (e) (see Supplementary Figs.\n1 and 2 for replicas 2 and 3). c, f, Conformational population analysis of the prereaction\nstates of W12M5 (c) and W15M4 (f) in the 100 ns MD simulation trajectories (three\njoint MD replicas). Quantum mechanics (QM)-predicted distances (distQM) and angles\n(angQM) of transition state geometry (Supplementary Figs. 3 and 4) for C12β-\nH/C15α-H abstraction are indicated by dashed lines (b, e) and black dots (c, f),\nrespectively.\n6. Optimized geometries (it seems key information is missing in Figure S4) and\nenergy profiles obtained with QM are missing.\nAnswer: Thank you for the valuable suggestion.\nThe QM energy profile for the Cpd I-mediated hydroxylation of progesterone is\nnow provided in Supplementary Fig. 3, and the missing key bond lengths/angles\nhave been added to Supplementary Fig. 4.\nSupplementary Figure 3. Potential energy profiles for the CYP68J5_fg-catalyzed\nC(12,15)-hydroxylation of 1. Abbreviations indicate the Cpd I and substrate PG complex\n(PG-Cpd I), the TS for hydrogen-atom transfer from PG to heme Fe=O species (TS ), the\nH\nintermediate including Fe-OH s species and the PG radical (PG-Cpd II), the TS for hydroxyl\nradical rebound from Cpd II to PG (TS ), and the hydroxylated substrate product intermediate\nOH\n(Prod).\nSupplementary Figure 4. QM-optimized transition-state structures for\nC(12,15)-H abstraction in 1. Key geometrical parameters are indicated with dashed\nlines. Asterisks indicate the atoms fixed in their X-ray positions.\n7. It is confusing to say “To obtain the optimal geometry for the transition state (TS),\nthe calculations were performed using unrestricted density functional theory (UDFT)\nwith the hybrid functional B3LYP as implemented in the Gaussian 16 program\npackage.”\nAnswer: Thank you for the constructive comments.\nTo eliminate confusion:\n(1) Consolidated all computational methods (UDFT/B3LYP, basis sets, CPCM, etc.)\ninto a unified preceding section;\n(2) Clarified the logical flow of our computational strategy. The updated content is\nnow: First, the enzyme-substrate complex was optimized in diverse electronic states.\nThe ground state has been found to be in the quartet state (Supplementary Tables 3 and\n4), which is consistent with previous research findings 10 demonstrating that the quartet\nC-H abstraction transition state (TS) consistently exhibits lower energy. Thus, all\nsubsequent calculations were conducted exclusively in the quartet state. P450-mediated\nhydroxylation involves the hydrogen atom abstraction from the C12/C15 position of\nPG by Cpd I, followed by a hydroxyl radical rebound. With computational results\nconfirming hydrogen abstraction as the rate-determining step (supplementary Fig. 3),\nits TS structure was used as a key reference to evaluate the prereactive conformations\nsampled in MD simulations.\n8. Instead of 6-31G(d,p)+, it is more common to use 6-31+G(d,p).\nAnswer: Thank you for pointing out the inaccuracies in the basis set format.\n“6-31G(d,p)+” has been corrected to the standard form “6-31+G(d,p)” in the\nSupplementary Information.\n9. More details may be supplied for how the binding conformations are obtained. For\nexample, whether the chosen conformation is the most favorable one?\nAnswer: Thank you for raising this important point.\nThe binding conformations were selected through molecular docking using\nLeDock software. For docking input files, the PG ligand structure obtained in SDF\nformat from the PubChem database was converted to mol2 format, while the receptor\nstructures, including the predicted CYP68J5_fg and its variants, were in PDB format.\nDocking poses were clustered based on a Root Mean Square Deviation (RMSD) cutoff\nof 1.0 Å. The space within 5 Å centered on the heme was designated as the docking\nbox using the GetBox plugin in PyMOL, which delineate the three-dimensional region\nwhere the ligand is expected to bind. The simulation was set to produce 20 distinct\nbinding poses, which were ranked by their predicted binding energy scores. From these\nenergy-ranked poses, catalytically competent conformations were identified by\nevaluating both the distance and orientation of the target C12-Hβ or C15-Hα bonds\nrelative to the ferryl oxygen of Compound I. Thus, the final selected conformation\nrepresents the pose that is both thermodynamically stable (most negative binding\nenergy) and mechanistically viable for C12/C15 hydroxylation. The relevant details\nhave now been added to the “Molecular Docking” section of the Supplementary\nInformation.\n10. What is the reference value (distQM) in Figure 4, the distance between Cpd I and\nhydrogen atoms or carbon atoms? Why use values of the transition state?\nAnswer: We thank the reviewer for this important clarification.\n(1) distQM denotes the QM-predicted distance between Cpd I and carbon atoms\nat the transition state, i.e., C12, C15(1)···O=Fe. For clarity, these definitions are\nexplicitly illustrated in the caption of Figure 4. Reference values for these distances are\nprovided in Supplementary Fig. 4.\n(2) The transition state (TS) geometry serves as the essential reference because it\nstructurally defines the reaction barrier—its energy and arrangement dictate both the\nrate and selectivity of C–H abstraction. A substrate conformation with bond lengths and\nangles closer to the TS requires less reorganization energy to reach the barrier, resulting\nin a lower activation energy and higher reactivity. Thus, proximity to the TS geometry\nis a direct predictor of reactivity. By comparing distances and angles sampled from MD\ntrajectories (prereactive states) with the QM-optimized TS values, we directly evaluate\nthe enzyme’s ability to preorganize the substrate. Conformations near the TS geometry\nindicate optimal positioning for hydrogen abstraction, enabling us to quantitatively\nexplain and predict regioselectivity and stereoselectivity.\nTo further emphasize this point, we have now included the following statement\nin the manuscript: “ The close agreement between these structural parameters and the QM-\noptimized transition state (TS) for C12β-H abstraction (4TS , Supplementary Fig. 4) is\nH,C12\nconsistent with the geometric requirements essential for efficient TS formation and reaction\nfeasibility.”\n11. The energy difference between doublet and quartet states is 3.2 kcal/mol in\nsupplementary Table 3. However, it is only 0.1 kcal/mol in supplementary Table 4.\nWhat is the reason?\nAnswer: We thank the reviewer for this insightful question regarding the energy\ndifferences between the doublet and quartet states in Supplementary Tables 3 and 4.\nThe small difference in the doublet-quartet energy splitting between pose 12 and\npose 15 (3.2 vs. 0.1 kcal/mol) is within the expected uncertainty of DFT calculations\nand does not indicate a fundamental change in electronic structure or reactivity. This\nvariation is attributed to subtle differences in the spatial arrangement and non-covalent\ninteractions between the substrate and Heme in the two binding poses. The weak\ninteraction allows minor conformational adjustments, such as slight changes in distance\nor orientation (J. Chem. Theory Comput. 2012, 8, 5, 1629-1640; Catal. Sci. Technol.,\n2015,5, 4946-4949; Annu. Rep. Prog. Chem., Sect. C: Phys. Chem., 2006,102, 203-226),\nto alter the electronic environment around the iron center, thereby modulating the\nrelative stability of the spin states. Fluctuations on the order of a few kcal/mol are\ncomputationally common and acceptable (Theor. Chem. Acc. 2010, 127, 429-442),\nwhereas larger differences (e.g., >8–10 kcal/mol) would be more unusual and indicative\nof a fundamental electronic change.\nThus, the observed differences between pose 12 and pose 15 are consistent with\nminor structural perturbations and do not affect the main conclusions of our work.\n12. According to Figure S5b, it is hard to explain the C15 reactivity of WT enzyme.\nAnswer: Thank you for raising this important point.\nWe agree that the original simulation results for the WT enzyme-substrate complex\ndid not adequately account for its C15 reactivity. To address this, we re-ran the MD\nsimulation specifically for the pose 15 binding conformation of CYP68J5_fg with\nsubstrate 1. Although the majority of the sampled conformations remain non-productive\n(revised Supplementary Fig. 6c), a minor population of poses in the WT enzyme\nadopts a geometry favorable for C15α-hydroxylation, with distance and angle\nparameters approaching those required for catalysis.\nTo better visualize the conformational distribution, we have also included a\nconformational population analysis in the revised Supplementary Fig. 6, which better\nillustrates the presence of these catalytically competent states in the WT enzyme.\nSupplementary Figure 6. MD simulations of CYP68J5_fg and its variants in the\nnon-productive position with 1. a, c, e, g, Distances determined between the oxygen\natom of the Fe=O and the C(12,15) atom of 1 (x axis) and angles formed by O(Fe=O)-\n(1)-H(C12,15)-(1)-C(12,15) (y axis) in CYP68J5_fg (Pose 12, 15) (a, c), W12M5 (Pose\n15) (e) and W15M4 (Pose 12) (g) along the whole simulation time. b, d, f, h,\nConformational population analysis of the prereaction states of CYP68J5_fg (b, d),\nW12M5 (f) and W15M4 (h) in the 100 ns MD simulation trajectories.\n13. It would be interesting to see what happened at 20 ns in Figure S5C.\nAnswer: We appreciate the reviewer’s insightful observation.\nAt around 20 ns in Supplementary Fig. 5c (now Supplementary Fig. 6c), the\nsubstrate undergoes a significant reorientation within the active site of variant\nW12M5, flipping nearly 90°. By 25 ns, the substrate has flipped almost 180° relative\nto its initial binding pose and repositioned into a C12-oriented binding mode\n(Supplementary Fig. 7). This spontaneous re-orientation reflects the instability of\nthe C15-bound conformation and confirms that the W12M5 variant specifically\npromotes the C12 hydroxylation of progesterone. This may be attributed to substrate\nbinding pocket of W12M5 that better accommodate the C12-binding pose while\ndisfavoring orientations for C15-hydroxylation.\nSupplementary Figure 7. Representative Snapshots from MD Simulations of the\nW12M5-1 Complex. Each snapshot illustrates the spatial arrangement of substrate\n1 (yellow) relative to the heme iron-oxygen (gray).\nReviewer #3: The C12β- or 15α-hydroxylated steroids are leading compounds in\ndeveloping steroid pharmaceuticals and medicines. However, it is a challenging role\nfor regioselective and stereoselective hydroxylation of steroid compounds either by\nchemical or biological approaches. Cytochrome P450 enzymes are known as an\nimportant biological hydroxylase for biosynthesis of steroid hormones in vivo. This\nmanuscript presents a groundbreaking work for engineering fungal P450\nCYP68J5_fg to achieve highly selective C12β- and C15α-hydroxylation of steroids.\nThe combination of structure-guided SSM and FRISM mutagenesis successfully\novercomes the classic activity-selectivity trade-off, yielding two variants (W12M5\nand W15M4) with exceptional regioselectivity (>97%). MD simulations revealed\nthat the key mutations reshape the substrate-binding conformation, thereby directing\nhydroxylation to distinct positions (C12β or C15α). The industrial relevance is\nsupported by unprecedented titers (up to 14.1 g/L) in P. pastoris fermentation. The\nmanuscript is suitable for publishing in Nature Communications after a further\nrevision.\n1. From the perspective of directed evolution, this is a surprisingly smooth job! The\nfirst step of directed evolution screening based on SSM strategy is a routine operation,\nbut I am a little concerned about calling the second stage of directed evolution\nFRISM. From the description in the text, it appears that this is somewhat different from\nthe classic FRISM. The author doesn't even need to perform a few limited tests at the\ntarget site, essentially just cross combining the single mutations obtained through the\nSSM strategy in the first round. Although the results may seem perfect, a more\naccurate description of this process should still be considered.\nAnswer: Thank you very much for your feedback and meticulous review of our\nmanuscript.\nRegarding whether the engineering approach employed in the manuscript\nqualifies as FRISM, we have reviewed the article of FRISM methodology (J. Am.\nChem. Soc. 2019, 141, 7934-7945; Mol. Catal. 2024, 553, 113755) and determined\nthat the method employed in this study aligns with the definition of FRISM (focused\nrational iterative site-specific mutagenesis).\nWe began by employing site-saturation mutagenesis (SSM) to identify key\nresidues and their corresponding beneficial mutations, followed by FRISM:\nFor the screening of C15α-hydroxylated products, SSM first identified four key\nresidues (Q112, D126, V299, A362) and their beneficial mutations (Q112A/M/T/S/C,\nD126T/V, V299I/L, A362M). Then, FRISM was performed: the variant V299L was\nselected as a template, and the beneficial mutations (D126T/V, A362M,\nQ112A/M/T/S/C) were individually introduced to generate 8 double variants. After\nscreening, the optimal double variant V299L/D126V was selected. Next, iterative\ncombinations with mutations A362M and Q112M/A/T/S/C produced 6 triple variants,\nthe subsequent screening led to the identification of the best triple variant\nV299L/D126V/A362M. Lastly, a final round of combination integrating the\nmutations Q112A/M/T/S/C generated 5 quadruple variants, and the catalytic\nefficiency test revealed V299L/D126V/A362M/Q112C to be the best-performing\nvariant.\nSimilarly, for screening C12β-hydroxylated products, by employing N295T as\nthe initial template, iterative combination with mutations at F107S, Q112R, V299T,\nand R368K efficiently yielded the optimal quintuple variant\nF107S/Q112R/N295T/V299T/R368K, after screening only 10 designed variants.\nIn summary, utilizing the FRISM approach, we constructed a focused yet\ncomprehensive library and successfully identified optimal variants after screening\nonly 29 mutants. This strategy significantly reduced experimental workload and\ncircumvented the need for exhaustive screening of all possible combinatorial\nmutations.\n2. In line 63, the authors describe that “fungal P450 enzymes demonstrate superior\nsuitability for industrial-scale production”, please provide more evidences or\nreferences.\nAnswer: Thank you very much for the comments.\nMicrobial hydroxylation of steroids is fundamentally mediated by enzymatic\ncatalysis. Current evidence indicates that steroid-hydroxylating enzymes in\nfilamentous fungi belong to the cytochrome P450 superfamily (KLINGENBERG M.\n1958, 75, 2, 376-386.). Numerous fungal industrial strains have been employed in\nsteroid drug synthesis.\nIndustrial applications of fungal P450-mediated steroid hydroxylation include:\n(1) Bayer's process employing Curvularia lunata mycelia containing P450\nenzymes to catalyze the 11β-hydroxylation of 11-deoxycortisol for hydrocortisone\nproduction (Process for the preparation of 11 β-hydroxy steroids. EP19800103478.\n1980).\n(2) Upjohn's biocatalytic route using Rhizopus arrhizus CYP509C12 to convert\nprogesterone to 11α-hydroxyprogesterone, thereby shortening the cortisol synthesis\npathway by >10 steps with significant cost reduction (Steroids. 1992, 57, 12, 593-\n616.).\nWe have cited these key references in our manuscript.\n3. As shown in Figure 2a, the dehydroxylated product 1c can be formed in this\nreaction. The direct dihydroxylation of steroids is also very important and interesting\n(see recent example, Wang et al. Catal. Sci. Technol., 2025, 4170-4178.). It is\nobserved that there is an unknown product by W12M1 in Figure 3d. Is this compound\na dihydroxylation product? I am wondered if the author has detected and identified\nthe generation of dihydroxy products during directed evolution. If so, it maybe your\nnext interesting paper.\nAnswer: Thank you very much for your comments and suggestions.\nIn Fig. 3d, the unknown product generated from 1 by mutant W12M1 is not the\ndihydroxylated product 1c. Compound 1c was previously identified in our earlier\nwork (“A Fungal P450 Enzyme from Fusarium graminearum with Unique 12β-\nSteroid Hydroxylation Activity.” Appl. Environ. Microbiol. 2023, 9, 3, e0196322.).\nDue to its dihydroxylated nature, 1c exhibits higher polarity than both the C15- and\nC12-monohydroxylated products, resulting in an earlier retention time during HPLC\nanalysis.\nRegarding the unknown product generated by W12M1 in Fig 3d, its structural\ncharacterization was not pursued owing to its low yield (approximately 3.8%) and\nthe fact that it disappeared upon further mutagenesis (not detectable in variants\ncatalyzed reactions).\n4. In Table 1, can the selectivity and conversion rate be accurately measured to two\ndecimal places?\nAnswer: Thank you very much for the suggestions. We will revise all the data in\nTable 1 and the main text to maintain precision using one decimal place.\n5. In line 329, the authors describe that “the low yield of 1a is potentially due to the\ncytotoxicity of 1a”, please provide the experiment details or references.\nAnswer: Thank you for your question.\nConsidering that the OD of the culture continued to increase alongside the\n600\naccumulation of 1a during the fermentation process, we have tentatively ruled out\ncytotoxicity as a contributing factor. Therefore, we suspect that the accumulation of\n1a may have caused product inhibition, leading to reduced substrate conversion. To\ntest this hypothesis, we conducted a new experiment, as shown in Supplementary\nFig. 12. As the concentration of 1a increases, the relative activity of W12M5 toward\nsubstrate 1 progressively decreases, indicating that high concentrations of the product\ninhibit the catalytic activity of the enzyme toward the substrate.\nWe have revised the mention of “cytotoxicity” in the main text manuscript\naccordingly and include this figure in the supplementary information.\nSupplementary Figure 12. Relative activity of GS115-W12M5 after 24 h reaction\nwith simultaneous addition of varying concentrations of 1a. The concentrations\non the x-axis represent the initial concentrations of compound 1a (0, 5, 10, 15,\n20 mM) added simultaneously with a fixed concentration (5 mM) of substrate 1 at the\nstart of the reaction.\n6. In line 382 and 383, the authors describe that “the integrated approach of SSM\ncoupled with FRISM strategy effectively modulates selectivity and enhances activity\nin these fungal-derived P450 enzymes”, to some extent, the approach may just\neffectively suitable for CYP68J5_fg, it would be better to state more precisely.\nAnswer: Thank you very much for your feedback and suggestion.\nWe have revised the sentence in the current line 387 (previously line 382 and\n383) by specifying “these fungal-derived P450 enzymes” as “CYP68J5_fg, with\npotential applicability to other fungal-derived P450 enzymes” to ensure greater\naccuracy.\n7. Except for substrate 6, the other four structurally similar substrates all showed\nexcellent selectivity and conversion rates. Did the author also examine for other\nsubstrates? If so, it is best to report the results, even if it is only included in the\nsupporting data. Because natural steroid hydroxylation P450 enzymes often have\nvery strict substrate limitations. Similar results are of great significance for the\ndevelopment of substrate-promiscuity P450 isoenzymes through protein\nengineerings.\nAnswer: Thank you very much for your feedback and suggestions.\nIn addition to substrates 2-6, we evaluated the enzyme variants against a panel\nof steroids with greater structural diversity, including lithocholic acid, pregnenolone,\n9(10)-dehydronandrolone, estrone, and D-ethylgonendione. While this screen\nconfirmed catalytic potential, all variants exhibited either severely diminished\nactivity or a complete loss of function against these substrates. Consequently, they\nwere excluded from the formal analysis. This initial activity, however, provides a\nfoundation for future protein engineering efforts aimed at improving both catalytic\nactivity and selectivity for structurally complex steroids.\n8. English language and style are required to fine spell check, such as “in line 175,\ncatalzyed should be catalyzed; in line 324, W1M4 should be W15M4.”\nAnswer: Thank you very much for the comments.\nWe have corrected “catalzyed” to “catalyzed” in the current line 172 (previously\nline 175) and “W1M4” to “W15M4” in the current line 326 (previously line 324).\nWe have further performed a thorough English language and style review of the\nmanuscript to ensure proper spelling and grammar."}, {"round": 2, "reviewer_comments": {"reviewer_1": "All of my concerns have been satisfactorily addressed. The manuscript is now suitable for publication. Congratulations to the\nauthors on this excellent work!", "reviewer_3": "I have carefully reviewed the revised manuscript. All of my concerns have been solved. I believe this is a nice work for regio\nand stereoselective hydroxylation of steroids compounds, thereby providing an efficient enzymatic approach for subsequent\ndevelopment of steroids pharmerceuticals, e.g., combining with chemical catalysis as discussed in the paper. This reviewer\nsuggests to publish the paper as is."}, "author_response": ""}]

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