Designing an oxidase toolbox for site-directed oxidation of taxanes

Domain: chemistry
Published: 2025-12-17
Editorial outcome: major_revision

Abstract
Paclitaxel is one of the most prominent drugs for cancer therapy, and its synthesis has long been pursued by scientific community. Although the biosynthetic pathway has been gradually unraveled, its synthetic efficiency remains severely constrained by the low activity and low product selectivity of the hydrolases involved. In this study, an artificial toolbox of oxidases derived from mutants of TteUPO (unspecific peroxygenase from Thielavia terrestris) is developed for site-directed oxidation of taxanes at the C-4, C-6, C-10, C-11, C-12, and C-13 positions. Furthermore, de novo biosynthesis of oxidized taxadiene products is achieved in an engineered E. coli chassis. These findings offer an unconventional strategy for developing an efficient route toward paclitaxel and constructing paclitaxel analogs with different structures for medicinal evaluation.

Introduction
Paclitaxel (Taxol®), a tetracyclic and functionally condensed diterpenoid, is renowned for its extensive anticancer efficacy. Its structural derivatives are widely utilized in the treatment of various malignancies (Fig. 1)1. Docetaxel and cabazitaxel, both classified alongside paclitaxel as first-generation taxane anticancer drugs, have therapeutic applications distinct from those of paclitaxel2,3. Next-generation taxoids feature modifications to the critical sites of paclitaxel, including the functional groups at C-10 and C-13. Notably, compounds such as SB-T-1214 have demonstrated remarkable efficacy against drug-resistant tumors4–6.

Paclitaxel is produced primarily via a semisynthetic method. Initial, 10-deacetyl-baclitatin III (10-DAB) is extracted from the needles of Taxus baccata L., after which its side chain is assembled through a 4-step chemical process7,8. Although this method has significantly improved the yield of paclitaxel compared with direct extraction from the bark of Taxus brevifolia Nutt.9–11, the clinical demand remains unfulfilled due to the long growth cycle and limited bioresources. The chemical synthesis of paclitaxel and its analogs has witnessed rapid advancements, yet challenges such as lengthy synthetic steps and low overall yields persist12–17.

In recent years, substantial progress has been made in elucidating and optimizing the natural biosynthetic pathway of paclitaxel, and all enzymes involved have been identified18–28. In 2010, the yield of taxadiene, the crucial paclitaxel precursor, produced by designed Escherichia coli cells was increased to 1 g/L18. The downstream biosynthetic of paclitaxel is severely limited by the low activity and low product selectivity of the cytochrome P450 monooxygenases (P450s) involved in native taxadiene oxidative derivation steps29–31. Despite the progress in elucidating the mechanism of taxadiene-5α-hydroxylase (T5αH), as the native enzyme responsible for the initial step in taxadiene oxidation, efforts to substantially enhance its catalytic efficiency and specificity via protein engineering have thus far proven unsuccessful32–34. To overcome the inherent limitations of T5αH, Xiao et al. investigated alternative taxadiene-oxidizing enzymes, finding that although CYP701A8 mediates C11-position oxidation, its application is severely constrained by pronounced allosteric effects35. Notably, Zhang et al. established the complete gene set requisite for heterologous paclitaxel biosynthesis and identified a taxadiene-epoxidizing enzyme that synergizes with T5αH to facilitate oxetane ring formation19. McClune et al. discovered FoTO1, an NTF2-like protein, can resolved the low product selectivity of T5αH, suppressing byproduct formation (e.g., OCT) and enabling the selective production of taxadiene-5α-ol (T5OH). This approach resulted in the isolation of baccatin III at ~8.7 μg/g DW from N. benthamiana36.

In the present study, we focus on identifying non-native taxadiene oxidases as alternatives to the native P450 enzymes. TteUPO (unspecific peroxygenase from Thielavia terrestris)37,38 is selected from 29 fungal-originated oxidases because of its highest activity toward taxadiene and excellent protein expression level in Pichia pastoris. We construct an enzyme toolbox comprising various TteUPO mutants, which exhibit distinct regioselectivities toward the taxadiene skeleton. Moreover, we engineer an E. coli BL21 (DE3) strain for the de novo biosynthesis of oxidized taxadiene products by introducing the taxadiene biosynthetic pathway and enabling the heterologous expression of a TteUPO mutant. These results lay a solid foundation for future development of artificial synthetic routes to paclitaxel and its analogs.

Results
Mining and characterization of taxane oxidases
Peroxidases and peroxygenases, both featuring heme coordinated by a cysteine ligand similar to that of P450s39–42, have been successfully utilized for the oxy-functionalization of carbon, sulfur, silicon, and nitrogen43–47. However, there are no reports of oxidases other than P450s that are capable of oxidizing taxanes. We initially screened 29 reported peroxidases and peroxygenases of fungal origin37,38,48–54, which were expressed in Pichia pastoris (syn. Komagataella phaffii). Among these, 13 oxidases exhibited activity toward taxadiene, producing identical oxidation products. TteUPO (unspecific peroxygenase from Thielavia terrestris)37,38 was chosen due to its highest activity and excellent protein expression level (Supplementary Note 1, Supplementary Tables 1, 2 and Supplementary Fig. 1, 2). TteUPO catalyzes the formation of hydroxylation products through the C-H activation of C-4 and C-6 positions in the taxadiene skeleton (Fig. 2A). Compounds 3 and 4 were identified as stereoisomers of the C-4 hydroxylation product ((S)-taxa-5(6),11(12)-dien-4-ol (3) and (R)-taxa-5(6),11(12)-diene-4-ol (4)), whereas 5 and 6 were found to be stereoisomers of the C-6 hydroxylation product ((R)-taxa-4(5),11(12)-diene-6-ol (5) and (S)-taxa-4(5),11(12)-dien-6-ol (6)). Additionally, during analysis of the products by gas chromatography (GC), elimination of the allylic products resulted in a diene compound, namely, taxa-4(20),5(6),11(12)-triene (2) (Fig. 2B and Supplementary Fig. 3, 4, 5). This phenomenon was also observed when compounds 3~6 were dissolved in CDCl3 for NMR experiments, indicating a facile elimination process that is enhanced by heat and acidic conditions.

In contrast to the oxidation pattern of T5αH17–20, TteUPO catalyzed the oxidation of taxadiene to generate various hydroxylation substances that were distinct from byproducts such as OCT, iso-OCT or other compounds that constituted a large proportion of the products generated by T5αH in previous investigations18,29,30 (Fig. 2C). The oxidation of taxadiene by TteUPO was localized to either the C4=C5 double bond or the allyl position of ring C. To elucidate the possible reaction pathway, taxadiene-4(5)-epoxide (7) was prepared by oxidation of taxadiene with DMDO55,56 (Fig. 2C and Supplementary Fig. 6). This unstable epoxy intermediate undergoes spontaneous rearrangement to form the products T5OH, OCT, and iso-OCT20,22,33,34. In TteUPO-catalyzed oxidation of taxadiene, taxadiene-4(5)-epoxide was not detected. Furthermore, epoxide 7 was not converted to compounds 3~6 by TteUPO. These observations led us to conclude that TteUPO-catalyzed taxadiene oxidation circumvents the taxadiene-4(5)-epoxide intermediate, thereby preventing the accumulation of byproducts (e.g., OCT). This finding also offers an alternative strategy to FoTO1 for addressing the McClune et al. discovered FoTO1, an NTF2-like protein, can resolved the low product selectivity of T5αH.

Protein engineering of TteUPO
Next, the catalytic efficiency was enhanced by protein engineering. Interestingly, the regioselectivity of TteUPO was also modified to enable oxidation at distinct sites of taxadiene. The crystal structure of TteUPO (PDB 9IZ8) was analyzed at a high resolution of 1.54 Å (Supplementary Fig. 7 and Supplementary Table 3). The conformation of substrate channel has been found to substantially influence the activity and selectivity of UPOs57–61. Molecular docking was performed via AutoDock Vina62, the fining docking structure indicates that the substrate channel of TteUPO is narrowed due to excessive steric hindrance of amino acids within the channel, thereby restricting the entry and binding of taxadiene (Supplementary Fig. 8). Furthermore, given the minimal polarity and strong hydrophobicity of taxadiene, which prevents hydrogen bond formation with surrounding amino acids, amino acids lining the substrate channel were mutated to alanine (Ala)—a sterically less hindered nonpolar amino acid. These mutations widen the channel and identify sensitive residues for further precise regulation of the channel geometry (Supplementary Tables 4 and 5).

The mutation of Leu208, which is located at the entrance of the substrate channel, to Ala, which results in less steric hindrance, widened the “bottlemouth”. This modification facilitated the entry of taxadiene into the active center (Fig. 3C, D). The L208A mutant exhibited a significant improvement in catalytic activity, with no alteration in product diversity (Fig. 3A, Table 1 and Supplementary Fig. 9A, 9C). The total turnover number (TON) increased from 82 to 4355, a marked 53-fold increase.

Notably, the L60A mutant exhibited enhanced activity and demonstrated low product selectivity compared with the WT (Fig. 3A, B, Table 1 and Supplementary Fig. 9B, 9C). Leu60, which is positioned above the heme porphyrin ring, was substituted with alanine, thereby enhancing substrate accessibility to the catalytic site (Fig. 3E, F) and thus increasing its oxidative activity (Table 1). Moreover, expansion of the inner side of the substrate channel increases the diversity of the substrate binding conformations, resulting in the formation of more than 6 oxidation products (Fig. 3A, B). In contrast to the WT, the L60A mutant tended to attack the C11=C12 double bond in ring A. In addition to the generation of hydroxylation products at the allylic positions (i.e., C-10 and C-13), epoxidation of the double bond in ring A was also observed, along with further oxidation of C-6 in ring C (Fig. 3A, B). Notably, the oxidation sites and configurations at C-10 and C-13 were identical to those in paclitaxel.

The L60S mutant exhibited activity and selectivity profiles comparable to those of L60A (Supplementary Fig. 9B). Although serine possesses a polar hydroxyl group not present in alanine, both residues impose similar steric constraints. This result further confirms that steric hindrance, rather than side-chain polarity, is the primary determinant of TteUPO’s activity and selectivity in taxadiene oxidation. Interestingly, the L60A mutant exhibits remarkable versatility, catalyzing oxidations at the C-4, C-6, C-10, C-11, C-12, and C-13 positions of taxadiene. This renders it an excellent starting point for constructing a mutant library tailored to the regioselective oxidation of taxadiene. Mutants generated through FLA scanning (replaced with Phe, Leu, or Ala) and STY scanning (replaced with Ser, Thr, or Tyr)63, along with site-directed saturation mutagenesis of amino acids within the substrate channel, were then specifically designed to enhance the regioselective oxidation capabilities of the mutant (Supplementary Fig. 10).

The combined mutant L60A/F156A enables catalysis of epoxidation at C-11 and C-12 and oxidation at C-6 on taxadiene, resulting in the formation of compound 16 (Table 1). Compared with the L60A/F156A mutant, the L60A/F156L mutant exhibits greater steric hindrance at position 156 due to the bulkier leucine residue replacing alanine. This increased steric clash further constrains the binding conformation of taxadiene and limits the positioning of ring C near heme-Fe. Additionally, the “concave surface” of taxadiene can only enter the active site with the concave side facing Leu156, positioning the double bond at ring A close to the heme-Fe (Fig. 3G and Supplementary Fig. 11C). As a result, the C-10 and C-13 hydroxylation products (compounds 11 and 12), along with the C-11 and C-12 epoxidation product (compound 9), dominate for over 90% of the products, while oxidation at C-6 occurs only rarely (Table 1).

Notably, the L60A/G159L mutant exhibited a significant increase in selectivity, specifically increasing the proportion of the C-10 (compounds 11) and C-13 (compounds 12) hydroxylation products to 66% and 19%, respectively, with a conversion of 80.2% (Table 1). In the presence of Leu159, which exhibits greater steric hindrance and thus constrains other substrate binding conformations, the substrate preferentially enters the channel with a parallel conformation relative to the heme plane. Consequently, ring B of taxadiene comes closest to heme-Fe, thereby favoring the hydroxylation product at C-10 of ring B. However, as the distance increases, the oxidation activity slightly deteriorates (Fig. 3H, Supplementary Fig. 11A, 11B, and Supplementary Table 6). To restore activity, the L60A/G159L mutant was combined with the activity-enhancing L208A mutation to generate the triple mutant L60A/G159L/L208A. This modification led to a significant performance improvement, achieving a turnover number (TON) of 757. Additionally, the selectivity for the hydroxylation product at C-13 was increased to 33% (Table 1). The L60A/G159L/L208A mutant also exhibited excellent scalability. In a 100 mL reaction system, the yield of 0.5 mM taxadiene catalyzed by 4 mg of enzyme reached 75.3% within 2 h.

Development of a taxane oxidase toolbox
Seven TteUPO mutants were employed to construct an engineered oxidase toolbox, enabling site-specific oxidation of taxanes (Fig. 4A). Eleven distinct oxidized products were structurally characterized, revealing six oxidizable sites on the taxane scaffold (Fig. 4B and Supplementary Fig. 12). Compounds 11 and 12 exhibit regioselectivity and stereoselectivity consistent with those of paclitaxel, making them potential building blocks for reconstructing the paclitaxel synthesis pathway. Notably, the L60A/F156A and L60A mutants exhibit poor regioselectivity in catalyzing the oxidation of taxadiene. However, when additional steric hindrance is introduced at C-10 on ring B or C-4 on ring C of taxadiene (compound 11 and 4), these mutants exhibit high regioselectivity in oxidizing taxanes, yielding compounds 17 and 18. This highlights the versatility of the toolbox, which not only has been proven effective for taxadiene but also demonstrates even greater performance for taxadiene derivatives. The oxidation sites of compounds 5, 9, 13, 15, 16, 17, and 18 differ from those of paclitaxel, and these distinct chemical modifications may result in diverse pharmacological activities, while oxidation at the C-6 position is associated with paclitaxel metabolism in the human body64.

The biosynthesis of paclitaxel involves eight oxidative modifications of the taxadiene core structure (Fig. 1). The L208A mutant, which exhibits the highest activity, was employed to probe the oxidation capabilities of this engineered enzyme toolbox toward higher-oxidation-state taxane derivatives (compounds 19/21/23) (Fig. 4 and Supplementary Fig. 12). The introduction of a C2-carbonyl group in compound 19 (vs. taxadiene) dramatically enhances both regioselectivity and stereoselectivity in L208A catalysis, with compound 20 accounting for over 99% of the total products. L208A selectively oxidizes the configurationally inverted isomer 21 to compound 22, but shows no activity toward its stereoisomer 23. We ultimately achieved the synthesis of tri-oxygenated taxanes, demonstrating the potential of our established enzyme toolbox to generate high oxidation state taxane derivatives.

In vivo biosynthesis of oxidized taxadiene
Finally, we attempted to construct a biosynthetic pathway for in vivo production of oxidized taxadiene derivatives. Building upon a high-yielding E. coli taxadiene production chassis established in our previous work65, we successfully achieved functional expression of the L208A mutant in E. coli through N-terminal signal peptide truncation (Supplementary Fig. 14). Subsequent integration of this artificial oxidase module into the taxadiene-producing chassis, coupled with exogenous H2O2 supplementation, achieved de novo biosynthesis of taxane oxidation products at a total titer of 14.2 mg/L (Fig. 5).

Methods
Strains, genes and vectors
The gene sequences of oxidases were codon-optimized for expression in Pichia pastoris (syn. Komagataella phaffii) and cloned into the pPICZA vector by GenScript (Shanghai, China). Plasmid cloning was carried out using Escherichia coli TOP10 (Novagen, Darmstadt, Germany). Pichia pastoris X-33 was employed for the expression of oxidases.

A 6×His tag was introduced after the signal peptide of TteUPO, with the specific primers detailed in Supplementary Table 1. All primers were synthesized by Tsingke Biotechnology Co., Ltd. (Shanghai, China).

All PCR reactions, including saturation mutation experiments, were conducted using PrimeSTAR® HS DNA polymerase (Takara, Shiga, Japan). The insertion of the 6×His tag was achieved utilizing homologous recombination assay kits (ClonExpress Ultra One Step Cloning Kit, Vazyme, China).

Shake flask cultivation of the recombinant P. pastoris X-33
Shake flask cultivation of the recombinant Pichia pastoris was used for the activity screening.

The recombinant P. pastoris X-33 strains were cultured for 24 hours in YPD medium containing 100 μg/L zeocin (30°C, 220 rpm). Subsequently, the strain was inoculated with 1% (vol/vol) of the inoculum into 50 mL MD medium (20 g/L peptone; 10 g/L yeast extract; 10 g/L glycerol) and incubated at 30°C for 16–18 h. After allowing it to stand at room temperature for 4 hours to separate the supernatant and precipitate, 50 mL of induction medium (20 g/L peptone; 10 g/L yeast extract; 13.4 g/L YNB; 100 mM potassium phosphate buffer pH 6.0; 400 μg/L biotin; 200 μM ALA (5-aminolevulinic acid), 3 mM MgSO4) was added to the precipitation.

The culture was then incubated with 1% methanol every 24 h for total 72 h.

To confirm that oxidases has been expressed, the activity of the expressed protein was assessed using the substrate ABTS (2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt). The assay reaction mixture, containing 1 mM ABTS, 2 mM H2O2, and citrate buffer (pH 4.5), was incubated at 30°C for 1 minute, and the absorbance change at 418 nm was measured (ε418 = 36,000 M−1 cm−1)48.

Screening of oxidases
29 reported oxidases, with protein sequences and signal peptides consistent with the published literatures, were screened. The expression vector was changed to pPICZA with the AOX1 promoter, and the enzymes were expressed in P. pastoris X-33. ABTS was utilized to confirm the correct expression of these oxidase. Subsequently, the expressed oxidases were employed to catalyze the oxidization of taxadiene, with 13 of them demonstrating activity against taxadiene and yielding the same oxidized products (Supplementary Fig. 1 and Supplementary Table 2). TteUPO was chosen due to its highest activity and excellent protein expression level.

The taxadiene oxidation reactions catalyzed by oxidases were conducted at 30°C in a citrate buffer solution (CPBS) with a pH of 5.5. The reaction mixture contained 0.5 mM taxadiene, 2 mM H2O2, 30% (vol/vol) acetonitrile, and 50 μL/500 μL concentrated crude enzyme solution was incubated for 1 hour at 200 rpm. Crude enzyme solution was concentrated 50-fold from the fermentation supernatant.

Batch feeding cultivation of recombinant P. pastoris X-33
Batch feeding cultivation of TteUPO and its mutants are used for products preparing.

Initial Fermentation Medium: Dissolve 1.14 g/L of CaSO4·2H2O, 14.3 g/L of K2SO4, 12.2 g/L of MgSO4·7H2O, 3.3 g/L of KOH, 21 mL/L of phosphoric acid, and 40 g/L of glycerol in deionized water and subject the medium to autoclaving at 121°C for 20 min. Prior to inoculation, adjust the fermentation pH to 6.0 using ammonia solution and supplement with 12 mL/L of PTM1.

Glycerol Feeding Medium: Dissolve 600 g/L of glycerol in deionized water to a total volume of 400 mL. And subject the medium to autoclaving at 121°C for 20 min. After cooling, add 4.4 mL/L of PTM1 in a sterile environment.

Methanol Feeding Medium: Add 12 mL/L of PTM1 to pure methanol.

PTM1: Dissolve the following in deionized water in the specified order: 0.2 g/L biotin, 6 g/L CuSO4·5H2O, 0.09 g/L KI, 3 g/L MnSO4·H2O, 0.2 g/L Na2MoO4·2H2O, 0.02 g/L H3BO3, 0.9 g/L CoCl2·6H2O, 42.2 g/L ZnSO4·7H2O, 5 mL/L sulfuric acid, and 65 g/L FeSO4·7H2O. Filter the medium using 0.22 μm filtration to sterilize.

Batch feeding cultivation of TteUPO and its mutants were performed in the following five stage.

Stage 1: Activation of preserved recombinant P. pastoris X-33 strains by streaking on YPD plates. Pick single colonies and inoculate into 4 mL of YPD culture medium for 24 h. Inoculate 1% into 250 mL of MD medium and incubate overnight at 30°C until OD600 = 2–6.

Stage 2: In a 5 L tank containing 2.5 L of liquid, use a 10% inoculation rate and incorporate 1 mM final concentration of Ampicillin, and culture at 28°C. Set up a dissolved oxygen (DO)-stirring linkage, increasing stirring speed when DO < 30% in the range of 200-1200 rpm. Approximately 17–18 h later, there will be a notable increase in DO. Following manually supplementing glycerol, DO will decrease, marking the start of automatic feeding. The DO-STAT system is configured to feeding when DO exceeds 30%, with observed fluctuations maintained around 30% DO. The aeration rate is controlled at 1-2 vvm.

Stage 3: When the OD600 reaches a value between 150 and 200, stop feeding. At this point, DO will rapidly increase to a plateau at around 100% and remain steady for about 1 h, ensuring complete consumption of glycerol to prevent any interference with the subsequent methanol induction.

Stage 4: Add 200 μL/L of 1 M ALA, and infuse methanol at a rate not exceeding 7 mL/L/h for a duration of 2 hours to acclimatize the cells to methanol metabolism. Following this acclimation period, configure the DO-STAT to automatically supplement methanol when DO exceeds 30%. The induction culture is maintained for a total of 72 h.

Stage 5: Centrifuge the fermentation broth at 12,000×g for 30 min and collect the supernatant from the fermentation.

Purification of TteUPO
Protein purification was carried out using Ni-NTA. Initially, impurities were eluted using buffer A (20 mM sodium dihydrogen phosphate, 500 mM sodium chloride, pH 7.4) with twice the column volume, followed by elution of TteUPO using buffer B (20 mM sodium dihydrogen phosphate, 500 mM sodium chloride, 50 mM imidazole, pH 8.0) with twice the column volume.

The protein content was determined using the Coomassie brilliant blue method.

Deglycosylation of TteUPO was performed with SpEndo H66 (mg TteUPO: mg SpEndo H=5:1). The deglycosylated product was separated by Ni-NTA. Finally, the results of protein purification and deglycosylation were analyzed using SDS-PAGE (Supplementary Fig. 2).

Crystallization and structure determination
The crystallization was carried out at 18°C in 96-well plates using sitting-drop vapor-diffusion with the commercial crystal condition (1.6% v/v MorpheusⅢ® Dipeptides Mix, 0.1 M Morpheus®-Buffer System 1 pH 6.5, 37.5% Precipitant Mix 4). The droplets consisted of 1 μL of 18 mg/mL purified TteUPO in buffer (10 mM potassium phosphate, pH 7.4) and 1 μL of the precipitant solution and were equilibrated against 50 μL of the precipitant solution. The crystals were flash-cooled in liquid nitrogen for data collection. Diffraction data were collected on beamlines BL10U2 and at Shanghai Synchrotron Radiation Facility. Data were processed with XDS and HKL2000 (Supplementary Fig. 7).

The structure was solved by molecular replacement with the structure of TteUPO predicted by AF267. Iterative model building and refinement were performed using WINCOOT 0.9.8.168 and PHENIX 1.21.1.526869, respectively (Supplementary Table 3).

GC-MS analysis
The analyses were performed with GCMS-QP2010 SE (Shimadzu, Kyoto, Japan), using an HP-5MS fused silica capillary column (30 m, 0.25 mm i.d., 0.50 μm film; Agilent). The oven temperature was from 80°C to 220°C (15°C min−1) and held for 3 min, then by 20 min−1 to 280°C, and held for 5 min. The scan range was set to m/z 35-400 and the electron ionization energy was 70 ev.

The reaction mixture was extracted with an equal volume of ethyl acetate, which contained 50 mg/L of octadecane as the internal standard for GC-MS analysis. Standard concentration curve of taxadiene is showed in Supplementary Fig. 3.

The quantitative analysis was performed using GC peak area integration, with the sample size set to n=3. MS was employed for compound identification.

Preparation of taxadiene
The preparation of taxadiene (1) follows the established method from our laboratory65. After 72 h of induction culture of recombinant Escherichia coli BL21 (DE3), the cells were harvested via centrifugation, separating the supernatant from the bacterial sediment. The supernatant was subjected to extraction with an equal volume of ethyl acetate, while the bacterial pellet was soaked in an appropriate volume of methanol overnight before being extracted with an equivalent volume of n-hexane. The resulting extract was then dried using an appropriate amount of anhydrous sodium sulfate, and the solvent was removed through rotary evaporation at −0.1 Mpa and 30°C. Preliminary purification of taxadiene was performed via silica gel column chromatography, followed by preparative HPLC to isolate taxadiene and its isomers. For the silica gel column chromatography, taxadiene was eluted by n-hexane. Detection was performed by TLC, developed with n-hexane, and observed by staining with phosphomolybdic acid. The preparative HPLC was conducted using an UltiMate 3000 chrome system (Thermo Fisher Scientific, USA) equipped with an Ultimate® XB-C18 column (Welch, China). The conditions were set as follows: 100% acetonitrile at a flow rate of 3 mL/min, with detection at 210 nm UV.

Preparation of taxanes
Compound 19, 21, and 23 were synthesized according to the protocol developed by the Baran’s group15,70.

The synthesis begins with the dibromocyclopropanation of a starting alkene using bromoform (CHBr3) and potassium tert-butoxide (t-BuOK) as base. This resulting cyclopropane intermediate undergoes thermal rearrangement in N,N-dimethylaniline (PhNMe2) to form a brominated alkene. In a separate branch, a cyclohexanone derivative is subjected to nucleophilic addition with vinylmagnesium bromide to introduce a vinyl group. The two segments are then converged. The vinyl-functionalized cyclohexanone is deprotonated with n-butyllithium (n-BuLi) and protected with trimethylsilyl chloride (TMSCl). A key copper(I)-catalyzed conjugate addition is subsequently carried out using methylmagnesium bromide (MeMgBr) in the presence of a copper(II) triflate/N-heterocyclic carbene (NHC) catalyst system. The resulting enolate is trapped in an aldol reaction with acrolein, and the product is oxidized with Dess-Martin periodinane (DMP) to yield an advanced intermediate. This intermediate is then treated with boron trifluoride diethyl etherate (BF3·OEt2), which triggers Diels–Alder cyclization reaction to form the core ring structure, yielding compound 23 and 21. The final step is a palladium-catalyzed Negishi coupling between compound 23 and dimethylzinc (Me2Zn), using tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4) as the catalyst. This coupling installs the final methyl group, affording the desired product (compound 19) (Supplementary Fig. 13).

Biotransformations
Unless otherwise specified, the biotransformations of taxanes were conducted at 30°C in a citrate buffer solution (CPBS) with a pH of 5.5. The reaction mixture contained 0.5 mM substrates, 2 mM H2O2, 30% (vol/vol) acetonitrile, and 40 μg/mL enzyme was incubated for one hour at 200 rpm. The addition of 5 mM 2-hydroxypropyl-β-cyclodextrin was found to be beneficial for the biotransformation of taxadiene.

The analytical-grade reaction is conducted in a 2 mL glass injection bottle for a 0.5 mL reaction, whereas the preparative-grade reaction takes place in a 2 L glass shake flask for a 500 mL reaction.

Isotope labeling
Utilizing H218O2 (Sigma-Aldrich®, Germany) as oxygen donor to oxidize taxadiene, under the conditions described in “Biotransformations” section, the molecular weight of compound 5 shifted from 288 to 290 (Supplementary Fig. 4). This suggests that H2O2 serves as the oxygen donor for unspecific peroxygenase during the oxidation of taxanes.

Product preparation and isolation
The reaction system was scaled up to 500 mL and conducted in a 2 L glass shake flask. The supernatant was extracted with ethyl acetate without an internal standard, dried with an appropriate amount of anhydrous sodium sulfate, and concentrated by rotary evaporation at −0.1 Mpa and 30°C. Subsequently, the resulting product underwent rough separation using silica gel column chromatography. The target compounds were then collected and further purified through preparative HPLC using an UltiMate 3000 chrome system (Thermo Fisher Scientific, USA) equipped with an Ultimate® XB-C18 column (Welch, China). For the silica gel column chromatography, compounds 2-16 were eluted by n-hexane: ethyl acetate = 30:1. Detection was performed by TLC, developed with a mixture of n-hexane: ethyl acetate in = 6:1, and observed by staining with phosphomolybdic acid. Compounds 17-22 were eluted by n-hexane: ethyl acetate = 10:1, and its detection was carried out by TLC with a development agent of n-hexane: ethyl acetate = 1:1, followed by staining with phosphomolybdic acid. The preparative HPLC conditions are as follows: 100% acetonitrile at a flow rate of 3 mL/min, with detection at 210 nm UV.

Analysis of catalytic mechanism
We synthesized taxadiene-4(5)-epoxide (7) using DMDO55,56, and analyzed it by GC-MS and 1H NMR as shown in Supplementary Fig. 6.

Synthesis procedure of taxadiene-4(5)-epoxide (7): NaHCO3 (12.00 g, 142.85 mmol) was dissolved in a mixture of H2O (10 mL) and acetone (15 mL). The reaction vessel was placed in an ice-water bath with stirring at 200 rpm. Oxone® (12.5 g total) was added batchwise within 1 min. After stirring for 30 min, the mixture was extracted with diethyl ether (20 mL). The resulting ethereal extract containing DMDO was stored in an ice-water bath. Taxadiene was dissolved in the DMDO/ether solution to a final concentration of 2 mM and reacted with stirring (200 rpm) in an ice-water bath for 1 h. The solvents were removed by rotary evaporation under reduced pressure. Taxadiene-4(5)-epoxide (7) was purified using silica gel column chromatography, eluted by n-hexane/ethyl acetate (30:1, v/v). Reaction progress and product fractions were monitored by TLC (silica gel, n-hexane/ethyl acetate = 6:1, v/v), with visualization achieved by staining with phosphomolybdic acid. The target compound was then collected and further purified through preparative HPLC using an UltiMate 3000 chrome system (Thermo Fisher Scientific, USA) equipped with an Ultimate® XB-C18 column (Welch, China).

Selection of amino acids within the substrate channel
L56, L60, F63, F156, G159, and L208 were selected for the first round of alanine scanning (Supplementary Fig. 8A). It is worth noting that E160 is a conserved site though to be involved in the oxidation reaction with H2O2, and saturation mutagenesis at this position has been significantly diminished the activity.

Building upon the L60A mutant, the following residues were selected for FLA and STY scanning: L56, L59, F63, A155, F156, G159, L208, A163, and the residues M20 and L21 located behind the heme (Supplementary Fig. 8B).

Construction of mutagenesis library
Site-directed mutagenesis was conducted by performing whole-plasmid PCR amplifications of the parental plasmids using PrimeSTAR HS (premix) from Takara along with the respective mutagenesis primers (Supplementary Table 4) carrying the designed mutations. The amplified product was then subjected to digestion with Dpn I (NEB) at 37°C for 2 h to selectively degrade the parental methylated strands. Subsequently, the digested product was transformed into Escherichia coli TOP10 cells. For site-directed mutagenesis, an Escherichia coli TOP10 colony of the designed mutant was selected from an agar plate and inoculated into 4 mL of LB medium (containing 5 g/L NaCl, 5 g/L yeast powder, and 10 g/L peptone) supplemented with 100 μg/mL zeocin in a glass tube, and cultivated at 37°C and 200 rpm. After 12 h of cultivation, the recombinant plasmid was extracted. Linearization of the recombinant plasmid was performed using SacⅠ for 3 h. Linearized fragments were then electro-transformed into P. pastoris X33 with a micropulser (BioRad, Hercules, CA, USA). The mutants were cultivated in 250 mL shake flasks containing 50 mL of media (as described in “Shake flask cultivation of the recombinant P. pastoris X-33” section).

Site-directed saturation mutagenesis involves mutating a specific site to each of the other 19 amino acids.

Screening of mutants
The mutants were cultivated in 250 mL shake flasks containing 50 mL of media and the secreted supernatant was subsequently purified using Ni-NTA (as described in “Purification of TteUPO” section). The purified enzymes were evaluated for their activity and selectivity through reactions with taxadiene, with the results analyzed via gas chromatography (GC) (as described in “GC-MS analysis” section) (Supplementary Tables 5, 6 and Supplementary Fig. 9-11).

MS, HR-MS and NMR spectroscopy
MS data were measured as described in “GC-MS analysis” section (Supplementary Fig. 15). HR-MS data were measured on a GCT Premier mass spectrometer (Waters Corporation, USA) (Supplementary Fig. 16). HR-MS analysis was performed using dried pure compounds, with a sample size of n=1. NMR spectra were recorded on a Bruker (Billerica, MA, USA) AV Neo 400 MHz, AV Neo 500 MHz, or AV Neo 600 MHz at ambient temperature. Spectra were referenced against solvent signals (1H-NMR, residual proton signal of C6D6: δ=7.16 ppm, CDCl3: δ=5.32 ppm; 13C-NMR, signal of C6D6: δ=128.06 ppm, CDCl3: δ=53.84 ppm), with a sample size of n=1. (Supplementary Tables 7–21 and Supplementary Figs. 17–121).

Construction and cultivation of recombinant E. coli
L208A codon optimization for E. coli was performed at GenScript (Shanghai, China), and the gene was synthesized into the plasmid pET-28a (+). After removal of the N-terminal signal peptide, L208A was successfully expressed in E. coli and exhibited activity against ABTS and taxadiene (Supplementary Fig. 14A–14F). By optimizing the concentration of the inducer IPTG to 0.1 mM during a 25-h induction, the expression level of L208A was enhanced by over two-fold (Supplementary Fig. 14G). H2O2 supplementation was initiated after the first 8 hours of induction and repeated every 8 h (total of 3 additions). When H2O2 concentration per addition was kept below 5 mM, bacterial growth was largely unaffected (Supplementary Fig. 14H). Based on these results, we transformed the recombinant plasmid carrying L208A into a taxadiene-producing E. coli chassis strain constructed previously in our laboratory (Supplementary Fig. 14I)65. The engineered E. coli chassis for taxadiene production was designed to overexpress two key upstream enzymes, 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and isopentenyl diphosphate isomerase (IDI), to enhance the supply of the precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). This elevated precursor pool then enabled the de novo biosynthesis of taxadiene through heterologous expression of geranylgeranyl pyrophosphate synthase (GGPPS) and taxadiene synthase (TS).

The recombinant E. coli strain was cultured under the following conditions: A 2% inoculum was transferred into 50 mL of Terrific Broth (TB) medium (12 g/L peptone, 24 g/L yeast extract, 5.2 g/L glycerol, 2.31 g/L KH2PO4, 12.54 g/L K2HPO4). Initial cultivation was carried out at 37°C and 200 rpm for 3 h, followed by temperature reduction to 20°C for 30 min. Subsequently, 0.1 mM IPTG was added, and cultivation continued for another 16 h. Then, 2.5 mM H2O2 was added, and cultivation proceeded for an additional 8 h. Finally, the fermentation broth was extracted with an equal volume of ethyl acetate for GC analysis.

Statistics and reproducibility
Unless otherwise specified, all statistical experiments were determined with n=3. No statistical method was used to predetermine sample size. No data were excluded from the analyses. The experiments were not randomized. The Investigators were not blinded to allocation during experiments and outcome assessment.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript presented by Yu group describes the C-H functionalization of taxadiene at different positions by unspecific peroxygenases (UPOs). This research first benchmarks several UPO enzymes to be potential candidates towards taxadiene oxidation. After selecting TteUPO as departure point, the enzyme was crystalized and subjected to rational design. As a result, up to 9 oxidation products were obtained opening a framework for future protein engineering. Without questioning the quality of the work, this reviewer feels that this study is not offering great novelty in the field. Over the years, P450s have been studied for taxadiene oxidation, and basically UPOs can achieve similar chemistries, yet only triggered by peroxide (instead of depending on auxiliary flavo proteins and expensive redox cofactors). The low TON obtained along with the absence of information about suicide inactivation -which could preclude the use of this system for practical purposes- are major issues that deserve further attention. Besides, new recent studies about P450/taxadiene are not mention (e.g. https://doi.org/10.1021/acs.orglett.4c00103). All in all, I am afraid I cannot support this manuscript for publication in Nat Comm and I would suggest to send it to a more specialized journal.", "reviewer_2": "The present manuscript titled as ‘Designing an Oxidase Toolbox for Site-Directed Oxidation of Taxanes’, developed a mutant library of TteUPO (a previously reported unspecific peroxygenase by other researchers) through site-directed mutagenesis, which have been systematically characterized to catalyze the oxidation of different sites of taxanes. This approach provided diverse novel taxol derivatives by in vitro reactions.\nComments:\n1.A study in 2016 (Orthogonal Assays Clarify the Oxidative Biochemistry of Taxol P450 CYP725A4, ACS Chem. Biol. 2016, 11, 1445.1451) has shown that taxadiene could be converted to OCT and other compound by H2O2. Why were there no products for negative control (no enzyme) in Fig.1A? Besides, please list the details of samples used for analyses in Figure legends (not only for this Figure) to make it easy to understand the details of experiments.\n2.These products generated by the mutants of TteUPO belong to low oxygenated level taxoids that are far away from paclitaxel analogs, which may take effects on their biological activities. Could these mutants of TteUPO catalyze the produced novel derivatives in this study to higher oxygenated level toxoids, for example, to produce taxanes with more than four oxidation modification? On the other hand, Could these mutants of TteUPO catalyze high oxygenated level toxoid?\n3.The biotransformation catalyzed by TteUPO and its mutants depend on the supply of high concentration H2O2 which is harmful or unfriendly for most of common microbial chassis cells e.g. yeast and E.coli. Did TteUPO and its mutants show catalytic activity toward taxadiene in vivo?\n4.The GC analysis of the L60A mutant of TteUPO revealed several new peaks, but many new peaks at retention times of approximately 19.0 min, 19.2 min, and between 17.5 min and 18 min (Figure 3A) were not identified. It is essential for the authors to identify and elucidate the structures of these new compounds.\n5.In Figure 3A, a new peak labeled 13 is mentioned, the structure of the corresponding compound should also be provided.\n6.The author should carefully review the Supporting Information (SI). It is inappropriate to include chemical structures with HMBC correlations in the COSY and HSQC NMR spectra. Instead of 2D structures, which make it difficult to interpret the mentioned NOE correlations, 3D-models of the newly identified compounds with highlighting key NOE interactions should be provided.\n7.The detail about the chemical synthesizing compound taxadiene-4(5)-epoxide (18) should be provided in SI.\n8.Based on the NMR spectra, Compound 11 is identified as the minor component, representing only 1/6 of the mixture. To ensure the accuracy of the structure presented in the manuscript, it is essential to provide the NMR spectra of the pure compound.\n9.In order to make the comparison of mutants, it is better to add GC analyses of taxadiene oxidation catalyzed by all mutants of TteUPO mention in the text, e.g. mutants L60A/F156A, L60A/F156L, and L60A/G159L/L208A.\n10.Some inaccurate descriptions about the background information in main part greatly weaken the quality of this manuscript,please check the accuracy of research background part:\n1)“However, the yield of paclitaxel produced in Nicotiana benthamiana is only 64.29 ng/g plant material. This extremely low yield is due mainly to the low activity and severe catalytic promiscuity of the cytochrome P450 monooxygenases (P450s) involved in native downstream taxadiene oxidative derivation steps”. In ref.26, Zhang et.al detected a low level of paclitaxel (64.29 ng/g) accumulation in the leaves of N. benthamiana by feeding baccatin III, in which the P450 might not be the only rate-limiting enzyme.\n2)‘Despite the progress in elucidating the mechanism of T5OH, the native enzyme in the initial step of taxadiene oxidation, attempts to substantially enhance its catalytic efficiency and specificity through protein engineering have thus far been unsuccessful’, in fact, a new study from bioRxiv (preprint doi: https://doi.org/10.1101/2024.11.06.622305) has overcame this problem. Please add the updated information.\n3)“In recent years, substantial progress has been made in elucidating the natural biosynthetic pathway of paclitaxel, and all enzymes involved have been identified18-32. However, some of these references (18-23) have no relationship with the elucidation of the natural biosynthetic pathway of paclitaxel. Besides, the contribution of professor Rodney Croteau should not be ignored. Please change the references with these papers from professor Rodney Croteau.\n4)“TteUPO (unspecific peroxygenase from Thielavia terrestris)36, 37 was chosen due to its highest activity and excellent protein expression level (Table S1, S2 and Figure S1, S2)”, however, Figure S2 has no relationship with the activity and protein expression level of TteUPO.", "reviewer_3": "The authors presented a review article on “Designing an Oxidase Toolbox for Site-Directed Oxidation of Taxanes” The topic is interesting and well within the aims and scopes of the Journal. Yet, the manuscript must be implemented in several parts especially the phytochemical parts. And the introduction\nThe things to revise and add are listed below one by one:\nWith the exception of compound 4, for which X-ray analysis is reported, the authors assign and present absolute stereochemistry for all the taxanes obtained. However, in my opinion, these stereochemical assignments have not been adequately investigated or verified. I recommend that experimental evidence or a rationale be provided in support of these assignments\".\nThe authors should briefly explain why alanine was chosen to generate the mutants.\nThe conclusions of the manuscript need to be improved. It would be important to include an indication of the timeline required to obtain the mutants of the protein, as well as the process for separating the compounds and related steps. Additional details on the practical aspects of obtaining and using these mutants would strengthen the conclusions of the manuscript\".", "reviewer_4": "The oxidation of taxanes has long posed a significant bottleneck in the elucidation of the biosynthetic pathway of paclitaxel, primarily due to the unresolved catalytic promiscuity of taxadiene hydroxylase (T5OH). In this study, the authors demonstrate the capability of unspecific peroxygenases (UPOs) to catalyze the oxidation of taxane compounds, thereby introducing a novel avenue for paclitaxel biosynthesis. Through targeted protein engineering, they not only substantially enhanced the oxidative activity of TteUPO toward taxadiene but also achieved precise control over its regioselectivity. Building on these findings, the authors developed a versatile enzyme toolbox and broadened the substrate spectrum to encompass a diverse array of taxanes. Collectively, this research holds significant scientific importance and demonstrates notable innovation, fully aligning with the publication standards of Nature Communications. To further elevate the manuscript's quality, the authors are encouraged to address the following aspects:\n1. Data Supplementation for the Gly159 Saturation Mutant Based on L60A:\n  It is strongly recommended to include the activity and selectivity data for all Gly159 saturation mutants. This would provide a complete dataset for evaluating the impact of amino acid substitutions at this position.\n  A systematic comparison of the performance of these mutants, particularly in terms of catalytic activity, regioselectivity, and steric effects, would offer deeper insights into the role of steric hindrance in modulating enzyme function. Such an analysis could be presented in a supplementary table or figure to enhance clarity and accessibility.\n2. Performance Characterization of the Oxidase Toolbox:\n  While the oxidation performance of each enzyme in the toolbox on taxadiene has been thoroughly documented in the main figures, the data for other taxanes are currently limited to chromatograms in the supplementary materials. To provide a more comprehensive evaluation, it is recommended to include quantitative metrics such as conversion rates and turnover numbers, indices for these substrates.\n  These quantitative data should be integrated into the main figures or supplementary tables to facilitate direct comparison. This addition would significantly strengthen the manuscript by demonstrating the versatility and applicability of the oxidase toolbox across a broader range of taxane substrates.\n3. Why did the authors prefer to select peroxidases and peroxygenases derived from fungi, rather than those from plants or bacteria? Additionally, what were the reasons for selecting Pichia pastoris as the host for heterologous expression? Was it due to its protein expression efficiency, post-translational modification capabilities, or other specific characteristics that make it an ideal expression system?\n4. P450s can also hydroxylate the C10 and C13 positions of taxadiene. What are the advantages of TteUPO compared to P450s? Does TteUPO exhibit higher catalytic efficiency, substrate specificity, or greater stability under specific conditions?\n5. The influence of Leu60 on selectivity is quite significant. Besides the steric hindrance effects described by the authors, are there any other possible influencing factors?\n6. Since the authors identified steric hindrance in the substrate channel as the influencing factor in the first round of mutations, why did they introduce polar amino acids (Ser, Thr, and Tyr) in the second round of mutations? Could the introduction of polar amino acids also help to fine-tune the enzyme's regioselectivity or activity by altering the local microenvironment of the active site?\n7. Compounds such as ABTs are widely used in high-throughput screening of UPOs, and the authors also mentioned this in the experimental methods. However, why not use ABTs for initial screening followed by taxadiene as a secondary screening substrate? This stepwise screening strategy could not only improve screening efficiency but also significantly reduce the workload. Why was it not adopted? Were there technical or scientific limitations?\n8. In Figure 3, the yield of compound 9 was not inferior and was even higher than that of compounds 11, 12, and 15. Given this, why was compound 9 not further prepared and structurally characterized? Was it due to its chemical instability, difficulties in separation and purification, or its low bioactivity or potential application value? Was this decision based on certain experimental considerations that were not explicitly stated?\n9. In addition to the target substrate in this work, what is the potential of this enzyme for oxidizing other compounds?\nBy addressing these points, the manuscript will achieve a higher level of rigor and provide readers with a more complete understanding of the scientific advancements presented."}, "author_response": "The point-by-point responses to Reviewer’s comment are presented as follows:\nReviewer #1 (Remarks to the Author):\nThe manuscript presented by Yu group describes the C-H functionalization of taxadiene at\ndifferent positions by unspecific peroxygenases (UPOs). This research first benchmarks\nseveral UPO enzymes to be potential candidates towards taxadiene oxidation. After selecting\nTteUPO as departure point, the enzyme was crystalized and subjected to rational design. As\na result, up to 9 oxidation products were obtained opening a framework for future protein\nengineering. Without questioning the quality of the work, this reviewer feels that this study is\nnot offering great novelty in the field. Over the years, P450s have been studied for taxadiene\noxidation, and basically UPOs can achieve similar chemistries, yet only triggered by peroxide\n(instead of depending on auxiliary flavo proteins and expensive redox cofactors). The low\nTON obtained along with the absence of information about suicide inactivation -which could\npreclude the use of this system for practical purposes- are major issues that deserve further\nattention. Besides, new recent studies about P450/taxadiene are not mention (e.g.\nhttps://doi.org/10.1021/acs.orglett.4c00103). All in all, I am afraid I cannot support this\nmanuscript for publication in Nat Comm and I would suggest to send it to a more specialized\njournal.\nResponse:\nThank you very much for your constructive comments. Regarding the contributions of this\nwork, we would like to highlight the following key aspects:\n1) Compared to P450s, TteUPO demonstrates superior engineering plasticity. While T5αH\nhas proven recalcitrant to protein engineering efforts aimed at improving its activity and\nselectivity, TteUPO's catalytic properties are readily tunable. The L208A variant exhibits\na 53-fold increase in turnover number (TON) over the wild-type enzyme, achieving a TON\nof 4,355 (Table 1). Notably, a taxadiene C10-oxidizing variant L60A/G159L/L208A\noutperformed the benchmark P450 T10βH, demonstrating a TON of 757 versus 33.8 (vide\nsupra).\n2) We developed an artificial enzyme toolbox enabling site-selective oxidation at six distinct\npositions on the taxane skeleton, leading to the generation of 15 novel taxane derivatives\n(Figures 1B, 2B, 3B). The exploration of these novel oxidative sites provides a structural\nfoundation for developing novel bioactive paclitaxel analogs with subsequently\nmodification in future.\nWe also noticed that several studies that have identified new P450s for oxidizing taxadiene,\nincluding the article cited by the reviewer (https://doi.org/10.1021/acs.orglett.4c00103). But\nthe identified CYP701A8 caused the carbocation rearrangement of taxadiene, generating two\nnovel taxane skeletons. These articles are cited on page 2 (Ref 36).\n(Org. Lett. 2024, 26, 8, 1640–1644)\nReviewer #2 (Remarks to the Author):\nThe present manuscript titled as ‘Designing an Oxidase Toolbox for Site-Directed Oxidation\nof Taxanes’, developed a mutant library of TteUPO (a previously reported unspecific\nperoxygenase by other researchers) through site-directed mutagenesis, which have been\nsystematically characterized to catalyze the oxidation of different sites of taxanes. This\napproach provided diverse novel taxol derivatives by in vitro reactions.\nComments:\n1. A study in 2016 (Orthogonal Assays Clarify the Oxidative Biochemistry of Taxol P450\nCYP725A4, ACS Chem.Biol. 2016, 11, 1445.1451) has shown that taxadiene could be\nconverted to OCT and other compound by H2O2. Why were there no products for negative\ncontrol (no enzyme) in Fig.1A? Besides, please list the details of samples used for analyses\nin Figure legends (not only for this Figure) to make it easy to understand the details of\nexperiments.\nResponse:\nThank you for the constructive comments. In the cited literature mentioned (ACS Chem. Biol.\n2016), taxadiene was oxidized under extremely high H₂O₂ concentrations and trace amounts\nof OCT and other products was detected after overnight reaction. The original text describes\nas follows: The peroxide experiments were conducted by first drying 50 μL of 1 g/L taxadiene,\nsuspended in hexanes, in a glass GC vial. Once dried, 50 μL of hydrogen peroxide was added\nand allowed to react overnight.\nIn contrast, the H₂O₂ concentration in our reaction was merely 2 mM with a reaction time of\n1 hour. Thus, it is reasonable that no OCT or related products were detected under our\nreaction condition. We also replicated this experiment with different H₂O₂ concentrations. The\nresults demonstrate that taxadiene undergoes oxidation by high-concentration H₂O₂ to form\nOCT and other products. Conversely, at a mere 2 mM H₂O₂ concentration, no OCT generation\nwas detected even after overnight incubation, confirming H₂O₂'s limited oxidative capability\ntoward taxadiene (Figure A).\nFigure A. GC analysis of H₂O₂-mediated taxadiene oxidation. Trace amounts of OCT and\nrelated products were detected in overnight reaction using 10 M H₂O₂. In contrast, no\ndetectable products formed when H₂O₂ concentration was reduced to 2 mM under identical\ncondition.\nAs suggested by the reviewer, we have refined the legends of all figures.\n2.These products generated by the mutants of TteUPO belong to low oxygenated level\ntaxoids that are far away from paclitaxel analogs, which may take effects on their biological\nactivities. Could these mutants of TteUPO catalyze the produced novel derivatives in this\nstudy to higher oxygenated level toxoids, for example, to produce taxanes with more than\nfour oxidation modification? On the other hand, Could these mutants of TteUPO catalyze high\noxygenated level toxoid?\nResponse:\nAccording to the reviewer's suggestions, we applied the most active mutant, L208A, to\ntaxanes with higher oxidation states (Compounds 19 and 21) (Figure 3A & B and Figure\nS11). This approach successfully yielded the tri-oxygenated taxane derivative (Compound\n22), demonstrating the potential of TteUPO for oxidizing higher oxidation-state taxanes. As\nfor taxanes with four or more oxidation states, further attempts will be made in future studies.\nDue to the difficulties in preparing and characterizing both the substrates and oxidation\nproducts, achieving this remains a longer-term objective.\n3.The biotransformation catalyzed by TteUPO and its mutants depend on the supply of high\nconcentrationH2O2 which is harmful or unfriendly for most of common microbial chassis cells\ne.g. yeast and E. coli. Did TteUPO and its mutants show catalytic activity toward taxadiene\nin vivo?\nResponse:\nIn the revised manuscript, we have supplemented experiments in this regard. First, we\nsuccessfully expressed the most active mutant, L208A, in the taxadiene-producing E. coli\nstrain previously constructed (Process. Biochem. 2022, 122, 129-136). (Figure S12A-F). The\nexperiments demonstrate that supplementing less than 5 mM H₂O₂ every 8 hours has no\nsignificant impact on the growth of recombinant E. coli (Figure S12H). Furthermore, with\nexogenous H₂O₂ supplementation, de novo synthesis of taxadiene oxidation products in E.\ncoli was successfully achieved with a total yield of 14.2 mg/L (Figure 4 and Page 6).\n4.The GC analysis of the L60A mutant of TteUPO revealed several new peaks, but many new\npeaks at retention times of approximately 19.0 min, 19.2 min, and between 17.5 min and 18\nmin (Figure 3A) were not identified. It is essential for the authors to identify and elucidate the\nstructures of these new compounds.\nResponse:\nThe L60A mutant exhibits pronounced catalytic promiscuity, generating multiple novel peaks,\nof which six new oxidation products have been identified thus far. In the scaled-up reactions,\nthe yields of additional peaks noted by the reviewer (Retention times of approximately 19.0\nmin, 19.2 min, and between 17.5 min and 18 min (Redesign, Figure 2A) were significantly\nreduced, rendering their preparation exceptionally challenging (Figure B and Figure S8D).\nWe have included essential GC-MS spectral data in the Supplementary Information to\nfacilitate future exploration (Figure S13).\nFigure B. GC analysis of the 1 L scale oxidation of 0.5 mM taxadiene by L60A. The result\nrevealed a substantial reduction of Compound 10 levels along with the appearance of multiple\nnew peaks within the 17.5-18.0 min and 18.5-19.2 min retention time window following\nreaction scale-up.\n5. In Figure 3A, a new peak labeled 13 is mentioned, the structure of the corresponding\ncompound should also be provided.\nResponse:\nThe peak for compound 13 (renamed as 14 in the revised manuscript) co-elutes with 12\n(renamed as 13). During the purification of 12 (rename 13), we detected trace amounts of 13\n(rename 14) but could not achieve definitive characterization due to its minute quantity. To\nuphold data integrity, we cannot solely assign the overlapping peak to 12 (rename 13) while\ndisregarding the presence of 13 (rename 14). The MS spectrum of 13 (rename 14) is provided\nin the Supplementary Information for reference purposes (Figure S13).\n6.The author should carefully review the Supporting Information (SI). It is inappropriate to\ninclude chemical structures with HMBC correlations in the COSY and HSQC NMR spectra.\nInstead of 2D structures, which make it difficult to interpret the mentioned NOE correlations,\n3D-models of the newly identified compounds with highlighting key NOE interactions should\nbe provided.\nResponse:\nIn response to the reviewer's suggestions, we have modified the presentation of NMR spectra,\nthe 2D structures in NOE spectra were converted to 3D formats, with key interaction sites\nexplicitly highlighted.\n7.The detail about the chemical synthesizing compound taxadiene-4(5)-epoxide (18) should\nbe provided in SI.\nResponse:\nAs suggested by the reviewer, we have supplemented the experimental methods with details\nof the chemical synthesis of epoxy products in the Methods and Supplemental Information\n(Figure S5).\n8.Based on the NMR spectra, Compound 11 is identified as the minor component,\nrepresenting only 1/6 of the mixture. To ensure the accuracy of the structure presented in the\nmanuscript, it is essential to provide the NMR spectra of the pure compound.\nResponse:\nWe appreciate the reviewer's suggestion. Although compounds 11 (rename 12) and 10\n(rename 11) exhibit very similar polarity, rendering separation challenging under current\nchromatographic conditions, their NMR spectra display distinct resolution. Crucially,\ndifferences in concentration allow unambiguous identification of both compounds from their\nmixture spectra. To facilitate verification of data accuracy, we have distinctly labeled 12-\nspecific peaks in the NMR spectra (Figure S64-70).\n9.In order to make the comparison of mutants, it is better to add GC analyses of taxadiene\noxidation catalyzed by all mutants of TteUPO mention in the text, e.g. mutants L60A/F156A,\nL60A/F156L, and L60A/G159L/L208A.\nResponse:\nIn accordance with the reviewers' recommendations, we have relocated selected mutant data\nfrom the Supplementary Information and incorporated it into Figure 2A within the Main Text.\n10.Some inaccurate descriptions about the background information in main part greatly\nweaken the quality of this manuscript, please check the accuracy of research background\npart:\n1)“However, the yield of paclitaxel produced in Nicotiana benthamiana is only 64.29 ng/g\nplant material. This extremely low yield is due mainly to the low activity and severe catalytic\npromiscuity of the cytochrome P450 monooxygenases (P450s) involved in native\ndownstream taxadiene oxidative derivation steps”. In ref.26, Zhang et.al detected a low level\nof paclitaxel (64.29 ng/g) accumulation in the leaves of N. benthamiana by feeding baccatin\nIII, in which the P450 might not be the only rate-limiting enzyme.\nResponse:\nThank for the reviewer's correction. We have revised description in the background section.\n“Notably, Zhang et al. established the complete gene set requisite for heterologous paclitaxel\nbiosynthesis and identified a taxadiene-epoxidizing enzyme that synergizes with T5αH to\nfacilitate oxetane ring formation.”\n2)‘Despite the progress in elucidating the mechanism of taxadiene-5α-hydroxylase, the native\nenzyme in the initial step of taxadiene oxidation, attempts to substantially enhance its\ncatalytic efficiency and specificity through protein engineering have thus far been\nunsuccessful’, in fact, a new study from bioRxiv (preprint\ndoi:https://doi.org/10.1101/2024.11.06.622305) has overcame this problem. Please add the\nupdated information.\nResponse:\nThe preprint manuscript mentioned by the reviewer has now been published in Nature\n(https://doi.org/10.1038/s41586-025-09090-z). This research elucidated seven novel genes\nand achieved the production of baccatin III in N. benthamiana. The study also uncovered a\ngroundbreaking discovery: a key regulatory factor named FoTO1, belonging to the NTF2-like\nprotein family, can form a protein complex with T5αH. This interaction reduces the generation\nof byproducts such as OCT and successfully addresses the long-standing challenge of\ncatalytic promiscuity in T5αH. We have updated the relevant section in the manuscript to\nreflect recent research progress in this field. “McClune et al. discovered FoTO1, an NTF2-\nlike protein, can resolved the catalytic promiscuity of T5αH, suppressing byproduct formation\n(e.g., OCT) and enabling the selective production of taxadiene-5α-ol (T5OH). This approach\nresulted in the isolation of baccatin III at ~8.7 μg/g DW from N. benthamiana.”\n3)“In recent years, substantial progress has been made in elucidating the natural biosynthetic\npathway of paclitaxel, and all enzymes involved have been identified18-32. However, some of\nthese references (18-23) have no relationship with the elucidation of the natural\nbiosynthetic pathway of paclitaxel. Besides, the contribution of professor Rodney Croteau\nshould not be ignored. Please change the references with these papers from professor\nRodney Croteau.\nResponse:\nIn response to the reviewers’ valuable feedback, we have revised the original statement to:\n\"In recent years, substantial progress has been achieved in both elucidating and optimizing\nthe paclitaxel biosynthetic pathway, with all associated enzymes now identified.\" (The phrase\n\"elucidating\" was expanded to \"elucidating and optimizing\" to better align with recent\nresearch advances.)\nAdditionally, we hold profound admiration for the pivotal contributions of Prof. Rodney B.\nCroteau to the field (Rod Croteau: 35 years of terrific terpene biochemistry, Special Issue of\nPhytochemistry dedicated to the celebration of the 60th anniversary of Rodney Croteau,\nhttps://doi.org/10.1016/j.phytochem.2006.06.009)—particularly his contributions on the\nfunctional characterization of key enzymes involved in paclitaxel biosynthesis. Consequently,\nwe have strategically cited Prof. Croteau’s landmark work characterizing enzymes involved\nin the biosynthetic pathway of paclitaxel (Ref 25-29).\n4)“TteUPO (unspecific peroxygenase from Thielavia terrestris)36, 37 was chosen due to its\nhighest activity and excellent protein expression level (Table S1, S2 and Figure S1, S2)”,\nhowever, Figure S2 has no relationship with the activity and protein expression level of\nTteUPO.\nResponse:\nIn accordance with the reviewer's suggestion, we have supplemented Table S2 with the\ncrude enzyme conversion rates of unspecific peroxygenases and included documentary\nphotographs validating UPO expression levels (Figure S1).\nReviewer #3 (Remarks to the Author):\nThe authors presented a review article on “Designing an Oxidase Toolbox for Site-Directed\nOxidation of Taxanes” The topic is interesting and well within the aims and scopes of the\nJournal. Yet, the manuscript must be implemented in several parts especially the\nphytochemical parts. And the introduction The things to revise and add are listed below one\nby one:\nWith the exception of compound 4, for which X-ray analysis is reported, the authors assign\nand present absolute stereochemistry for all the taxanes obtained. However, in my opinion,\nthese stereochemical assignments have not been adequately investigated or verified. I\nrecommend that experimental evidence or a rationale be provided in support of these\nassignments\".\nResponse:\nOur analytical workflow establishes the structure of the oxidation products, including both the\noxidation site(s) and stereochemistry. First, the oxidation state is deduced from MS data,\nwhile 13C-NMR and 1H-NMR chemical shifts identify the functional groups (e.g. hydroxyl,\ncarbonyl, or epoxide). Subsequently, the precise location(s) of these modification and the\ncomplete molecular structure are then resolved using 2D NMR techniques (HSQC, HMBC,\nand COSY). Finally, NOE correlations assign the stereochemistry. The stereochemical\nconfiguration of Compound 4 was determined using this method (Table S9 and Figure S29-\nS35) and validated through its X-ray structure (Figure 1B), demonstrating the robustness of\nour methodology for identifying stereochemical structures.\nComplete spectra are provided in the Supporting Information.\nThe authors should briefly explain why alanine was chosen to generate the mutants.\nResponse:\nThank you for the reviewer's suggestion. We have elaborated on this part in the main text.\n“Furthermore, given the minimal polarity and strong hydrophobicity of taxadiene, which\nprevents hydrogen bond formation with surrounding amino acids. Amino acids within the\nsubstrate channel were mutated to sterically less hindered nonpolar amino acid—alanine\n(Ala). These mutations widen the channel and identify sensitive residues for further precisely\nregulating the geometry of the channel.”\nThe conclusions of the manuscript need to be improved. It would be important to include an\nindication of the timeline required to obtain the mutants of the protein, as well as the process\nfor separating the compounds and related steps. Additional details on the practical aspects\nof obtaining and using these mutants would strengthen the conclusions of the manuscript\".\nResponse:\nThank you for the reviewer's suggestion. We have thoroughly revised the Conclusion section.\nIn the revised version, the logic and flow of the experimental work are presented with\nenhanced clarity and coherence.\nReviewer #4 (Remarks to the Author):\nReviewer comments: The oxidation of taxanes has long posed a significant bottleneck in the\nelucidation of the biosynthetic pathway of paclitaxel, primarily due to the unresolved catalytic\npromiscuity of taxadiene hydroxylase (T5OH). In this study, the authors demonstrate the\ncapability of unspecific peroxygenases (UPOs) to catalyze the oxidation of taxane\ncompounds, thereby introducing a novel avenue for paclitaxel biosynthesis. Through targeted\nprotein engineering, they not only substantially enhanced the oxidative activity of TteUPO\ntoward taxadiene but also achieved precise control over its regioselectivity. Building on these\nfindings, the authors developed a versatile enzyme toolbox and broadened the substrate\nspectrum to encompass a diverse array of taxanes. Collectively, this research holds\nsignificant scientific importance and demonstrates notable innovation, fully aligning with the\npublication standards of Nature Communications. To further elevate the manuscript's quality,\nthe authors are encouraged to address the following aspects:\n1. Data Supplementation for the Gly159 Saturation Mutant Based on L60A:\nIt is strongly recommended to include the activity and selectivity data for all Gly159\nsaturation mutants. This would provide a complete dataset for evaluating the impact of amino\nacid substitutions at this position.\nA systematic comparison of the performance of these mutants, particularly in terms of\ncatalytic activity, regioselectivity, and steric effects, would offer deeper insights into the role\nof steric hindrance in modulating enzyme function. Such an analysis could be presented in a\nsupplementary table or figure to enhance clarity and accessibility.\nResponse:\nThank you for the reviewer's suggestion. We have incorporated the relevant data, the activity\nand selectivity data for all Gly159 saturation mutants, into Supplementary Information (Table\nS6), as suggested.\n2. Performance Characterization of the Oxidase Toolbox:\nWhile the oxidation performance of each enzyme in the toolbox on taxadiene has been\nthoroughly documented in the main figures, the data for other taxanes are currently limited to\nchromatograms in the supplementary materials. To provide a more comprehensive evaluation,\nit is recommended to include quantitative metrics such as conversion rates and turnover\nnumbers, indices for these substrates.\nThese quantitative data should be integrated into the main figures or supplementary tables\nto facilitate direct comparison. This addition would significantly strengthen the manuscript by\ndemonstrating the versatility and applicability of the oxidase toolbox across a broader range\nof taxane substrates.\nResponse:\nIn response to the reviewer's suggestion, we have supplemented Figure S11 in the\nSupplementary Information with oxidation data of taxanes by the mutants not listed in Table\n1.\n3. Why did the authors prefer to select peroxidases and peroxygenases derived from fungi,\nrather than those from plants or bacteria? Additionally, what were the reasons for selecting\nPichia pastoris as the host for heterologous expression? Was it due to its protein expression\nefficiency, post-translational modification capabilities, or other specific characteristics that\nmake it an ideal expression system?\nResponse:\nUPOs (unspecific peroxygenases) are our primary targets, and all currently characterized\nUPOs originate from fungi. During our screening process, we noted that peroxidases and\nperoxygenases share a conserved structural motif: a heme prosthetic group coordinated by\na cysteine residue. This structural homology prompted us to include both enzyme classes in\nour screening pipeline. All the oxidases selected for this study were a literature-derived, with\ntheir heterologous expression conducted in either Saccharomyces cerevisiae or Pichia\npastoris. Notably, P. pastoris demonstrates superior performance in high-density\nfermentation compared to S. cerevisiae. Given that our biosynthetic approach requires\nsubstantial enzyme quantities for novel product formation, this operational requirement\nestablishes P. pastoris as the more appropriate expression host for our purposes.\n4. P450s can also hydroxylate the C10 and C13 positions of taxadiene. What are the\nadvantages of TteUPO compared to P450s? Does TteUPO exhibit higher catalytic efficiency,\nsubstrate specificity, or greater stability under specific conditions?\nResponse:\nThank you for the reviewers' suggestion. In response, we have expanded the main text with\na comparative analysis of P450 enzymes (Figure 1C). In addition, conventional protein\nengineering approaches have proven ineffective in overcoming the inherent limitations of\ntaxadiene-5α-hydroxylase—particularly its low catalytic activity (Proc. Natl. Acad. Sci. U. S.\nA. 2016, 113, 3209-3214; Nat. Commun. 2019, 10, 4850). In this study, we implemented a\nstructure-guided protein engineering strategy to redesign the substrate channel of TteUPO.\nThis approach enabled precise tuning of its activity and regioselectivity, resulting in an\nengineered enzyme toolbox capable of site-specific oxidation at six distinct positions on\ntaxadiene. Our results demonstrates that TteUPO exhibits superior engineerability and\ngenerates a more diverse array of taxadiene oxidation products compared to P450 systems.\nNotably, the C10-oxidizing variant L60A/G159L/L208A engineered in this study achieves a\nTotal Turnover Number (TON) of 757—representing a >20-fold enhancement over the taxane\noxidase T10βH (TON = 33.8).2\n5. The influence of Leu60 on selectivity is quite significant. Besides the steric hindrance\neffects described by the authors, are there any other possible influencing factors?\nResponse:\nBased on the reviewer's suggestion, we have conducted a more thorough investigation into\nthe saturation mutagenesis at Leu 60, identified as the single residue exerting the most\nsignificant influence on selectivity in our engineered TteUPO variants. “The L60S mutant\nexhibited activity and selectivity profiles comparable to those of L60A (Figures S8B).\nAlthough serine possesses a polar hydroxyl group absent in alanine, both residues impose\nsimilar steric constraints. This result further confirms that steric hindrance—rather than side-\nchain polarity—is the primary determinant of TteUPO’s activity and selectivity in taxadiene\noxidation.”\n6. Since the authors identified steric hindrance in the substrate channel as the influencing\nfactor in the first round of mutations, why did they introduce polar amino acids (Ser, Thr, and\nTyr) in the second round of mutations? Could the introduction of polar amino acids also help\nto fine-tune the enzyme's regioselectivity or activity by altering the local microenvironment of\nthe active site?\nResponse:\nDuring our initial round of engineering, we observed the formation of over-oxidation products\n(carbonyl derivatives) and dioxygenated species. Recognizing that hydroxylated products\ncontain hydroxyl groups capable of forming hydrogen bonds with the enzyme active site, we\nintroduced polar amino acid substitutions in subsequent designs. Through iterative\noptimization, we developed an engineered enzyme platform capable of oxidizing diverse\ntaxane derivatives beyond taxadiene. By strategically incorporating polar residues, we\nexpanded substrate scope and achieved regio-diversified oxidative functionalization across\nmultiple positions of taxane scaffolds.\n7. Compounds such as ABTs are widely used in high-throughput screening of UPOs, and the\nauthors also mentioned this in the experimental methods. However, why not use ABTs for\ninitial screening followed by taxadiene as a secondary screening substrate? This stepwise\nscreening strategy could not only improve screening efficiency but also significantly reduce\nthe workload. Why was it not adopted? Were there technical or scientific limitations?\nResponse:\nWhile ABTS serves as an effective high-throughput assay for general\nperoxidase/peroxygenase activity (used here to confirm proper UPO expression, as\ndocumented in Table S2), it cannot resolve the regio- and stereoselectivity critical for\ntaxadiene oxidation. Our screening for taxadiene oxidation therefore employs gas\nchromatography (GC) analysis, which provides the necessary resolution to distinguish\nbetween isomeric oxidation products that colorimetric assays like ABTS fundamentally\ncannot differentiate.\n8. In Figure 3, the yield of compound 9 was not inferior and was even higher than that of\ncompounds 11, 12, and 15. Given this, why was compound 9 not further prepared and\nstructurally characterized? Was it due to its chemical instability, difficulties in separation and\npurification, or its low bioactivity or potential application value? Was this decision based on\ncertain experimental considerations that were not explicitly stated?\nResponse:\nDuring scale-up preparation, the yield of Compound 9 (rename 10) decreased significantly\n((Figure B and Figure S8D). The amount obtained is currently insufficient for isolation and\nstructural characterization.\n9. In addition to the target substrate in this work, what is the potential of this enzyme for\noxidizing other compounds?\nResponse:\nBased on the reviewer's suggestion, we applied the most active mutant, L208A, to oxidized\ntaxane derivatives with higher oxidation states (Figure 3). This approach successfully yielded\ntaxanes bearing up to three oxidative modifications, highlighting the potential of TteUPO for\nconstructing complex taxane analogues through multi-step enzymatic functionalization.\nThe point-by-point responses to Reviewer’s comment are presented as follows:\nReviewer #5 (Remarks to the Author):\nNot all questions of reviewer 2 could be answered, but the reasons are understandable.\nAdditional information to points 4 (why additional peaks is smaller after reaction at a higher\nscale) and 7 (synthesis procedure need to be included) would improve the quality of the\nmanuscript.\nI found the manuscript very interesting. it contains many new important results. Notable\nachievements include the establishment of an E. coli strain capable of synthesising the\noxidised taxadiene derivatives, and the resolution of the crystal structure of a UPO produced\nin P. pastoris. My only criticism is that no regioselective mutant could be engineered. All\nmutant produce multiple products, but difficult product isolation has not been discussed.\nAdditionally, I would advise the authors against using the term 'high catalytic promiscuity'\ninstead of 'low regioselectivity’ or ‘low product selectivity'.\nResponse:\nWe appreciate your positive feedback on the manuscript and the constructive suggestions\nyou have provided. As detailed below, we have provided a point-by-point response to your\nquestions and suggestions.\n1. The L60A mutant exhibited low product selectivity, generating a range of peroxidated\nderivatives, such as compound 13, 15, and 16. These initial, single-step oxidation products\nof taxadiene are susceptible to further oxidation, yielding compounds with higher oxidation\nstates. Compound 10 (MW 286) is hypothesized to be either a carbonyl product formed via\noxidation of a hydroxylated taxadiene intermediate (MW 288), or a dehydration product (MW\n304 - 18 = 286) of a dihydroxylated precursor (MW 304) formed under GC heating conditions.\nSupporting this, oxidation of a mixture of compounds 11 and 12 (which are difficult to separate)\nwith L60A resulted in the formation of compound 10, along with several new peaks (retention\ntime: 17.50–19.00 min), three of which exhibited a molecular weight of 304 (Figure S8E).\nThis suggests that compound 10 may originate from oxidation of either the C10 or C13\nposition of taxadiene, or alternatively, result from thermal degradation a dihydroxylated\nintermediate during GC analysis. We then proposed that scaling up this multi-step enzymatic\noxidation compromised oxygen mass transfer, thereby reducing oxidation efficiency, which in\nturn led to a lower yield of compound 10.\nFigure S8E. Oxidation of compounds 11 and 12 by L60A yielded compound 10 and multiple\nnew peaks in the retention time range of 17.50–19.00 min. Molecular weights were\ndetermined by GC-MS (Figure S13).\n2. The detailed synthetic procedure for compound 7 has been relocated to Figure S5\n(Supporting Information). Appropriate citations have been added in both the main text and\nthe Methods section to facilitate easy access to this information.\n“NaHCO (12.00 g, 142.85 mmol) was dissolved in a mixture of H O (10 mL) and acetone\n3 2\n(15 mL). The reaction vessel was placed in an ice-water bath with stirring at 200 rpm. Oxone®\n(12.5 g total) was added batchwise within 1 min. After stirring for 30 min, the mixture was\nextracted with diethyl ether (20 mL). The resulting ethereal extract containing DMDO was\nstored in an ice-water bath. Taxadiene was dissolved in the DMDO/ether solution to a final\nconcentration of 2 mM and reacted with stirring (200 rpm) in an ice-water bath for 1 h. The\nsolvents were removed by rotary evaporation under reduced pressure. Taxadiene-4(5)-\nepoxide (7) was purified using silica gel column chromatography, eluted by n-hexane/ethyl\nacetate (30:1, v/v). Reaction progress and product fractions were monitored by TLC (silica\ngel, n-hexane/ethyl acetate = 6:1, v/v), with visualization achieved by staining with\nphosphomolybdic acid. The target compound was then collected and further purified through\npreparative HPLC using an UltiMate 3000 chrome system (Thermo Fisher Scientific, USA)\nequipped with an Ultimate ® XB-C18 column (Welch, China).”\n3. In the work, TteUPO demonstrates superior engineering plasticity compared to P450s.\nL60A/G159L variant, for instance, achieved site-directed oxidation of taxadiene at C10 and\nC13, matching the oxidation sites found in paclitaxel. Our findings indicates that\nregioselectivity is primarily governed by the steric hindrance within the substrate channel,\nsuggesting that its further precise regulation as a viable strategy for regioselectivity\nenhancement—a direction we plan to pursue.\nAdditionally, we found that the structure of taxane substrates also influences the\nregioselectivity of TteUPO (Figures 3B and Table A), implying that the oxidase toolbox\ndeveloped in this study is not limited to taxadiene but also enables site-directed oxidation of\nother highly oxidized taxanes.\nTable A. Oxidation of taxanes using TteUPO mutants in the developed oxidase toolbox.\nPeak area ratio (%)\ncatalyst substrate\nC4 C6 C10 C11 + C12 C13\nWT 1 11 89 0 0 0\nL60A 1 1 36 14 41 8\nL60A/G159L 1 0 5 74 0 21\nL60A/G159L/L208A 1 0 3 54 0 43\nL60A 4 0 0 0 100 0\nF156A 5 0 100 0 0 0\nL60A/F156A 11 0 100 0 0 0\nL208A 19 0 100 0 0 0\nL208A 21 0 0 0 0 100\n4. The detailed preparation and isolation procedures for all identified products are provided\nin the \"Product Preparation and Isolation\" section of the Methods.\n5. Thank you for your correction. We have changed the term “high catalytic promiscuity” to\n“low product selectivity”."}, {"round": 2, "reviewer_comments": {"reviewer_1": "Despite I still consider that the manuscript is not getting superb results in terms of TTNs and mostly in novelty (as P450s have been already deeply explored for this biotransformation), I do appreciate the effort done by the authors in the reviewing process addressing most of the queries from the other reviewers. Accordingly, I am keen to accept the final decision of this manuscript by the main editor.", "reviewer_3": "The authors have adequately addressed my questions", "reviewer_4": "All concerns and question have been thoroughly addressed through textual modifications or supplementary experiments, resulting in a more robust presentation of this work. These newly supplemented data have significantly strengthened this work, demonstrating the considerable potential of synthetic pathway reconstruction for paclitaxel and its structural analogs.", "reviewer_5": "Not all questions of reviewer 2 could be answered, but the reasons are understandable. Additional information to points 4 (why additional peaks is smaller after reaction at a higher scale) and 7 (synthesis procedure need to be included) would improve the quality of the manuscript.\nI found the manuscript very interesting. it contains many new important results. Notable achievements include the establishment of an E. coli strain capable of synthesising the oxidised taxadiene derivatives, and the resolution of the crystal structure of a UPO produced in P. pastoris. My only criticism is that no regioselective mutant could be engineered. All mutant produce multiple products, but difficult product isolation has not been discussed. Additionally, I would advise the authors against using the term 'high catalytic promiscuity' instead of 'low regioselectivity’ or ‘low product selectivity'."}, "author_response": "The point-by-point responses to Reviewer’s comment are presented as follows:\nReviewer #5 (Remarks to the Author):\nNot all questions of reviewer 2 could be answered, but the reasons are understandable.\nAdditional information to points 4 (why additional peaks is smaller after reaction at a higher\nscale) and 7 (synthesis procedure need to be included) would improve the quality of the\nmanuscript.\nI found the manuscript very interesting. it contains many new important results. Notable\nachievements include the establishment of an E. coli strain capable of synthesising the\noxidised taxadiene derivatives, and the resolution of the crystal structure of a UPO produced\nin P. pastoris. My only criticism is that no regioselective mutant could be engineered. All\nmutant produce multiple products, but difficult product isolation has not been discussed.\nAdditionally, I would advise the authors against using the term 'high catalytic promiscuity'\ninstead of 'low regioselectivity’ or ‘low product selectivity'.\nResponse:\nWe appreciate your positive feedback on the manuscript and the constructive suggestions\nyou have provided. As detailed below, we have provided a point-by-point response to your\nquestions and suggestions.\n1. The L60A mutant exhibited low product selectivity, generating a range of peroxidated\nderivatives, such as compound 13, 15, and 16. These initial, single-step oxidation products\nof taxadiene are susceptible to further oxidation, yielding compounds with higher oxidation\nstates. Compound 10 (MW 286) is hypothesized to be either a carbonyl product formed via\noxidation of a hydroxylated taxadiene intermediate (MW 288), or a dehydration product (MW\n304 - 18 = 286) of a dihydroxylated precursor (MW 304) formed under GC heating conditions.\nSupporting this, oxidation of a mixture of compounds 11 and 12 (which are difficult to separate)\nwith L60A resulted in the formation of compound 10, along with several new peaks (retention\ntime: 17.50–19.00 min), three of which exhibited a molecular weight of 304 (Figure S8E).\nThis suggests that compound 10 may originate from oxidation of either the C10 or C13\nposition of taxadiene, or alternatively, result from thermal degradation a dihydroxylated\nintermediate during GC analysis. We then proposed that scaling up this multi-step enzymatic\noxidation compromised oxygen mass transfer, thereby reducing oxidation efficiency, which in\nturn led to a lower yield of compound 10.\nFigure S8E. Oxidation of compounds 11 and 12 by L60A yielded compound 10 and multiple\nnew peaks in the retention time range of 17.50–19.00 min. Molecular weights were\ndetermined by GC-MS (Figure S13).\n2. The detailed synthetic procedure for compound 7 has been relocated to Figure S5\n(Supporting Information). Appropriate citations have been added in both the main text and\nthe Methods section to facilitate easy access to this information.\n“NaHCO (12.00 g, 142.85 mmol) was dissolved in a mixture of H O (10 mL) and acetone\n3 2\n(15 mL). The reaction vessel was placed in an ice-water bath with stirring at 200 rpm. Oxone®\n(12.5 g total) was added batchwise within 1 min. After stirring for 30 min, the mixture was\nextracted with diethyl ether (20 mL). The resulting ethereal extract containing DMDO was\nstored in an ice-water bath. Taxadiene was dissolved in the DMDO/ether solution to a final\nconcentration of 2 mM and reacted with stirring (200 rpm) in an ice-water bath for 1 h. The\nsolvents were removed by rotary evaporation under reduced pressure. Taxadiene-4(5)-\nepoxide (7) was purified using silica gel column chromatography, eluted by n-hexane/ethyl\nacetate (30:1, v/v). Reaction progress and product fractions were monitored by TLC (silica\ngel, n-hexane/ethyl acetate = 6:1, v/v), with visualization achieved by staining with\nphosphomolybdic acid. The target compound was then collected and further purified through\npreparative HPLC using an UltiMate 3000 chrome system (Thermo Fisher Scientific, USA)\nequipped with an Ultimate ® XB-C18 column (Welch, China).”\n3. In the work, TteUPO demonstrates superior engineering plasticity compared to P450s.\nL60A/G159L variant, for instance, achieved site-directed oxidation of taxadiene at C10 and\nC13, matching the oxidation sites found in paclitaxel. Our findings indicates that\nregioselectivity is primarily governed by the steric hindrance within the substrate channel,\nsuggesting that its further precise regulation as a viable strategy for regioselectivity\nenhancement—a direction we plan to pursue.\nAdditionally, we found that the structure of taxane substrates also influences the\nregioselectivity of TteUPO (Figures 3B and Table A), implying that the oxidase toolbox\ndeveloped in this study is not limited to taxadiene but also enables site-directed oxidation of\nother highly oxidized taxanes.\nTable A. Oxidation of taxanes using TteUPO mutants in the developed oxidase toolbox.\nPeak area ratio (%)\ncatalyst substrate\nC4 C6 C10 C11 + C12 C13\nWT 1 11 89 0 0 0\nL60A 1 1 36 14 41 8\nL60A/G159L 1 0 5 74 0 21\nL60A/G159L/L208A 1 0 3 54 0 43\nL60A 4 0 0 0 100 0\nF156A 5 0 100 0 0 0\nL60A/F156A 11 0 100 0 0 0\nL208A 19 0 100 0 0 0\nL208A 21 0 0 0 0 100\n4. The detailed preparation and isolation procedures for all identified products are provided\nin the \"Product Preparation and Isolation\" section of the Methods.\n5. Thank you for your correction. We have changed the term “high catalytic promiscuity” to\n“low product selectivity”."}, {"round": 3, "reviewer_comments": {"reviewer_5": "The authors have answered my new questions and addressed the previous issues as well. They explain everything very well. The manuscript can be accepted for publication in its current state."}, "author_response": ""}]

Decision letter