Metabolic engineering of doxorubicin biosynthesis through P450-redox partner optimization and structural analysis of DoxA

Domain: neuroscience
Published: 2026-02-04
Editorial outcome: minor_revision

Abstract
Doxorubicin, a widely used chemotherapy drug, is produced by Streptomyces peucetius ATCC 27952. The biosynthesis relies on the cytochrome P450 monooxygenase DoxA, which catalyzes three consecutive late-stage oxidation steps. However, conversion from daunorubicin to doxorubicin is inefficient, necessitating semi-synthetic industrial manufacturing. Here, we address key limitations in DoxA catalysis. We identify the natural redox partners ferredoxin Fdx4 and ferredoxin reductase FdR3 by transcriptomic analysis. We discovered the vicinal oxygen chelate family protein DnrV to prevent product inhibition by binding doxorubicin. Structural analysis of DoxA and density functional theory (DFT) calculations reveal that inefficient C14 hydroxylation results from the unfavorable anti-conformation of the methyl ketone side chain of daunorubicin. We harness these advances for rational strain engineering, leading to an 180% increase in doxorubicin yields and an improved production profile. This study provides singular insights into enzymatic constraints in anthracycline biosynthesis and facilitates cost-effective manufacturing to meet the growing global demand for doxorubicin.

Introduction
Anthracyclines are an important class of microbial natural products that have been a cornerstone in clinical anticancer chemotherapy for several decades1. These molecules consist of a linear tetracyclic 7,8,9,10-tetrahydro-5,12-naphthacenoquinone scaffold, which is decorated with one or more carbohydrate moieties2. The best-known anthracyclines are daunorubicin (DNR) and doxorubicin (DXR), which are first-choice chemotherapeutic agents administered to more than one million cancer patients each year in the treatment of lymphomas, carcinomas, and sarcomas3. The biological activity of anthracyclines is mediated primarily via interactions with DNA, where the polyphenolic tetracyclic aglycone intercalates between base pairs and the carbohydrate unit at C7 is positioned in the minor groove of DNA1. Intercalation to DNA promotes poisoning of topoisomerase II, leading to DNA double strand brakes and histone eviction, both of which lead to cell death4,5.

DNR was originally discovered in Streptomyces peucetius ATCC 27952 cultures and later clinically introduced for treating acute leukemia and lymphoma3. DXR was identified serendipitously during strain development of S. peucetius var. caesius, when a C14 hydroxy derivative of DNR accumulated in the culture medium following random mutagenesis of the parental strain6. DXR exhibited enhanced cytotoxic activity and a higher therapeutic index than DNR. However, DXR-producing strains typically co-produce substantial amounts of DNR, complicating industrial-scale biosynthetic production. The structural similarity of DXR and DNR poses challenges for chromatographic separation and downstream processing. Consequently, DXR and related anthracyclines in clinical use—such as epirubicin, pirarubicin, and idarubicin—are manufactured semi-synthetically from DNR1.

The biosynthesis of DNR and DXR has been intensively investigated due to their clinical relevance2. Sequencing of the biosynthetic gene cluster (Fig. 1A) clarified that a canonical type II polyketide synthase system generates the aglycone from one propionyl-CoA and nine malonyl-CoA molecules via iterative Claisen condensations (Fig. 1B)7. In addition, the biosynthesis of the TDP-daunosamine carbohydrate and attachment at C7 has been fully elucidated8. After glycosylation, the tetracyclic core is modified in extensive tailoring reactions, which include C15 demethylation, C10 decarboxylation, C4 O-methylation, and C11 hydroxylation9. In the context of DXR biosynthesis, the most intriguing steps are the three C-H oxidation reactions catalyzed by a single heme-containing cytochrome P450 enzyme DoxA (Fig. 1B)8. The enzyme performs sequential conversion of 13-deoxydaunorubicin (DOD) to 13-dihydrodaunorubicin (DHD), further C13 oxidation to DNR, and finally catalyzes C14 hydroxylation to generate DXR10. Enzyme kinetic studies of DoxA revealed that the Vmax values varied significantly for the three substrates and were 520-folds lower for DNR than for DHD10. In addition, early in vivo experiments identified that the gene dnrV, encoded directly upstream of doxA, positively influences doxorubicin production8. DnrV is annotated as a member of the vicinal oxygen chelate (VOC) enzyme family, which is typically not associated with P450 enzymes, and the precise role of this protein in doxorubicin biosynthesis remains unknown.

P450 enzymes have been extensively investigated for their important role in various biocatalytic processes11. One of the challenges in engineering P450 systems is the requirement for a complex co-factor-mediated electron transport chain to promote activation of the heme co-factor (Fig. 1C). Classical microbial P450 redox systems are composed of a flavin-dependent ferredoxin reductase (FdR) that utilizes NAD(P)H for the reduction of ferredoxin (Fdx). In turn, Fdx catalyzes the single electron reduction of the heme Fe3+ iron to Fe2+, which enables a reaction with molecular oxygen and formation of the catalytic high-valent iron-oxo-heme species that is capable of oxidizing unactivated C-H bonds of substrates12. The correct pairing of the FdR-Fdx-P450 components is vital for the performance of P450 biocatalysts13, but identification of the natural redox partners of DoxA has been challenging14,15. The genome harbors six fdx and seven fdr genes, which are not co-localized within the daunorubicin biosynthetic genes. In addition, even though attempts have been made to identify the redox partners of DoxA by predictive molecular modeling, the improvements in catalysis have been modest14.

Here, we identify three factors that limit DXR biosynthesis in S. peucetius ATCC 27952 and overcome these bottlenecks in strain engineering. The work includes (i) identification of the natural redox partners of DoxA from transcriptomics data, (ii) elucidation of the role of DnrV as a drug-binding protein that improved catalysis by preventing product inhibition of DoxA, and (iii) determination of the ternary crystal structures of DoxA in complex with ligands, which reveal the molecular basis for poor catalytic turnover of DNR. These advances guide our rational metabolic engineering efforts in the creation of a strain with a significantly improved DXR production profile and yield.

Results
Identification of DoxA redox partners by comparative metabolome, genome and transcriptome analyses
We performed comparative time-course analysis of metabolite production by wild-type S. peucetius ATCC 27952 and two industrial strains generated by random mutagenesis. The industrial strains included S. peucetius G001 and X121 (Fig. 2A), which produce high yields of DNR and DXR, respectively. Anthracycline production is under carbon catabolite repression in the wild-type strain16 and, in agreement, we observed the initiation of DNR production on day 6. In contrast, anthracycline production could already be observed during the high-exponential growth phase on day 2 in G001 and X121. Both G001 and X121 produced DXR mainly in the exponential and early stationary growth phases, reaching DXR yields of approximately 40 mg/L. In contrast, DNR was primarily produced in the late stationary growth phase, particularly in G001 (Fig. 2A).

Genome and transcriptome sequencing data clarified several parameters related to the early onset of anthracycline production in G001 and X121 (Fig. 2B, Supplementary Table 1). The regulation of the DNR biosynthetic gene cluster is under a three-tier transcriptional activator system1. The TetR-family transcriptional regulator dnrO binds to all glycosylated products of the pathway and controls the activation of dnrN. In turn, DnrN is a pseudo-response regulatory protein that governs the expression of dnrI, which directly controls the transcription of the biosynthetic and resistance genes by binding to the promoter regions within the dnr cluster17. Both G001 and X121 harbored a deletion mutation in the bidirectional promoter region of dnrO and dnrN (Fig. 2C), which may have contributed to early anthracycline biosynthesis. Consistently, transcriptomics data revealed 3.4- and 2.6-fold overexpression of dnrN in G001 and X121, respectively, compared to the wild-type on day 1 (Fig. 2C, Supplementary Table 1). Moreover, we identified a mutation in the drrD/dauW gene, which has been suggested to negatively regulate DNR production18. Finally, ATP-glucokinase19 was downregulated in the mutants G001 and X121 on day 1, thereby decreasing carbon catabolite repression and promoting early onset of DNR and DXR production.

Analysis of the biosynthetic gene cluster did not provide insight into the production ratio of DNR/DXR. Neither G001 nor X121 had accumulated any mutations in the doxA gene, and the upregulation of doxA was consistent with the overall overexpression of the dnr biosynthetic genes (Fig. 2B, Supplementary Table 1). Therefore, we surmised that the higher DXR yields DXR in G001 and X121 could be indirectly influenced by the earlier onset of production due to differential expression of endogenous fdx/fdr partners during the Streptomyces growth cycle. Comparative transcriptomics revealed significant upregulation at two genetic loci in X121 compared to G001 and the wild-type (Supplementary Table 1). We observed overexpression of the suf pathway20, which included 3.4-fold upregulation of the ferredoxin fdx4 and six genes related to iron-sulfur cluster assembly in X121 (Fig. 2B, D, Supplementary Table 1). The regulatory gene sufR contained a point mutation leading to a Gly235Asp substitution in X121, possibly resulting in overexpression of the suf operon21. We inactivated sufR in S. peucetius ATCC 27952ΔsufR, which resulted in activation of the suf pathway and 4.4-fold overexpression of fdx4 (Supplementary Fig. 1). The second operon upregulated in X121 encoded the ferredoxin fdx5 and the tau pathway22 (Supplementary Table 1) responsible for the acquisition of sulfur from taurine. However, we did not consider fdx5 as the correct redox partner, since transcription of the tau pathway was exceedingly low in G001 during high-yield production of DXR in days 2-4. The fdx/fdr pairs previously studied in conjunction with DoxA14 were transcribed at a basal level and did not show differential expression between any strain combinations (Supplementary Table 1).

The ferredoxin SpFdx4 and ferredoxin reductase SpFdR3 are the natural redox partners of DoxA
To evaluate the various SpFdx and SpFdR redox partners from S. peucetius ATCC 27952, we established an in vitro enzymatic activity assay. DoxA was expressed and purified from E. coli (Supplementary Fig. 2), and an active heme prosthetic group was confirmed spectrophotometrically (Supplementary Fig. 3). Under optimized conditions, spinach Spinacia oleracea ferredoxin reductase (SoFdR) and spinach ferredoxin (SoFdx) converted a small amount of DOD to DHD (11%) based on HPLC analysis (Fig. 3A), while putidaredoxin reductase PdR and putidaredoxin Pdx from Pseudomonas putida performed better with the production of both DHD (69%) and DNR (18%) (Fig. 3A). All activity assays required rigorous optimization to determine the correct ratio of the P450-Fdx-FdR components, since an excess of ferredoxin reductases e.g. PdR led to reductive deglycosylation (Supplementary Fig. 4A). As noted previously23, the deglycosylation reaction is dependent on direct reduction of anthracyclines by ferredoxin reductases, which we observed with DHD and PdR even in the absence of Pdx and DoxA (Supplementary Fig. 4B). Preparative scale reactions with DNR and SoFdR verified the reaction product as 7-deoxydaunorubicinone (Supplementary Table 2, Supplementary Figs. 5-10), which is likely formed from DNR under anoxic conditions via a quinone methide intermediate (Supplementary Fig. 4C)23.

To study native redox partners from S. peucetius ATCC 27952, recombinant SpFdx4 from the suf cluster was first produced and purified from E. coli. Based on sequence analysis (Supplementary Fig. 11), SpFdx4 is a Rieske-type Cys2His2Fe2S2 ferredoxin containing two histidine and two cysteine residues for coordination of the [2Fe—2S] cluster, similarly to BphA324 and TDO-F25 ferredoxins affiliated with biphenyl and toluene dioxygenase systems, respectively. Therefore, SpFdx4 is not evolutionarily related to the Cys4Fe2S2 plant-type SoFdx26 or adrenodoxin-type Pdx27. Spectrophotometric analysis revealed canonical UV/Vis maxima for SpFdx4 and Pdx (Supplementary Fig. 3B), including expected differences reported for the two ferredoxin subfamilies28,29.

Our comparative transcriptomics data did not reveal obvious FdR candidate proteins, presumably due to sufficient FdR activity at basal expression levels in the industrial strains. Genome mining and phylogenetic analysis revealed that S. peucetius ATCC 27952 harbored five putidaredoxin reductase-type and two adrenodoxin reductase-type enzymes, but no plant-type ferredoxin reductases were detected (Fig. 3B)30. Five of the seven SpFdR proteins could be produced in soluble form in E. coli (Supplementary Fig. 2), which allowed the use of SpFdR1, SpFdR2, SpFdR3, SpFdR4, and SpFdR7 in comparative activity assays with SpFdx4, DoxA, and DOD as a substrate. One of the proteins, SpFdR3, mediated efficient conversion of the DOD substrate into DHD (88%) and DNR (8%) (Fig. 3C). By contrast, low amounts of DHD formation (4–22%) was detected with the other four candidate reductase proteins SpFdR1, SpFdR2, SpFdR4 and SpFdR7 (Fig. 3C). Interestingly, the non-native ferredoxin reductase SoFdR converted nearly all of the substrate DOD to DNR when paired with SpFdx4 and DoxA (Fig. 3C).

DnrV is an anthracycline resistance determinant that aids DoxA catalysis by preventing product inhibition
None of the reactions resulted in the formation of DXR, which is in line with previous reports on the in vitro activity of DoxA, where only trace quantities of DXR were observed14,15. Hence, we turned our attention to dnrV, which is located in the same operon as doxA and encodes a protein belonging to the vicinal oxygen chelate (VOC) enzyme family31. Addition of purified DnrV to reactions with SpFdx4, SoFdR, and DoxA with any of the substrates DOD, DHD or DNR led to concentration-dependent improvement in catalysis (Fig. 4A). We achieved full conversion to DXR without formation of deglycosylated shunt products through use of stoichiometric DNR:DnrV concentrations up to 100 μM DNR substrate (Fig. 4B).

The improved activity assay and the discovery of the beneficial effect of DnrV led us to revisit previous observations that DoxA catalysis is limited by product inhibition10. We investigated the influence of DXR in catalysis by titration of the DoxA reaction with DXR (0 μM–10 μM), which revealed concentration-dependent reduced activity (Fig. 4C). In the absence of DXR, a control reaction using DOD as a substrate led to the formation of 59% DXR and 41% DNR, but addition of 10 μM DXR resulted in decreased activity, and the main products were 49% DHD and 30% DNR.

Proteins from the VOC enzyme family have not previously been associated with P450 enzymes, and we hypothesized that DnrV may have an indirect role in enhancing DoxA activity. We observed that DnrV shares sequence similarity with mitomycin-resistant determinant (MDR)31,32, which is a non-metalloprotein member of the VOC superfamily. This indicated that DnrV may be an intracellular anthracycline drug-binding protein. In agreement, we performed binding studies exploiting the fluorescent properties of anthracyclines to demonstrate that DnrV binds DOD, DNR and DXR at low micromolar Kd values (Fig. 4D). As a positive control, we measured binding of anthracyclines to DoxA, which also occurred at a low micromolar range for the substrates DOD and DNR, but at lower affinity to the product DXR (Fig. 4E). In a negative control reaction, we demonstrated that titration of SpFdx4 with DXR did not alter the fluorescent properties of the anthracyclines (Fig. 4F). The activity appeared to be specific for DnrV, since the related mitomycin MDR did not bind anthracyclines (Supplementary Fig. 12A) or enhance the catalytic rate of DoxA (Supplementary Fig. 12B). These experiments suggest that DnrV indirectly improves DoxA activity by sequestering DXR, which prevents product inhibition from reducing catalysis by DoxA.

Structural basis for DoxA catalysis
To investigate the molecular basis for C13 and C14 hydroxylation, we solved the crystal structures of DoxA in complex with DOD, DHD, and DNR (Supplementary Table 3). The structure models reveal a crystallographic dimer (Fig. 5A) of typical class I P450 enzymes33, and monomers of all three structures align well with rmsd values < 1 Å. In the asymmetric unit, two monomers (Fig. 5A) interact on a site close to the heme group. Although the buried site (approximately 750 Å2) does not indicate a biologically relevant interaction, the region overlaps significantly with the likely proximal site identified for electron delivery to the heme from Fdx (Supplementary Fig. 13A)34–36.

For each ternary complex, electron density for both the heme and the substrates is well-defined, unequivocally placing these molecules in the structures (Fig. 5B). The substrates bind along the central I-helix (amino acids 249–279, Supplementary Fig. 13B). The active site is sealed off in the substrate-bound state, but the high B-factors in the B-C loop (amino acids 69–83) and the C-terminal loop (amino acids 407–410) indicate flexibility (Supplementary Fig. 13C). This flexibility is most prominent in the F and G helices (amino acids 190–207), where the F helix extends further over the bound substrate contributing to hydrophobic packing.

In all substrate-bound structures, the C13 atom preferred for hydroxylation is situated within 4.4 Å of the heme center. The anthracycline aglycone extends towards the N-terminus of the central I-helix and is surrounded by mainly hydrophobic side chains (Ala261, Phe260, Gly257), with Tyr253 ‘capping’ off the binding site near the C4 methoxy group (Fig. 5B). Other residues anchoring the aromatic part of the substrate are in the 310-helix in the B-C loop region (amino acids 94-96) and Leu189 from the F-helix, while Val305 and Leu309 create a hydrophobic cavity for C14 near the active site (Fig. 5C). The substrate binding mode was investigated through mutation of Val305 to Leu or Ile, which led to loss-of-activity (Supplementary Fig. 13D). The L-daunosamine sugar moiety is coordinated through hydrogen bonds with water molecules and polar residues Lys185 and Thr411, as well as the main chain of Gly82 and a salt bridge with Asp84 (Fig. 5B). The axial water ligand is well defined in the electron density among the different substrate complexes. This axial water is also near the conserved Thr265 on the I-helix (Fig. 5C), and implicated in proton delivery via the acid-alcohol pair with Asp264 and bridging water molecules that are resolved in the complex structures33. Mutation of Thr265 to Ser was highly detrimental to the enzymatic activity in comparison to the wild-type (Supplementary Fig. 13D).

The ternary structures shed light on the relatively high conversion of DOD and DHD to their respective products compared to the low-level catalysis of DNR to DXR. In the structure models, C13 lies in a favorable position with respect to the heme center, while the C14 methyl is oriented away from the heme iron (Fig. 5C). Because the electron density at this resolution does not permit differentiation of the O13 ketone from the C14 methyl of DNR, we performed DFT calculations for both syn- and anti-conformers of C13, as this rotational barrier could, in part, inform the low rates of C14 C-H hydroxylation by DoxA (Supplementary Table 4). The calculations revealed that the anti-conformation between O9—O13 is favored by 2.3 kcal/mol over the corresponding syn conformer (Fig. 5D). This computational result is supported by the high-resolution crystal structures of DNR37 and carminomycin38,39, where the same anti-conformation was observed.

Other factors may contribute to attenuated C14 hydroxylation, namely the energetic barriers for the respective radical intermediates at C13 versus C14, but these do not obviate the requirement for proper substrate orientation in catalysis. Therefore, we performed 1000 ns molecular dynamics simulations with the DOD and DNR complex structures and the iron(IV)-oxo species (Compound I)33 modeled in place of the heme iron (Supplementary Fig. 14) in triplicate (Supplementary Table 5). These simulations revealed stable substrate binding throughout the simulation, where poses matching the crystallographic orientation were predominant. In the DOD complex, C13 was consistently oriented toward the iron(IV)-oxo (average 43% of frames, Fig. 5E). For DNR, C14 occasionally sampled productive binding poses (average 9% frames) either by rotation about the C13—C14 bond or by repositioning the anthracycline core in the active site (Fig. 5F). Further structural analysis of the MD simulations showed that DNR C13 carbonyl consistently forms a hydrogen bond with the Thr265 side chain (average 28% of frames, Supplementary Fig. 15), further stabilizing DNR in an unreactive pose for C14 oxidation. Together, these data support the observation that DoxA is highly predisposed for C13 oxidation, as opposed to C14 where low levels of C-H hydroxylation are explained by the structural and computational studies.

Metabolic engineering for improved production of DXR
To maximize production of DXR, we introduced all relevant genes into G001 stepwise using two vectors pSET15240 and pEN-SV141 integrating to the attB sites of phages ΦC31 and SV1, respectively, under the control of constitutive promoters kasOp*42, sp44 and the gapdh43 (Fig. 6A). As the first step, the DNA repair gene snorO from the nogalamycin pathway44 was introduced under the control of kasOp* in pEN-SV1 to improve the stability of the strain with initial production of 61 mg/L DXR and 154 mg/L DNR. Notably, the addition of doxA alone as a second gene in the same operon did not influence the production profile, indicating that the P450 enzyme was not rate-limiting. The subsequent introduction of the fdx4-fdr3 redox system as a single operon under the control of sp44 using pSET15 shifted the metabolic profile towards DXR with a 1.8-fold increase in production of DXR to 112 mg/L. Surprisingly, overexpression of the dnrV gene as a third gene in the operon did not significantly improve DXR yields in the first development cycle.

In a second development round, we considered the importance of the correct stoichiometry of the Fdx-FdR-P450 partners for efficient catalysis in the in vitro reactions (Fig. 4B), where the best results were obtained with high ferredoxin and low ferredoxin reductase concentrations. Early literature reports have also highlighted the importance of retaining the natural dnrV-doxA genetic organization in expression studies8. Expression of fdx4-dnrV-doxA as a multigene operon using the strong synthetic sp44 promoter resulted in a 2.8-fold increase in production of DXR to 166 mg/L. Interestingly, the addition of fdr3 under the control of the moderately active gapdh promoter was detrimental to the formation of DXR, demonstrating that the correct stoichiometry of the redox partners was also relevant in vivo.

Cultivation of the engineered strains was conducted with LXA1180 resin to bind anthracyclines and alleviate toxicity issues. However, the use of the resin resulted in the appearance of an unexpected compound (Fig. 6C), which was identified as ε-rhodomycinone (Fig. 1) based on LC-MS and NMR analysis (Supplementary Table 6, Supplementary Fig. 16–S18). We optimized the culture conditions and found that the hydrophobic pathway intermediate did not accumulate in cultures enriched with XAD7 resin (Fig. 6C), where carbon flux to the end products DXR and DNR was further improved. Anthracycline production by the best performing strain G001/pSnorO+pFdx4_DnrV_DoxA was subsequently evaluated in a bioreactor in 20 L volume (Fig. 6D). Time-course analysis revealed production of 336 mg/L DXR with an 81:19 DXR:DNR ratio, a 180% increase in doxorubicin production, enhancing its biotechnological feasibility.

In summary, the anthracycline DXR was discovered in 1969 from cultures of S. peucetius ATCC 27952 during industrial strain development6. However, despite decades of work and the importance of the compound for the pharmaceutical industry, DXR is still manufactured predominantly by semi-synthesis from DNR1. Here, we have uncovered several independent factors that limit the formation of DXR. First, we demonstrate that the activity of the P450 enzyme DoxA is highly dependent on the correct Fdx-FdR redox engine. Our transcriptomics data using industrial production strains uncovered fdx4 as the preferred electron donor to DoxA. In turn, phylogenetic analysis and biochemical characterization revealed that the SpFdR3 ferredoxin reductase is the optimal natural reductant of SpFdx4. Future research focusing on the use of non-native or artificial redox partners may lead to further catalytic improvements13, since wild-type S. peucetius does not naturally produce DXR and therefore evolution has not optimized the natural redox partners of DoxA. Second, we clarified the role of DnrV in the metabolic pathway and propose that it is an intracellular drug-binding protein, which may prevent the interaction of anthracyclines with DNA and subsequent toxic effects. The protein is indirectly vital for the biosynthesis of DXR due to the inherent product inhibition displayed by DoxA. However, the observation that DnrV also protects the anthracycline substrates from deglycosylation indicates a multifaceted role and warrants further investigations into the protein family. Third, the structure of DoxA in complex with DNR demonstrates that C13 of the substrate is positioned ideally for catalysis above the heme group. The preferred anti-conformation of DNR positions the C14 carbon in a small hydrophobic pocket pointing away from the heme unit. While other factors, such as electron transfer rate and the oxygen activation mechanism, have been noted to strongly influence P450 catalysis33,36, the slow conversion rate to DXR suggests that structure-based engineering of DoxA may be required to further improve catalysis. Despite these caveats, we were able to utilize the results presented here for rational metabolic engineering to generate a high-yield strain, paving the way for industrial manufacturing of biosynthetic DXR.

Methods
Genome assembly and annotation
Chromosomal DNA of the DNR and DXR over producing mutants, G001 and X121, respectively, were extracted and their genomes were sequenced. The strains were cultivated (30 °C, 300 rpm) in 50 ml GYM medium consisting of glucose 4 g/L, yeast extract 4 g/L, malt extract 10 g/L, and supplemented with 0.5% glycine for 3 days. The cells were collected by centrifugation (3000 x g, 5 min). Genomic DNA was extracted using the method from Nikodinovic et al.45 with minor modifications. The DNA was transferred to the Finnish Functional Genomics Centre (Turku, Finland) for PCR-free shotgun library preparation (Illumina) and sequenced using MiSeq v3, generating 2×300 bp paired-end reads (Illumina). The quality of the reads was manually checked before and after trimming and error correction using FASTQC (v0.11.2)46. Subsequently, the reads were error corrected and de novo assembled using A5-miseq (v20150522)47. Assembly of contiguous sequences was performed with ABACAS (v1.3.1)48 using S. peucetius ATCC 27952 (CP022438.1) as the reference49. Gaps were filled using IMAGE (v2.4.1)50. The final assembly was annotated using RAST51. All programs were run with the default parameters on the CSC–IT Center for Science’s Taito super-cluster (Espoo, Finland). The final assembly was deposited in the National Center for Biotechnology Information (NCBI) database under the accession numbers JBLNLL000000000 for G001 and JBLNLK000000000 for X121.

Transcriptomics
Wild-type, G001 and X121 strains were cultivated (30 °C, 300 rpm) in quadruplicates in 50 ml of E1 medium (glucose 20 g/L, starch 20 g/L, farmamedia 5 g/L, yeast extract 2.5 g/L, K2HPO4 x 3H2O 1.3 g/L, MgSO4 x 7H2O 1 g/L, NaCl 3 g/L, CaCO3 3 g/L). After cultivation, 1 ml of culture from each respective cultivation was pooled. Immediately after pipetting the samples into the tubes, 445 μl of STOP solution (95% EtOH, 5% phenol) was added. The tubes were vortexed for 5-10 s at full speed and centrifuged (4000 x g, 10 min). The supernatants were discarded, and the pellets were stored at -80 °C after freezing in liquid nitrogen. The cells were lysed with a mortar and pestle under liquid nitrogen. Total RNA was isolated using a RNeasy Mini Kit (Qiagen) with DNase treatment. Total RNA was sent to Finnish Functional Genomics Centre (Turku, Finland) for quality control (Agilent Bioanalyzer 2100), library preparation (Illumina), and sequencing with HiSeq 2500 (Illumina) to produce 1×50 bp reads. All analyses were performed using the Chipster platform52. The reads were manually checked using FASTQC46, and trimming was performed using TRIMMOMATIC53. The trimmed reads were aligned to the genome using BowTie254 and counted using HTSeq55. Differential expression was performed using edgeR56. The RNA-Seq data have been deposited in GEO (GSE289319) for WT, G001, and X121, respectively.

Phylogenetic analysis
The evolutionary history was inferred using the UPGMA method. The bootstrap consensus tree inferred from 888 replicates was taken to represent the evolutionary history of the taxa analyzed57. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The evolutionary distances were computed using the JTT matrix-based method58 and are in the units of the number of amino acid substitutions per site. The rate variation among sites was modeled with a gamma distribution (shape parameter = 3). This analysis involved 15 amino acid sequences. All positions with less than 88% site coverage were eliminated, i.e., fewer than 12% alignment gaps, missing data, and ambiguous bases were allowed at any position (partial deletion option). There were a total of 276 positions in the final dataset. Evolutionary analyses were conducted in MEGA1159. UniProt accession numbers for BphA4, PdR, SmFdR, AtFdR, MtFdR, ZmFdR, SoFdR, and PsFdR are Q52437, P16640, Q5S3I2, Q8W3L1, P9WIQ3, Q9SLP6, P00455, P10933, respectively.

Protein production and purification
The proteins used in the in vitro assays were produced as N-terminally 6×His-tagged recombinant proteins in Escherichia coli TOP10. Genes were ordered as synthetic DNA strands from either GeneWiz or Eurofins Genomics, cloned into the plasmid vector pBADHisBΔ60 with the restriction enzymes BglII and HindIII, and the expression construct was transformed into TOP10 by heat shock. Cells were grown in 330 ml of 2× tryptone yeast extract medium (2xTY) until the cultures reached an OD600 of 0.6—0.8, at which point protein production was induced by adding 0.02% L-arabinose. When producing iron-containing enzymes and DoxA, 0.1 mM of (NH₄)₂Fe(SO₄)₂ · 6H₂O and 0.25 mM aminolevulinic acid, respectively, were added at the time of induction. Protein was produced overnight, and cells were collected by centrifugation, suspended in wash buffer (20 mM sodium phosphate buffer, pH 7.4, 150 mM NaCl, 5 mM imidazole, 10% (v/v) glycerol), and disrupted by sonication. Cell debris was removed by centrifugation, and TALON Superflow resin (GE Healthcare) was added to the crude extract. In the case of the reductases, 10 μM of FAD was also added to the crude extract during this step. After 1 h of incubation, the resin was moved to a column and washed with additional A buffer and then eluted with B buffer (20 mM sodium phosphate buffer, pH 7.4, 150 mM NaCl, 200 mM imidazole, 10% (v/v) glycerol). PD-10 columns (GE Healthcare) were used to change the buffer to buffer C (40 mM sodium phosphate buffer, pH 7.4, 300 mM NaCl, 10% (v/v) glycerol), and the protein was concentrated with Amicon Ultra-4 10K centrifugal filters (Merck Millipore) down to 300–600 μl. Glycerol was added to 40% (v/v), and the proteins were stored at -20 °C. The purity and molecular weight of the proteins were checked by SDS-PAGE (Supplementary Fig. 2), and the concentration was estimated by the Bradford method61. Spinach (Spinacia oleracea) ferredoxin SoSfx and ferredoxin—NADP+ reductase SoFdR were obtained from Sigma.

For crystallization purposes, the doxA gene was cloned into the in-house vector pCPF3.11, a pET derivative containing an N-terminal enterokinase-cleavable His6-tag. The protein was expressed using BL21 (DE3) Rosetta 2 cells by growing fresh transformants in 2xYT supplemented with Ampicillin (100 μg mL−1) and chloramphenicol (30 μg mL−1) to OD600 1.4 before inducing with 0.5 mM Isopropyl β-D-1-thiogalactopyranoside. Expression was performed at 25 °C for 3 hours and cells were harvested by centrifugation for 20 min at 4kG, and the resulting pellets were stored at −20 °C. DoxA-expressing cell pellets were resuspended in buffer HisA (50 mM Tris pH 8.0, 300 mM NaCl, 20 mM imidazole) and lysed using sonication with these and all following steps conducted at 4 °C. The supernatant after centrifugation for 40 min at 30kG was loaded on a 5 mL HisTrap FF crude column (Cytiva) preequilibrated in buffer HisA, and the DoxA protein was eluted using a gradient of buffers HisA and HisB (HisA with 500 mM Imidazole). Fractions containing DoxA were combined and diluted two-fold using buffer Q-A (50 mM Tris pH 8.0) before being loaded on an equilibrated HiTrap Q column (Cytiva) and eluted with a gradient of buffer B (Q-A with 1 M NaCl). DoxA-containing fractions were pooled and divided in 200 nmole aliquots before each was incubated with a substrate (if required) for 1 hr. The resulting mix was run on a Superdex75 16/60 (Cytiva) gel filtration column using 20 mM Tris pH 7.4 and 150 mM NaCl, with extra absorbance monitoring on 482 nm for anthracycline monitoring. Complex-containing fractions were analyzed on gel, pooled, and concentrated to at least 12 mg mL−1 before being flash frozen or directly setup for crystallization.

CYP450 CO-binding assay
The integrity of the heme group and the concentration of active DoxA were measured with a P450 CO-binding assay62. DoxA was diluted into a solution of 80 mM sodium phosphate buffer, 1 mM EDTA, and 10% glycerol, and the difference in absorption at 420 nm and 450 nm was measured before and after the addition of CO gas and sodium dithionite to the sample. The concentration was then calculated using the extinction coefficient 91,000 M−1 cm−1.

Enzymatic assays
Enzymatic assays to measure DoxA activity were performed in vitro in reaction volumes of 200 μl. For analysis of FdR enzymes, DoxA (6 μM), ferredoxin (10 μM), ferredoxin reductases (10 μM), catalase (2.5 U), sodium phosphate buffer pH 7.5 (≈ 20 mM) and substrate (13-DOD, 13-DHD, DNR, ≈ 15 μM) were mixed with a NAD(P)H recycling system consisting of glucose (4 mM) and glucose dehydrogenase (0,5 U). For the commercially acquired spinach SoFdx and SoFdR, 8.8 μg and 0.02 U, respectively, were used in the reactions. For the reactions with PdR, only 0.5 μM reductase and an incubation time of 2 h were used, since higher concentrations led to degradation of the substrate. Optimized reaction conditions for full conversion to DXR included 10 μM DoxA, 20 μM SpFdx4, 0.1 μM SoFdR, 20 μM DnrV, 4 mM Glucose, 1 U GDH, and 2,5 U catalase, 0.5 mM NADP+. The reactions were started via the addition of NAD(P)H and NAD(P)+ (0.5 mM) and left to react at room temperature overnight. The reactions were extracted with a 4:1 mixture of chloroform and methanol, dried in vacuo, dissolved in pure methanol, and analyzed by HPLC (SCL-10Avp/SpdM10Avp system with a diode array detector (Shimadzu)) using a C18 Kinetex column (2.6 μM, 100 Å, 4.6 × 100 mm (Phenomenex)) at room temperature. The gradient run parameters included 0-2 min, 0% B; 2–20 min, 0–60% B; 20-24 min, 100% B; 24–29 min, 0% B. Solvent A consisted of 15% acetonitrile in 0.1% HCOOH, and solvent B of 100% acetonitrile. The absorbance of the samples was recorded at 490 nm.

Crystallization of DoxA
All DoxA crystals were grown at 20 °C by sitting drop vapor diffusion by mixing mother liquor and protein solution in a 1:1 volumetric ratio. Crystals of DoxA in complex with DNR were grown at 20 °C by sitting drop vapor diffusion in Intelli-Plate 96-2 shallow well plates (Hampton Research) by combining 1 μL of protein solution containing 12 mg mL-1 DoxA and 1 mM DNR with 1 μL of well solution composed of 1.65 M ammonium sulfate, 0.1 M Bis-Tris pH 5.5, 2% trehalose. The droplets were seeded immediately by streaking with a cat whisker charged with previously grown DoxA crystals. The crystals were cryoprotected by the direct addition of 8 μL of a cryoprotectant solution composed of 1.65 M ammonium sulfate, 0.1 M Bis-Tris pH 5.5, 18% trehalose, 10 mM Hepes pH 7.5, 50 mM NaCl, 2% glycerol, 1 mM DNR, and flash frozen in liquid nitrogen. Diffraction data was collected at the University of Michigan Center for Structural Biology using an Excillum MetalJet-D2+ 70 kV, 250 W X-ray source equipped with an Eiger 2R 4M hybrid detector. The data were integrated and scaled using XDS63 and the structure was solved by molecular replacement using Phaser MR64 with CYP154C2 (pdb: 7CL9) as the search model. The initial model was subjected to iterative rounds of manual building and refinement using Coot65 and Phenix.refine66. For the crystals of complexes with DOD and DHD, DoxA was pre-incubated for 1 hr with the substrate before being subjected to gel filtration. Peak fractions were concentrated to 16 mg/mL and setup in MRC 2-well plates using an NT8 robotic crystal screen dispenser (Formulatrix) on a 200 nL scale. Crystals for DoxA-DOD were found in conditions containing 0.1 M citrate pH 5.0 and 20% (w/v) PEG 6000, whilst for DHD-complex a mother liquor solution with 0.1 M MES pH 6.0, 0.2 M NH4Cl and 20% (w/v) PEG 6000 yielded diffracting crystals. Single crystals were cryoprotected with 30% ethylene glycol before being flash-frozen. Diffraction data were collected at the European Synchrotron Radiation Facility, in particular the MASSIF beamlines, and processed using DIALS67. Data were loaded in the CCP468 pipeline: data were reduced using Aimless69 and the structure was solved using PHASER64 by molecular replacement with the DoxA AlphaFold model (AF-A0A2D3UDU4). The resulting model was finalized using iterative rounds of refinement with REFMAC70 and model building in COOT65.

Quantum mechanics
All QM calculations were performed with Gaussian 1671. Geometry optimizations and frequency calculations were performed on the proposed crystal structure conformations at the B3LYP level with 6-31G(d). Optimized structures contained zero negative frequencies.

Molecular dynamics simulations
The heme iron(IV)-oxo complex involved in the cytochrome-catalyzed oxidation (compound I) was used to model the active form of the heme cofactor. Simulations were performed using the Amber 12 package72, and the Amber-compatible parameters73 were used for compound I and its axial Cys ligand. Parameters for anthracycline ligands (DOD and DNR) were generated within the antechamber module in AMBER force field (gaff), with partial charges set to fit the electrostatic potential generated at the HF/6-31G(d) level by the RESP model74. The charges were calculated according to the Merz-Singh-Kollman scheme75,76 using Gaussian 16. DoxA was immersed in a pre-equilibrated truncated cuboid box with a 10 Å buffer of TIP3P77 water molecules using the tleap module. All subsequent calculations were done in Amber/ff99SBildn force field78. We then performed 1000 ns simulations starting from the crystal structure of DoxA bound to deoxydaunorubicin (DOD) and daunorubicin (DNR). The system was optimized, gradually heated to 300 K, and equilibrated before 1000 ns simulation runs. Simulations were analyzed using Chimera79, and figures were made using PyMol (The PyMOL Molecular Graphics System, Schrödinger, LLC).

Generation of sufR mutant strain
The disruption of the sufR gene was carried out via homologous recombination using the unstable multicopy pWHM3-based vector80, pWHM3-oriT. The disruption cassette contained the apramycin resistance gene, aac(3)IV, flanked by 1 kb regions upstream and downstream of sufR. This cassette was synthesized as synthetic DNA and cloned into HindIII and XbaI restriction sites of the multicopy pWHM3-oriT vector. The resulting construct was introduced into E. coli ET12567/pUZ8002 strain80 and conjugated with S. peucetius ATCC 27952 strain. Mutants were generated through several passages of exconjugants under non-selective conditions on MS plates. Double-crossover mutants were screened based on apramycin resistance and the loss of thiostrepton resistance. Finally, PCR and gel electrophoresis confirmed the deletion of the target gene (Supplementary Table 7, Supplementary Fig. 1).

Construction of plasmids and strains
The doxA, dnrV, fdx4, and fdr3 genes from S. peucetius ATCC 27952 and snorO gene from S. nogalater ATCC 2745137, along with their corresponding ribosome binding sites, were ordered as synthetic fragments from GeneWiz. The dnrV-doxA sequence was synthesized as fragments from the genome while keeping their native sequence intact. The integrative vectors pSET15240, pSET154BB81, and pEN-SV141 were used to express biosynthetic genes. The E. coli TOP10 strain was utilized for cloning and plasmid propagation. DNA fragments were assembled with vectors using corresponding restriction sites. The snorO gene, flanked with EcoRI/PstI restriction sites, was cloned into the pEN-SV vector. Then, for the first round of constructs, the doxA gene, flanked with XbaI/PstI, was added downstream of the snorO gene in the pEN-SV1 vector. The pSET152 vector was constructed to harbor the fdx4 and fdr3 genes, flanked with EcoRI/PstI and SpeI/PstI, respectively, followed by the introduction of the dnrV gene with PstI/NsiI. For the second round of constructs, fdx4 gene, the dnrV-doxA fragment and fdr3 gene were assembled using BioBricks and cloned in pSET154BB with EcoRI/PstI. The fd phage terminator82 was introduced after dnrV-doxA fragment. Promoters and RBSs are indicated in Fig. 6. The final constructs were integrated into the genome of S. peucetius G001 with conjugation using standard protocol. The E. coli ET12567/pUZ8002 strain was used for conjugation.

Culture conditions and metabolite extraction
For comparison of WT, G001, and X121, strains were cultivated in triplicate in 50 ml of E1 medium for 13 days. Anthracyclines aglycones were extracted using acid hydrolysis. Specifically, 2 mL of culture samples were collected, mixed with 2 mL of 1 M HCl, and boiled for 20 minutes. After cooling, 2 mL of chloroform was added, and the samples were mixed for 30 minutes. The lower phase was then dried in a concentrator and dissolved in 200 μL of methanol before HPLC analysis. The S. peucetius G001 strain and its mutants were cultivated in triplicate in 30 mL of E1 medium with the addition of 20 g/L of either LXA1180 (Sunresin) or XAD7 HP (Supelco) resin. After 5 days, the resin was collected, washed, and the produced compounds were extracted with 25 mL of methanol. For the best doxorubicin-producing mutant, 1 mL of culture was collected at various time points, the resin was washed from the cells, and the produced compounds were extracted with 1 mL of methanol. The HPLC analysis of samples was performed at room temperature using a Shimadzu Nexera X3 system with a photodiode array detector and a C18 column (2.6 μM, 100 Å, 4.6 × 100 mm Kinetex column (Phenomenex)). A total of 10 μL was injected. The gradient run parameters included 0-2 min, 0% B; 2–20 min, 0–60% B; 20-24 min, 100% B; 24–29 min, 0% B. Solvent A consisted of 15% acetonitrile in 0.1% HCOOH, and solvent B of 100% acetonitrile. Absorbance was measured at 490 nm, and the reaction products were identified by comparison to standard compounds. The yield of compounds was calculated using doxorubicin and daunorubicin standard curves.

For large-scale production, strain G001/pSnorO+pFdx4_DnrV_DoxA was cultivated in a 42 L total volume Bioengineering LP bioreactor (Bioengineering AG). Seed cultures were prepared in 250 mL flasks using 50 mL of E1 medium and incubated at 30 °C and shaking at 250 rpm. A 10% (v/v) inoculum was transferred into the bioreactor containing 20 L of E1 medium supplemented with XAD-7 resin (20 g/L) at the start of fermentation. The fermentation was carried out for 8 days at 30 °C with an airflow rate of 15 NL/min. The dissolved oxygen (DO) setpoint was maintained at 40% by controlling agitation in a cascade mode, from a baseline of 250 rpm up to 300 rpm. Polypropylene glycol 2000 was added as an antifoam agent as needed. Samples were collected every 24 hours for compound extraction and subsequent HPLC analysis as previously described.

Purification and identification of compounds
For the structure elucidation of ε-rhodomycinone, methanol extracts from LXA1180 resin were purified by silica chromatography using high-purity grade silica (pore size 60 Å, 230-400 mesh particle size) and 250 mL of an eluent at 49.5:49.5:1 MeOH/DCM/HCOOH and 100 mL of an eluent at 59.4:39.6:1 MeOH/DCM/HCOOH. Fractions from silica chromatography were further purified by semi-preparative HPLC using a LC-20AP/CBM-20A system with a diode array detector (Shimadzu) and Phenyl-Hexyl, 5 μm, 100 Å, 250 × 21.2 mm Kinetex column (Phenomenex) The gradient run parameters included 0–2 min, 0% B; 2–22 min, 0–100% B; 22–24 min, 100% B with a flow rate of 20 mL/min. Solvent A consisted of 15% acetonitrile in 0.1% HCOOH, and solvent B of 100% acetonitrile. Fractions containing ε-rhodomycinone were extracted with chloroform, dried using a rotary evaporator and desiccator, and resuspended in deuterated solvents for NMR measurements. High-resolution electrospray ionization mass spectra were recorded by Waters ACQUITY RDa Detector using an XBridge BEH C18 Column, 130 Å, 5 μm, 4.6 × 30 mm (Waters). NMR spectra were recorded by a 500 MHz Bruker AVANCE-III system with a liquid nitrogen-cooled Prodigy BBO cryoprobe. All NMR spectra were processed in Bruker TopSpin 3.7.0 version, and the signals were internally referenced to the solvent signals.

For the structure elucidation of 7-deoxydaunorubicinone, 10.5 mg of daunorubicin was subjected to biocatalytic reduction by spinach ferredoxin reductase SoFdR. The reaction was performed overnight at 28 °C in a degassed buffer with the following components: 2 mM daunorubicin, 1 mM NADP+, 5 mM glucose-6-phosphate, 1 U/μL glucose-6-phosphate dehydrogenase, 10 μM spinach ferredoxin reductase, 1% DMSO, 10% glycerol, 50 mM potassium phosphate, pH 7.4. Precipitation of the aglycone was observed after incubation. The reaction was extracted four times with an equal volume (10 mL) of chloroform. The first solvent addition produced an emulsion that was treated by bath sonication and centrifugation at 1000 x g for 5 min. The combined organic layers were washed twice with an equal volume of brine, dried over Na2SO4, and concentrated by rotary evaporation to afford 7-deoxydaunorubicinone in quantitative yield. HR-MS analysis was performed on Agilent 6545 Q-TOF. NMR spectra were recorded by Varian 600 MHz NMR with nitrogen cooled Prodigy cryoprobe. All spectra were processed with MNova software.

Statistics and reproducibility
No statistical method was used to predetermine sample size. Sample sizes were chosen based on standard practice in the field and on the number of independent biological replicates required to ensure reproducibility. No data were excluded from the analysis. Bacterial cultivations for metabolite production analysis were performed in independent biological triplicates (n = 3) unless otherwise stated in the text. Results of experiments were presented as mean ± standard deviation (SD), as indicated in the legends of the figures. Molecular dynamics simulations were performed in triplicate (n = 3). Differential expression analysis for transcriptomics was performed using the statistical methods implemented in edgeR.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this paper, the natural electron transfer chaperone FDX4-FDR3 was systematically identified and validated for the first\ntime by comparing transcriptomic data, which significantly enhanced the catalytic efficiency of DoxA and solved the key\nproblem of the P450 enzyme, which has long been an “unknown source of electrons”. Subsequently, the authors revealed\nthat DnrV, as the first drug-binding protein in the VOC superfamily that interacts with P450, indirectly promotes DoxA\ncatalysis by preventing product inhibition through binding to DXR, thus expanding the understanding of P450 enzyme\nregulation. By resolving the crystal structure of DoxA complexed with three substrates, combined with molecular simulations,\nthe conformational mechanism (anti-conformation advantage) of DNR being difficult to be hydroxylated was elucidated,\nwhich provides a theoretical basis for the subsequent structural modification. Based on the above discoveries, the authors\nhave constructed an engineered strain with high yield of DXR, which can increase the yield by 180%, and has the value of\npractical industrialization application. The paper covers multi-dimensional means, such as histological analysis, protein\nexpression purification, in vitro enzymology, structural biology, computational chemistry and metabolic engineering, etc.,\nwhich are cross-validated, logical, and highly convincing. However, the manuscript needs to be improved for publication in\nNature Communications. Some comments should be addressed below.\nMajor points:\nThe exploration of the dynamics of the crystal structure of DoxA is still insufficient, although the authors have performed\n1000 ns molecular dynamics simulations, and it is recommended to supplement the statistical analysis of conformational\nchanges induced by different substrates (e.g., changes in the volume of the active pocket, RMSF of key residues, etc.) to\nexplain the differences in substrate selectivity in a more specific way.\nThe article focuses on the role of FDX4/FDR3, but no comparative experiments with other potential FDX/FDR combinations\n(e.g., fdx5 mentioned in the article) were performed, which may affect the generalizability of the conclusions. It is\nrecommended to supplement the in vitro activity comparison of different FDX/FDR combinations to verify whether\nFDX4/FDR3 is the optimal choice.\nThe molecular mechanisms (e.g., binding sites, dynamic interactions) of DnrV binding to DXR have not been validated by\nco-crystallization or mutagenesis experiments. It is recommended to utilize targeted mutagenesis or structural biology to\nclarify the key residues of DnrV binding to DXR and to verify its regulatory role on product inhibition.\nThe percentage of DNR “productive poses” in the molecular dynamics simulation was only 14%, which needs to be further\nstatistically verified for significance. It is recommended to increase the number of simulation repetitions or adopt enhanced\nsampling techniques (e.g. Metadynamics) to improve the reliability of conformational sampling.\nData on long-term passaging stability of engineered strains, scale-up of fermentation (e.g., bioreactor level), and economic\nanalysis (e.g., carbon source utilization efficiency) are lacking. It is recommended to supplement the results of continuous\nculture experiments and pilot-scale fermentation to assess its industrialization feasibility.\nSome of the experimental steps are described briefly (e.g. DoxA purification conditions, crystallization conditions\noptimization), which may affect the reproducibility. It is suggested to add key parameters (e.g., buffer pH, temperature, protein\nconcentration) and quality control data (e.g., standard curve for enzyme activity assay).\nOther potential factors (e.g., electron transfer rate, oxygen activation mechanism) for the inefficiency of DoxA-catalyzed DNR\nto DXR were not adequately discussed. It is recommended to include in the discussion an exploration of possible rate-\nlimiting steps and to suggest future research directions (e.g. cofactor engineering or dynamic regulatory strategies).\nGraphical and Data Presentation: Error calculation methods: Specify the type of error bars (standard deviation SD or\nstandard error SEM) in the legend or methods section, and indicate the number of replicates (e.g., “n=3 biological\nreplicates”). If there are batch-to-batch differences, supplement the data points of a single experiment with a boxplot or\nscatterplot. Detailed description of the HPLC gradient program (e.g., “0-2 min, 0% B; 2-20 min, 0-60% B”) with mobile phase\ncomposition (e.g., “A: 0.1% formic acid aqueous solution, B: acetonitrile”) in the Methods section. Add the detection\nwavelength (e.g., “λ = 490 nm”) and column temperature (e.g., “25°C”).", "reviewer_2": "• What are the noteworthy results?\n1. The natural redox partners ferredoxin FDX4 and ferredoxin reductase FDR3 were identified through transcriptomic\nanalysis, which are crucial for DoxA catalysis.\n2. Structural analysis and density functional theory (DFT) calculations revealed that the inefficient C-14 hydroxylation of\ndaunorubicin is due to the unfavorable anti-conformation of its methyl ketone sidechain.\n3. Advances in rational strain engineering led to a 180% increase in doxorubicin yields, improving the production profile.\n• Will the work be of significance to the field and related fields? How does it compare to the established literature? If the work\nis not original, please provide relevant references.\nThere is some progress, but it is not significant. The main achievement is obtaining the three-dimensional structures of the\ncomplexes formed by the DoxA enzyme and the substrates DNR, DHD, and DOD.\n• Does the work support the conclusions and claims, or is additional evidence needed?\nAdditional evidences are needed. For example, high-resolution three-dimensional structure of apo DoxA, and direct use of\nDNR as substrate to verify the product inhibition of DXR, etc.\n• Are there any flaws in the data analysis, interpretation and conclusions? Do these prohibit publication or require revision?\nYes, I have attached a file. I believe these should prohibit publication.\n• Is the methodology sound? Does the work meet the expected standards in your field?\nNo, I do not believe the methodology is sound, and the work does not meet the expected standards in the field.\n• Is there enough detail provided in the methods for the work to be reproduced?\nThe substrate DOD is not readily available. Additionally, reproducing the other data as a whole is relatively straightforward.\n\n[ATTACHED REVIEWER REPORT]\n Is the CC (1/2) value for DoxA apo reasonable? It is significantly lower\nthan 0.5. The Rpim value is 0.902, which is much greater than 0.5. The\nresolution is too low, resulting in lower reliability for the overall apo\nstructure.\n Is \"Na SO \" written incorrectly in Figure S3? Additionally, is the\n2 4\nformatting consistent with academic standards?\n The compound 7-deoxydaunoribicinone does not appear anywhere in\nthe main text of the manuscript.\n Line 67: V values writing specification?\nmax\n Line 143-145:\nThe manuscript states that \"FDX4 is a 2Fe-2S ferredoxin similarly to\nSFX and PDR,\" but later concludes that FDX4 is a Rieske-type ferredoxin\nwhile PDR is classified as a non-Rieske [2Fe-2S] ferredoxin (Figure S3).\nThis raises two concerns:\n1. Logical inconsistency: If FDX4 shares similarity with PDR, their\nclassifications as Rieske-type and non-Rieske ferredoxins, respectively,\nappear contradictory.\n2. Functional misclassification: The description of PDR as a\nferredoxin may require re-evaluation. Ferredoxin reductases (e.g., PDR\nfamily proteins) and ferredoxins (e.g., SFX family) are functionally distinct\nredox partners, not homologous proteins. This mischaracterization\nundermines the comparative analysis.\n Line 149-150:\nThe statement that \"Five of the six FDR proteins related to PDR\nwere produced in E. coli\" (R1, R2, R3, R4, R7) raises methodological\nconcerns:\n1. Unclear selection criteria:\nThe manuscript does not define which six FDR proteins are\nconsidered \"related to PDR\" or specify the criteria for their selection. The\nphylogenetic tree (Figure 3B) does not clearly demarcate a clade\ncontaining exactly six FDR proteins. For instance, the purple-clade region\n(putative PDR-related group) appears to contain more than six members.\nThe rationale for excluding one protein from heterologous\nexpression requires justification. Was this due to failed\nexpression/purification, or evolutionary divergence?\n2. Phylogenetic inconsistencies:\nThe claimed PDR-related FDRs (R1, R4, R7) cluster within a\nsubclade distinct from other FDRs, yet R2 and R3 appear evolutionarily\ndistant from this group. This contradicts the implied functional\nrelationship.\nIf evolutionary proximity to PDR is the basis for selection, the six\ncandidates should be explicitly annotated on the phylogenetic tree with\nbootstrap support values.\n Enzyme activity assay section: In Fig. 4A, using DOD as the\nsubstrate, only a small amount of DXR is produced at a DnrV concentration\nof 10 μM, while at 20 μM, the yield of DXR is somewhat higher, but still\nlower than that of DNR. However, in Fig. 4C, when examining the effect\nof DXR concentration on the reaction, DOD is again used as the substrate,\nand at a DnrV concentration of 10 μM, the amount of DXR produced in\nthe absence of added DXR is significantly higher than in Fig. 4A, even\nexceeding the yield of DNR. This presents a contradiction, as indicated by\nthe red box in the figure below.\n Line 168-170:\nWhy was DOD used as the substrate in the experiment examining the\neffect of DXR concentration on the reaction, rather than DHD or DNR?\nHow can we determine whether the inhibition of the DoA reaction is due\nto the DXR product or if DoxA exhibits catalytic region selectivity?\n Line 400: 16 mg mL-1. Pay attention to format specifications.", "reviewer_3": "This is an excellent study on engineering a P450 system for improved production of doxorubicin, an important\nchemotherapeutic agent. The authors have improved catalysis by finding the natural redox partners for DoxA and by using\nan anthracycline binding protein to block product inhibition of the P450. I have only a few minor comments.\n1. Somewhere in the paper the author should give the correct CYP name for DoxA.\n2. In the figure legend to figure 4 they refer to panel “f” as “e”.\n3. The MD doesn’t add much especially using compound I. The lifetime of compound I is on the order of picoseconds, as\nestimated from radical clocks, so a 1000 ns simulation really is not of much relevance. A better model would be the oxy-\ncomplex. There are a few good P450-oxy-complex structures as a basis for modeling.\n4. In the MD word they only report the percentage of frames where C14 is in position for hydroxylation. They should also\nreport the percentage of frames for C13."}, "author_response": "RESPONSES TO REVIEWER COMMENTS:\nReviewer #1 (Remarks to the Author):\nIn this paper, the natural electron transfer chaperone FDX4-FDR3 was systematically identified and\nvalidated for the first time by comparing transcriptomic data, which significantly enhanced the catalytic\nefficiency of DoxA and solved the key problem of the P450 enzyme, which has long been an “unknown\nsource of electrons”. Subsequently, the authors revealed that DnrV, as the first drug-binding protein in the\nVOC superfamily that interacts with P450, indirectly promotes DoxA catalysis by preventing product\ninhibition through binding to DXR, thus expanding the understanding of P450 enzyme regulation. By\nresolving the crystal structure of DoxA complexed with three substrates, combined with molecular\nsimulations, the conformational mechanism (anti-conformation advantage) of DNR being difficult to be\nhydroxylated was elucidated, which provides a theoretical basis for the subsequent structural modification.\nBased on the above discoveries, the authors have constructed an engineered strain with high yield of DXR,\nwhich can increase the yield by 180%, and has the value of practical industrialization application. The paper\ncovers multi-dimensional means, such as histological analysis, protein expression purification, in vitro\nenzymology, structural biology, computational chemistry and metabolic engineering, etc., which are cross-\nvalidated, logical, and highly convincing. However, the manuscript needs to be improved for publication in\nNature Communications. Some comments should be addressed below.\nRESPONSE: Thank you for the overall evaluation.\nMajor points:\nThe exploration of the dynamics of the crystal structure of DoxA is still insufficient, although the authors\nhave performed 1000 ns molecular dynamics simulations, and it is recommended to supplement the\nstatistical analysis of conformational changes induced by different substrates (e.g., changes in the volume\nof the active pocket, RMSF of key residues, etc.) to explain the differences in substrate selectivity in a more\nspecific way.\nRESPONSE: We have analyzed the major hydrogen bond contacts between DoxA and either DOD or DAU\nthrough 1uS simulations run in triplicate, and this data is summarized in Figure S15. We note the\ndifferences between DOD and DAU, specifically that of DAU with Thr265 in line “240-242” of the main text.\nThe article focuses on the role of FDX4/FDR3, but no comparative experiments with other potential\nFDX/FDR combinations (e.g., fdx5 mentioned in the article) were performed, which may affect the\ngeneralizability of the conclusions. It is recommended to supplement the in vitro activity comparison of\ndifferent FDX/FDR combinations to verify whether FDX4/FDR3 is the optimal choice.\nRESPONSE: Thank you for the suggestions. As explained in more detail below, we were not able test all\nFDX/FDR combination as many of the proteins were not soluble. In the case of ferredoxins, our\ntranscriptomics data revealed overexpression of only two genes fdx4 and fdx5. We expressed both of these\ngenes in E. coli for production of recombinant proteins, but Fdx5 could not be produced in soluble form.\nBased on sequence analysis, Fdx5 contains a [Fe4-S4] cluster that is likely to be highly sensitive to molecular\noxygen, but protein production was not improved even under anoxic conditions. In the case of ferredoxin\nreductases, the transcriptomic data did not identify any strong ferredoxin reductase candidates likely to\nfunction with Fdx4 and DoxA. We did clone all seven fdr genes that were identified from the genome of S.\npeucetius but were able to only produce five enzymes in soluble form in E. coli. As also noted by Reviewer\n#2, this was stated too briefly in the original submission, and we have re-phrased the text in line 159. For\nthese reasons, we believe that we have completed the in vitro activity comparison with all protein pairs\nthat could be feasibly tested and the data is presented in Figure 3B.\nThe molecular mechanisms (e.g., binding sites, dynamic interactions) of DnrV binding to DXR have not been\nvalidated by co-crystallization or mutagenesis experiments. It is recommended to utilize targeted\nmutagenesis or structural biology to clarify the key residues of DnrV binding to DXR and to verify its\nregulatory role on product inhibition.\nRESPONSE: We agree that the understanding of the molecular mechanism of DnrV is highly important and\nthe topic has been the target of our investigations for several years. The research has expanded beyond\nanthracycline biosynthesis and has uncovered that DnrV is a member of a novel subgroup of VOC enzyme\nwhose genes are often found co-located with P450 partner genes in numerous metabolic pathways. The\ncommon feature appears to be that the P450/VOC enzyme combinations are found from pathways where\nthe substrates contain quinone units, such as in the biosynthesis of the vitamin K precursor menadione. The\nstudies have expanded to such a degree that we feel that inclusions of structural studies of DnrV in this\nsubmission is challenging. A preprint of the DnrV work containing the three-dimensional structure,\nstructure/function studies, more detailed biochemical characterization and enzyme kinetics is available at:\nhttps://chemrxiv.org/engage/chemrxiv/article-details/688a543623be8e43d6e90942\nHowever, we do recognize the need to reinforce our findings on DnrV in this work. Thus, we have expanded\nour analysis of the role of DnrV in the revised manuscript as follows. In the original submission, we only\nbriefly mentioned the formation of deglycosylation shunt products. This is a previously known\nphenomenon that occurs even in the absence of P450 enzymes through direct reduction of anthracycline\nquinones by, for instance, spinach ferredoxin reductase (Fisher et al, 1985)). In the revision, we now\npresent a new control reaction depicting formation of deglycosylation products under suboptimal reaction\nconditions under excess Pdx/PdR (lines 142 and Figure S4). This was done to highlight the role of DnrV in\nthe protection of anthracycline substrates from degradation (revised Figure 4B). We have expanded the\ndiscussion on this point (line 173) and confirmed that substrate protection is specific for DnrV. As a\nnegative control, we demonstrate that the related VOC enzyme, the Mitomycin Resistance Determinant\n(MRD), does not bind anthracyclines and therefore the reaction leads to formation of deglycosylated shunt\nproducts discussed in line 191 and shown in a new Figure S12.\nOverall, we conclude that DoxA and improving doxorubicin production should be the focus of the current\nmanuscript, while the focus of our follow-up manuscript will be on DnrV and the role of VOC enzymes more\nbroadly on P450 catalysis.\nThe percentage of DNR “productive poses” in the molecular dynamics simulation was only 14%, which\nneeds to be further statistically verified for significance. It is recommended to increase the number of\nsimulation repetitions or adopt enhanced sampling techniques (e.g. Metadynamics) to improve the\nreliability of conformational sampling.\nRESPONSE: Thank you for this suggestion, we have repeated our MD simulations in triplicate and reported\nthese values in a new Table S5.\nData on long-term passaging stability of engineered strains, scale-up of fermentation (e.g., bioreactor\nlevel), and economic analysis (e.g., carbon source utilization efficiency) are lacking. It is recommended to\nsupplement the results of continuous culture experiments and pilot-scale fermentation to assess its\nindustrialization feasibility.\nRESPONSE: We agree that demonstrating the potential of our engineering efforts for industrial DXR\nmanufacturing is important. In this revision, we have scaled the production of DXR to a 20L volume in a\nbioreactor, which retained the overproduction of DXR over DNR (81:19 ratio) with slightly increased yields\n336 mg / L DXR. This information has been added to the revised manuscript (lines 274) and replaces the\ntime-course analysis of DXR production that was previously conducted in flask cultures (Figure 6D).\nIn terms of strain stability, we performed seven rounds of continuous cultivations in 250 mL flasks to\nobserve product formation. Each flask was inoculated on day 3 from the previous generation and product\nformation was monitored on day 7. Based on the figure below, we observed increased DXR production in\nour engineering strain (left) throughout the experiments over the parental control strain (right). However,\nour data also demonstrated that anthracycline yields decreased after each cultivation round. Thus, our\nconclusion from this data is that the overproducing strain G001 is not suitable for industrial production, but\nthat our P450 redox engineering appears to be stable as demonstrated by the DXR/DNR production\nprofiles. We have decided not to include these data to the manuscript due to the loss of productivity.\nIn terms of economic analysis, we were severely hampered by lack of public information on industrial strain\nyields and costs of semi-synthesis of DXR from DNR, which are highly guarded trade secrets. Our\nconsultation with industry representatives suggested that the semi-synthesis consists of five steps and\nsynthesis yields of 50% are considered sufficient. In terms of carbon utilization, the fermentations were\nperformed in an industrial medium that was utilized for anthracycline manufacturing in the early 2000s, but\nthat product yields of 1-2 g / L of DNR are required for commercialization. When DNR to DXR semi-\nsynthesis yields of 50% are taken into consideration, our engineering strains appear to be slightly below the\ntarget value. We would like to highlight that in our view this does not diminish the value of our P450 redox\nengineering, since transfer of our expression system to a contemporary industrial host strain should retain\nhigh DXR:DNR ratio with improved yields. However, we have not been able to acquire current industrial\nproduction strains for analysis. We have not added any discussion on the economic analysis to the\nmanuscript, since we feel that unofficial discussions with industry representatives do not meet the rigor\nrequired for scientific publication.\nSome of the experimental steps are described briefly (e.g. DoxA purification conditions, crystallization\nconditions optimization), which may affect the reproducibility. It is suggested to add key parameters (e.g.,\nbuffer pH, temperature, protein concentration) and quality control data (e.g., standard curve for enzyme\nactivity assay).\nRESPONSE: Agreed and amended. We have improved the Methods section and added details for DoxA\npurification and crystallization (starting at line 411).\nWe also agree that addition of quality control data for enzymatic activity (standard curves mentioned\nabove, boxplots or scatterplots mentioned below) would improve the rigor of the work. However,\ngenerating this data has proven to be highly challenging, and time-demanding. The key issue is that\noptimization of the multienzyme reaction conditions is the result of years of work. The challenge has been\nthat formation of deglycosylation shunt products occurs readily if the Fdx-FdR-DoxA-substrate ratio in not\noptimized. This key point is discussed in the revised manuscript at line 140 and displayed in Figure S4. We\nhave identified the following factors that influence product yields and formation of shunt products:\n- We have noted that each Fdx-FdR-DoxA combination requires individual optimization for best\nperformance\n- We have observed activity loss in storage for DoxA and Fdx4\n- Heterologous expression of Fdx4 in E. coli has given variable yields of holoprotein generating batch-\nto-batch variation\n- We have observed loss in activity with FdR with repeated freeze thaw cycles.\n- We have found that inclusion of catalase is required, even with DnrV, indicating that some\nhydrogen peroxide is formed even under optimal reaction conditions\nThese issues have prevented us from presenting further quality control data. We would like to highlight\nthat due to the challenges in the enzymatic assays, all data presented in any single figure in the manuscript\nalways originate from parallel enzymatic reactions that were conducted at the same time on the same day.\nWe deemed that this approach was necessary to prevent influence from batch-to-batch variation and\nstorage stability issues. Reaction outcome data presented in the revision are based on optimized conditions\nproviding the highest conversion rates.\nOther potential factors (e.g., electron transfer rate, oxygen activation mechanism) for the inefficiency of\nDoxA-catalyzed DNR to DXR were not adequately discussed. It is recommended to include in the discussion\nan exploration of possible rate-limiting steps and to suggest future research directions (e.g. cofactor\nengineering or dynamic regulatory strategies).\nRESPONSE: Agreed and amended. Discussion on the implications of our findings was limited in our original\nsubmission due to length restraints (<3500 words). However, we fully agree that it is important to address\nthese issues in the Conclusions section. Thus, we have briefly outlined possible future research directions\nfor each of the three major findings (DoxA redox engine; role of DnrV; structure of DoxA) in the revised\nmanuscript.\nGraphical and Data Presentation: Error calculation methods: Specify the type of error bars (standard\ndeviation SD or standard error SEM) in the legend or methods section, and indicate the number of\nreplicates (e.g., “n=3 biological replicates”). If there are batch-to-batch differences, supplement the data\npoints of a single experiment with a boxplot or scatterplot. Detailed description of the HPLC gradient\nprogram (e.g., “0-2 min, 0% B; 2-20 min, 0-60% B”) with mobile phase composition (e.g., “A: 0.1% formic\nacid aqueous solution, B: acetonitrile”) in the Methods section. Add the detection wavelength (e.g., “λ =\n490 nm”) and column temperature (e.g., “25°C”).\nRESPONSE: Agreed and amended. We have added error bar descriptions to Figure 1A and Figure 6B. As\ndetailed above, presentation of the results in boxplots or scatterplots is challenging due to large variation in\nthe formation of deglycosylation shunt products. As such, the plots would either have been illogical if all\nreactions were included or highly biased if only a selection of reactions were presented. We have added\ndetails of HPLC experimental profiles to the enzymatic activity assays in the Methods sections (the details\nwere already present in the section describing analysis of culture extracts). Information on wavelength and\nHPLC column temperature is included in the revised manuscript Methods section.\nReviewer #2 (Remarks to the Author):\n• What are the noteworthy results?\n1. The natural redox partners ferredoxin FDX4 and ferredoxin reductase FDR3 were identified through\ntranscriptomic analysis, which are crucial for DoxA catalysis.\n2. Structural analysis and density functional theory (DFT) calculations revealed that the inefficient C-14\nhydroxylation of daunorubicin is due to the unfavorable anti-conformation of its methyl ketone sidechain.\n3. Advances in rational strain engineering led to a 180% increase in doxorubicin yields, improving the\nproduction profile.\n• Will the work be of significance to the field and related fields? How does it compare to the established\nliterature? If the work is not original, please provide relevant references.\nThere is some progress, but it is not significant. The main achievement is obtaining the three-dimensional\nstructures of the complexes formed by the DoxA enzyme and the substrates DNR, DHD, and DOD.\n• Does the work support the conclusions and claims, or is additional evidence needed?\nAdditional evidences are needed. For example, high-resolution three-dimensional structure of apo DoxA,\nand direct use of DNR as substrate to verify the product inhibition of DXR, etc.\n• Are there any flaws in the data analysis, interpretation and conclusions? Do these prohibit publication or\nrequire revision?\nYes, I have attached a file. I believe these should prohibit publication.\n• Is the methodology sound? Does the work meet the expected standards in your field?\nNo, I do not believe the methodology is sound, and the work does not meet the expected standards in the\nfield.\n• Is there enough detail provided in the methods for the work to be reproduced?\nThe substrate DOD is not readily available. Additionally, reproducing the other data as a whole is relatively\nstraightforward.\nRESPONSE: Thank you for the overall evaluation. We have addressed the concerns of the reviewer below in\nthe appropriate sections describing the specific issues that were raised. We would especially grateful to the\nreviewer for noting inconsistencies and typographical errors in our use of the abbreviations\nFDX/FDR/PDX/PDR. In this revised version, we have reverted to a more suitable nomenclature, where first\nthe species is mentioned (Sp for Streptomyces peucetius and So for Spinacia oleracea) followed by\nabbreviation of the enzyme (Fdx or FdR for ferredoxin or ferredoxin reductase, respectively). However, we\nhave retained the original abbreviations Pdx and PdR for enzymes from Pseudomonas putida, since these\noriginate from the words “putidaredoxin” and “putidaredoxin reductase”, respectively.\n• Is the CC (1/2) value for DoxA apo reasonable? It is significantly lower than 0.5. The Rpim value is 0.902,\nwhich is much greater than 0.5. The resolution is too low, resulting in lower reliability for the overall apo\nstructure.\nRESPONSE: We agree that improved statistics for this apo dataset would be preferable. However, it is likely\nthat this reflects a relatively flexible or unstructured protein. In the original submission, we included the\ndataset to address minor issues, particularly to highlight flexibility in the F-G loop, which is well-known for\nP450 enzymes. The B-factors of the other structures already indicate this sufficiently.\nWe concur with the reviewer that the dataset quality does not meet the necessary standard. Because the\nstructure does not provide additional mechanistic insight, we have removed it from the manuscript and\ninstead emphasized the B-factors and the F–G loop to clarify the proposed substrate entry pathway.\n• Is \"Na2SO4\" written incorrectly in Figure S3? Additionally, is the formatting consistent with academic\nstandards?\nRESPONSE: Agreed and amended. We have corrected this typo in the revised manuscript.\n• The compound 7-deoxydaunoribicinone does not appear anywhere in the main text of the manuscript.\nRESPONSE: We did not show the chemical structure of 7-deoxydaunoribicinone in the original submission\nas it is a well-known shunt product. In the revised manuscript, we discuss the formation of the compound\nin more detail and the name “7-deoxydaunoribicinone” (line 145). In addition, the chemical structure is\nshown in Figure S4 together with the NMR data of the molecule (Figures S5-S10).\n• Line 67: Vmax values writing specification?\nRESPONSE: We sought to avoid including exact Vmax values for previously conducted studies since the\nconditions in enzyme assays have a substantial influence on the values. This point is discussed in the\nintroduction, and we elected to use fold difference to relay conversion rates of the various DoxA\nsubstrates. Furthermore, the previous studies were conducted in the absence of DnrV, which we show here\nto have a significant influence on the catalytic values.\n• Line 143-145: The manuscript states that \"FDX4 is a 2Fe-2S ferredoxin similarly to SFX and PDR,\" but later\nconcludes that FDX4 is a Rieske-type ferredoxin while PDR is classified as a non-Rieske [2Fe-2S] ferredoxin\n(Figure S3).\nRESPONSE: Thank you for point out these errors in the fluency of the text. In the revised manuscript, we\nstate that Fdx4 belongs to a class of Rieske enzymes and further describe known members of the family\nbased on evidence supported by a multiple sequence alignment (new Figure S11). We also explain how\nFdx4 differs from SoFdx and Pdx (line 152). We also explain that each of these ferredoxins contain [2Fe-2S]\nclusters, but that the coordinating amino acids residues are unique in FDX4 (2 x His, 2 x Cys) compared to\nPDX/FDX (4 x Cys).\nThis raises two concerns:\n1. Logical inconsistency: If FDX4 shares similarity with PDR, their classifications as Rieske-type and non-\nRieske ferredoxins, respectively, appear contradictory.\nRESPONSE: Agreed and amended. The low sequence similarity does not warrant comparison of the\nsequences of SpFdx4 and Pdx and we have revised the text accordingly.\n2. Functional misclassification: The description of PDR as a ferredoxin may require re-evaluation. Ferredoxin\nreductases (e.g., PDR family proteins) and ferredoxins (e.g., SFX family) are functionally distinct redox\npartners, not homologous proteins. This mischaracterization undermines the comparative analysis.\nRESPONSE: Agreed and amended. We noticed two typographical errors where the putidaredoxin was\nincorrectly abbreviated as PDR instead of PDX.\n• Line 149-150:\nThe statement that \"Five of the six FDR proteins related to PDR were produced in E. coli\" (R1, R2, R3, R4,\nR7) raises methodological concerns:\n1. Unclear selection criteria: The manuscript does not define which six FDR proteins are considered \"related\nto PDR\" or specify the criteria for their selection. The phylogenetic tree (Figure 3B) does not clearly\ndemarcate a clade containing exactly six FDR proteins. For instance, the purple-clade region (putative PDR-\nrelated group) appears to contain more than six members. The rationale for excluding one protein from\nheterologous expression requires justification. Was this due to failed expression/purification, or\nevolutionary divergence?\nRESPONSE: Thank you for noticing these inconsistencies. We have corrected the text in the revised\nmanuscript, which now reads “Five of the seven FdR proteins”. Each of the seven genes homologous to\nferredoxin reductases were cloned, but only five of the proteins could be produced in soluble form in E.\ncoli. The selection criteria including protein expression is described in the revised manuscript (line 160).\n2. Phylogenetic inconsistencies:\nThe claimed PDR-related FDRs (R1, R4, R7) cluster within a subclade distinct from other FDRs, yet R2 and R3\nappear evolutionarily distant from this group. This contradicts the implied functional relationship. If\nevolutionary proximity to PDR is the basis for selection, the six candidates should be explicitly annotated on\nthe phylogenetic tree with bootstrap support values.\nRESPONSE: Thank you for your valuable feedback. In response to your comments, we have performed a\nnew phylogenetic analysis to provide a more accurate and robust representation of the relationships\namong the uncharacterized ferredoxin reductase proteins from Streptomyces peucetius.\nTo this end, we have constructed a new phylogenetic tree incorporating a broader and more representative\nset of reference sequences from established ferredoxin reductase families. This approach enabled us to\nmore precisely determine the evolutionary relationships and assign the functions of the Streptomyces\nproteins with high confidence. This revised analysis has resulted in a well-resolved and statistically\nsupported tree, as evidenced by high bootstrap values at the key nodes defining the major clades.\nThe revised tree (Figure 3B) clearly shows the three distinct evolutionary and functional families of\nferredoxin reductases:\n- Plant-type (pink): This clade represents the plant-type ferredoxin reductases, which are typically\ninvolved in photosynthesis. The absence of this protein type in our Streptomyces species is\nconsistent with its non-photosynthetic, heterotrophic metabolism.\n- Mitochondrial-type (blue): This clade contains the mitochondrial-type ferredoxin reductases\n(adrenodoxin reductase-like) and includes our Streptomyces proteins, SpFdR2 and SpFdR3. This\ngrouping is consistent with their likely role in supporting P450-mediated metabolic pathways.\n- Putidaredoxin Reductase-like (purple): This clade contains the putidaredoxin reductase-like\nferredoxin reductases, which forms a distinct family. The remaining five Streptomyces proteins\ncluster robustly within this group, indicating their evolutionary and functional relationship to this\nenzyme family.\n• Enzyme activity assay section: In Fig. 4A, using DOD as the substrate, only a small amount of DXR is\nproduced at a DnrV concentration of 10 μM, while at 20 μM, the yield of DXR is somewhat higher, but still\nlower than that of DNR. However, in Fig. 4C, when examining the effect of DXR concentration on the\nreaction, DOD is again used as the substrate, and at a DnrV concentration of 10 μM, the amount of DXR\nproduced in the absence of added DXR is significantly higher than in Fig. 4A, even exceeding the yield of\nDNR. This presents a contradiction, as indicated by the red box in the figure below.\nRESPONSE: We agree that inconsistencies in product yields are present in different figures in the activity\nassays. As explained to Reviewer #1, these fluctuations are due to the challenges in establishing a robust\nenzymatic assays system; the development and optimization of the system took two years. We have\nobserved variation in the activities of the enzymes both between different purification batches, depending\non storage time in the freezer. In addition, even minor changes such as a new vial of the co-substrate\nNADP+ or new stocks for catalase for removal of H O or glucose dehydrogenase for regeneration of\n2 2\nNADPH had an influence in the product yields. However, all of the data presented in the various manuscript\nfigures are derived from experiments that were conducted at the same time to ensure consistency and\nscientific rigor.\n• Line 168-170:\nWhy was DOD used as the substrate in the experiment examining the effect of DXR concentration on the\nreaction, rather than DHD or DNR? How can we determine whether the inhibition of the DoA reaction is\ndue to the DXR product or if DoxA exhibits catalytic region selectivity?\nRESPONSE: Thank you for the comment. DOD was used as a substrate because it has the highest relative\nturnover rate for the three DoxA-catalyzed reactions. We reasoned that use of this substrate would provide\nthe clearest signal for decreased product formation upon titration with DXR. DHD could have been another\nfeasible option since the conversion of DHD to DNR is high. DNR was not selected as a substrate due to the\nrelatively poor reaction turnover, and the significant data variance between individual samples.\nOur hypothesis for DoxA product inhibition is supported by the observation that DoxA binds DXR (see\nFigure 4E). We do not observe that product inhibition and catalytic C-H oxidation regio-selectivity are\nmutually exclusive in the case of DoxA. The reaction that is most interesting from an applied industrial\nperspective is the conversion of DNR to DXR, which does not occur in the wild type S. peucetius strain.\nTherefore, it is apparent that wt DoxA has high catalytic selectivity for DOD and DHD, but not for DNR.\n• Line 400: 16 mg mL-1. Pay attention to format specifications.\nRESPONSE: Agreed and amended.\nReviewer #3 (Remarks to the Author):\nThis is an excellent study on engineering a P450 system for improved production of doxorubicin, an\nimportant chemotherapeutic agent. The authors have improved catalysis by finding the natural redox\npartners for DoxA and by using an anthracycline binding protein to block product inhibition of the P450. I\nhave only a few minor comments.\nRESPONSE: Thank you for the overall evaluation.\n1. Somewhere in the paper the author should give the correct CYP name for DoxA.\nRESPONSE: Agreed and amended. We now refer to DoxA as “a heme containing cytochrome P450” enzyme\n(line 65).\n2. In the figure legend to figure 4 they refer to panel “f” as “e”.\nRESPONSE: Agreed and amended.\n3. The MD doesn’t add much especially using compound I. The lifetime of compound I is on the order of\npicoseconds, as estimated from radical clocks, so a 1000 ns simulation really is not of much relevance. A\nbetter model would be the oxy-complex. There are a few good P450-oxy-complex structures as a basis for\nmodeling.\nRESPONSE: While the oxy-complex persists across a longer timeframe within the P450 reaction cycle, it is\nthe heme iron-oxo (compound I) that is the primary reactive oxygen species responsible for the C-H\nhydrogen atom abstraction that leads to substrate oxidation (Munro et al, 2013; Shaik et al, 2010). In\naddition, while 1000ns is a much longer timescale than the presence of the iron-oxo experimentally,\n(Munro et al, 2013) there is ample precedent for using long-scale simulations with the iron-oxo species to\nmodel P450 reactivity (Caddell Haatveit et al, 2019; Shende et al 2023; Narayan et al 2015). Therefore, we\nbelieve that use of long-scale simulations of substrates in complex with compound I is appropriate for this\nstudy.\n4. In the MD word they only report the percentage of frames where C14 is in position for hydroxylation.\nThey should also report the percentage of frames for C13.\nRESPONSE: Response: Thank you for this suggestion, we have repeated our MD simulations in triplicate and\nreport these values in revised Table S5 as noted above.\nREFERENCES:\nCaddell Haatveit, K.; Garcia-Borràs, M.; Houk, K. N. Computational Protocol to Understand P450\nMechanisms and Design of Efficient and Selective Biocatalysts. Front. Chem. 2019, 6, 663.\nhttps://doi.org/10.3389/fchem.2018.00663.\nFisher, J., Abdella, B. R. J. & McLane, K. E. Anthracycline antibiotic reduction by spinach ferredoxin-NADP+\nreductase and ferredoxin. Biochemistry 24, 3562–3571 (1985). https://doi.org/10.1021/bi00335a026\nMunro, A. W.; Girvan, H. M.; Mason, A. E.; Dunford, A. J.; McLean, K. J. What Makes a P450 Tick? Trends\nBiochem. Sci. 2013, 38 (3), 140–150. https://doi.org/10.1016/j.tibs.2012.11.006.\nNarayan, A. R. H.; Jiménez-Osés, G.; Liu, P.; Negretti, S.; Zhao, W.; Gilbert, M. M.; Ramabhadran, R. O.; Yang,\nY.-F.; Furan, L. R.; Li, Z.; Podust, L. M.; Montgomery, J.; Houk, K. N.; Sherman, D. H. Enzymatic Hydroxylation\nof an Unactivated Methylene C–H Bond Guided by Molecular Dynamics Simulations. Nat. Chem. 2015, 7 (8),\n653–660. https://doi.org/10.1038/nchem.2285.\nShaik, S.; Cohen, S.; Wang, Y.; Chen, H.; Kumar, D.; Thiel, W. P450 Enzymes: Their Structure, Reactivity, and\nSelectivity—Modeled by QM/MM Calculations. Chem. Rev. 2010, 110 (2), 949–1017.\nhttps://doi.org/10.1021/cr900121s.\nShende, V. V.; Harris, N. R.; Sanders, J. N.; Newmister, S. A.; Khatri, Y.; Khatri, Y.; Movassaghi, M.; Houk, K. N.;\nSherman, D. H. Molecular Dynamics Simulations Guide Chimeragenesis and Engineered Control of\nChemoselectivity in Diketopiperazine Dimerases. Angew. Chem. Int. Ed. n/a (n/a).\nhttps://doi.org/10.1002/anie.202210254."}, {"round": 2, "reviewer_comments": {"reviewer_2": "Agree to proceed with the publication of the article.", "reviewer_3": "The authors have addressed my main comments regarding molecular dynamics. They’ve included more runs which gives\nbetter statistics. They still argue that long simulations of compound I are justified because everyone does it this way. That\nmay be so, but it doesn’t mean it’s correct thing to do. In the long run it would only be significant if the substrate wandered far\naway over the long simulation but that’s not the case here so everything is okay."}, "author_response": ""}]

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