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

Figures

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  Fig. 1. The biosynthesis of daunorubicin (DNR) and doxorubicin (DXR). A Organization of the daunorubicin biosynthetic gene cluster. The key genes doxA and dnrV are highlighted in blue and annotated to encode cytochrome P450 and vicinal oxygen chelate (VOC) enzymes, respectively. B Abbreviated biosynthetic scheme for formation of DXR. Polyketide biosynthesis results in the formation of ε-rhodomycinone, which is glycosylated by attachment of L-daunosamine, leading to the formation of rhodomycin D. Polyketide tailoring results in the formation of the intermediate 13-deoxydaunorubicin (DOD) important for this study. The P450 enzyme DoxA catalyzes three consecutive hydroxylation reactions that convert DOD to 13-dihydrodaunorubicin (DHD), DNR, and finally to DXR. C The redox cofactors of P450 enzymes consist of ferredoxin reductases (FDR) that utilize NADPH to convert oxidized flavin (FADox) to reduced flavin (FADred). In turn, FADred is reduced by the [2Fe-2S] centre of ferredoxins (FDX), which enables donation of two single electrons to the heme of DoxA. Reduction of heme-Fe3+ allows a reaction with molecular oxygen O2 and formation of a high valent heme-Fe4+=O iron oxo species, which is…

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  Fig. 2. Comparative metabolome, genome, and transcriptome analysis. A Metabolite production profiles of S. peucetius ATCC 27952 wild-type (WT), and mutant strains G001 and X121. The experiments were conducted in triplicate (n = 3 biological replicates). Data are presented as mean values ± SD. B Average day 2 differential expression of genes in the dnr BGC (top) and suf cluster (bottom). C Cluster situated regulation of the daunorubicin and doxorubicin BGC. The red arrow shows the mutation in the bidirectional promoter of dnrO and dnrN, resulting in the overexpression of dnrN and subsequently the overexpression of the BGC, in G001 and X121. D Biosynthetic scheme for the formation of Fe-S clusters by the suf pathway and upregulation of the ferredoxin fdx4. The strain X121 harbored a mutation in the negative regulatory gene sufR that leads to activation of the cluster. Source data are provided as a Source Data file.

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  Fig. 3. Identification of the natural redox partners of DoxA. A DoxA reactions with non-native redox partners PDX + PDR from P. putida and SFX + SFR from spinach using DOD as a substrate, B Phylogenetic tree of putative FDR enzymes, including putidaredoxin-type (purple), adrenodoxin-type (blue), and plant-type ferredoxin reductases (pink) identified from the genome of S. peucetius ATCC27952. Reference sequences for putidaredoxin reductase-type (BphA4, Pseudomonas sp.; PdR, Pseudomonas putida; SmFdR, Stenotrophomonas maltophilia), adrenoredoxin reductase-type (AtFdR, Arabidopsis thaliana; MtFdR, Mycobacterium tuberculosis), and plant ferredoxin reductase-type (ZmFdR, Zea mays; SoFdR, Spinacia oleracea; PsFdR, Pisum sativum) were used to guide the tree. Proteins marked with an asterisk (*) could be produced in soluble form and were used in enzyme activity assays, C DoxA reactions with FDX4 and various ferredoxin reductase partners using DOD as a substrate.

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  Fig. 4. DnrV improves catalysis of DoxA by binding to anthracyclines. A Increasing the concentration of DnrV improves DXR formation with all three substrates, DOD, DHD, and DNR. Reactions included 10 µM DoxA, 5 µM FDX4, 0.1 µM SFR, 0-20 µM DnrV, 4 mM Glucose, 1U GDH and 2.5 U catalase, 0.5 mM NADP + . No NADP+ was added to the control reactions. B Stoichiometric DNR: DnrV ratios enabled full conversion to DXR at 100 µM substrate concentration. This optimized reaction used 20 µM FDX4 instead of 5 µM. C Increasing the presence of DXR prevents catalysis by DoxA. Reactions included 20 µM DOD, 7 µM DoxA, 10 µM DnrV, 5 µM FDX4, 0.1 µM SFR, 4 mM glucose, 1 U Glucose dehydrogenase, 0.5 mM NADP+ and 2 U catalase. D Anthracycline binding assay based on quenching of the anthracycline fluorophore upon binding to proteins due to changes in the chemical environment. DnrV binds to the anthracyclines DOD, DNR, and DXR at low micromolar concentrations. E DoxA binds to the anthracyclines DOD and DNR at low micromolar concentrations and DXR at higher micromolar concentrations. F The ferredoxin FDX4 does not interact with anthracyclines such as DXR. The anthracycline fluorophores were excited at 470 nm, and…

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  Fig. 5. Structural basis for DoxA catalysis. A The overall structure of DoxA complexed with DOD demonstrates a crystallographic dimer (green, purple) with the heme (grey) and substrate (cyan) stacking perpendicular to each other. B The active site of DoxA showing amino acid residues in the vicinity of the substrate DOD, which is contoured with the electron density at 1 σ, highlights the proximity of the substrate to the heme group. C Residues Val305 and Leu309 create a hydrophobic cavity for C14, which positions the adjacent C13 carbon close to the axial water molecule coordinated to the heme. D DFT calculations demonstrated an energetic preference for anti- conformation. E Superposition of the top five most occupied DOD clusters in 1000 ns simulation with DoxA. DOD is stably oriented for C13 oxidation by compound I. F Superposition of the top five most occupied DNR clusters in 1000 ns simulation with DoxA. DNR infrequently samples conformation required for C14 oxidation.

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  Fig. 6. Metabolic engineering of S. peucetius G001 for improved production of DXR. A Schematic presentation of constructs for overexpression of snorO, doxA, fdx4, fdr3 and dnrV. A two-plasmid system was utilized in the expression studies. B Production of DNR and DXR by the engineered strains. The experiments were conducted in triplicate (n = 3 biological replicates). Data are presented as mean values ± SD. C HPLC production profile of G001/pSnorO+pFdx4_DnrV_DoxA with LXA1180 and XAD7 resins. D Time-course analysis for production of DXR and DNR by G001/pSnorO+pFdx4_DnrV_DoxA in a bioreactor. Source data are provided as a Source Data file.