Metabolic engineering of Streptomyces peucetius for biosynthesis of N,N-dimethylated anthracyclines

Abstract

Introduction: Daunorubicin and doxorubicin, two anthracycline polyketides produced by S. peucetius, are potent anticancer agents that are widely used in chemotherapy, despite severe side effects. Recent advances have highlighted the potential of producing improved derivatives with reduced side effects by incorporating l-rhodosamine, the N,N-dimethyl analogue of the native amino sugar moiety. Method: In this study, we aimed to produce N,N-dimethylated anthracyclines by engineering the doxorubicin biosynthetic pathway in the industrial Streptomyces peucetius strain G001. To achieve this, we introduced genes from the aclarubicin biosynthetic pathway encoding the sugar N-methyltransferases AclP and AknX2. Furthermore, the native gene for glycosyltransferase DnrS was replaced with genes encoding the aclarubicin glycosyltransferases AknS and AknT. Additionally, the gene for methylesterase RdmC from the rhodomycin biosynthetic pathway was introduced. Results: A new host was engineered successfully, whereby genes from the aclarubicin pathway were introduced and expressed. LC-MS/MS analysis of the engineered strains showed that dimethylated sugars were efficiently produced, and that these were incorporated ino the anthracycline biosynthetic pathway to produce the novel dimethylated anthracycline N,N-dimethyldaunorubicin. Further downstream tailoring steps catalysed by the cytochrome P450 monooxygenase DoxA exhibited limited efficacy with N,N-dimethylated substrates. This resulted in only low production levels of N,N-dimethyldaunorubicin and no N,N-dimethyldoxorubicin, most likely due to the low affinity of DoxA for dimethylated substrates. Discussion: S. peucetius G001 was engineered such as to produce N,N-dimethylated sugars, which were incorporated into the biosynthetic pathway. This allowed the successful production of N,N-dimethyldaunorubicin, an anticancer drug with reduced cytotoxicity. DoxA is the key enzyme that determines the efficiency of the biosynthesis of N,N-dimethylated anthracyclines, and engineering of this enzyme will be a major step forwards towards the efficient production of more N,N-dimethylated anthracyclines, including N,N-dimethyldoxorubicin. This study provides valuable insights into the biosynthesis of clinically relevant daunorubicin derivatives, highlighting the importance of combinatorial biosynthesis.

Keywords: Streptomyces; anthracyclines; anticancer; biosynthesis; doxorubicin; metabolic engineering.

FIGURE 1

Chemical structures and BGCs of anthracyclines described in this work. (A) Chemical structures of daunorubicin, doxorubicin, N,N-dimethyldaunorubicin (11), N,N-dimethyldoxorubicin (12), rhodomycin B and aclacinomycin A (aclarubicin). (B) The BGCs of daunorubicin/doxorubicin, rhodomycin and aclarubicin were aligned and visualised using clinker (Gilchrist and Chooi, 2021). To achieve biosynthesis of N,N-dimethyldaunorubicin (11) and N,N-dimethyldoxorubicin (12), several genes from the rhodomycin and aclarubicin BGCs were introduced to Streptomyces peucetius, as highlighted in bold.

FIGURE 2

Biosynthetic pathway of doxorubicin and modifications required for biosynthesis of N,N-dimethylated daunorubicin and doxorubicin. Doxorubicin biosynthesis occurs in three stages: biosynthesis of TDP-l-daunosamine from D-glucose-1-phosphate, biosynthesis of ɛ-rhodomycinone (16) from one propionyl-CoA and nine malonyl-CoA units, followed by tailoring steps of the aglycone. For biosynthesis of N,N-dimethylated daunorubicin and doxorubicin, the pathway should be modified in three steps: 1) N,N-dimethylation of TDP- l-daunosamine to TDP-l-rhodosamine, 2) glycosylation of ɛ-rhodomycinone (16) with l-rhodosamine instead of l-daunosamine, and 3) the further tailoring steps should be performed when l-rhodosamine is attached.

FIGURE 3

Expression of aclarubicin methyltransferases and glycosyltransferases genes in G001 results in the attachment of l-rhodosamine to ɛ-rhodomycinone (A) Schematic representation of the relevant genotype of the strains used in this experiment, with heterologous genes indicated by a diagonal striped pattern. (B) LC-MS analysis of crude extracts of G001, MAG301, MAG302 and MAG303 cultivated in E1 medium. Extracted ion chromatograms showing the mass peaks [M + H]⁺ of compounds 3–7 and 13–15. (C) Schematic representation of the engineered doxorubicin pathway. Introduction of the N-methyltransferases (aclP/aknX2) and glycosyl transferases (aknS/aknT) genes from the aclarubicin BGC resulted in incorporation of l-rhodosamine onto ɛ-rhodomycinone (16), forming ɛ-rhodomycin T (7). ɛ-Rhodomycin T (7) was converted to 4-methoxy-ɛ-rhodomycin T (13) by DnrK. Additionally, a minor mass peak of daunorubicin (5) was detected in MAG301, which was completely abolished in MAG303 where the native glycosyltransferase gene dnrS was deleted.

FIGURE 4

Expression of rhodomycin methylesterase gene in G001 results in the production of N,N-dimethyldaunorubicin. (A) Schematic representation of the relevant genotype of the strains used in this experiment, with heterologous genes indicated by a diagonal striped pattern. (B) LC-MS analysis of crude extracts of G001, MAG304 and MAG305 cultivated in E1 medium. Extracted ion chromatograms showing the mass peaks [M + H]⁺ of compounds 3–7 and 9–12. (C) Schematic representation of the proposed biosynthetic pathway for N,N-dimethyldoxorubicin (12). Introduction of the 15-methylesterase gene rdmC from the rhodomycin BGC resulted in the production of N,N-dimethyl-13-deoxydaunorubicin (9) and N,N-dimethyl-13-dihydrodaunorubicin (10). A minor peak was detected for N,N-dimethyldaunorubicin (11), but N,N-dimethyldoxorubicin (12) could not be detected.

FIGURE 5

DoxA is the bottleneck for biosynthesis of N,N-dimethyldoxorubicin. (A) MS-based quantitative proteomics analysis of MAG304 cultivated in E1 medium for 3 days. Histogram showing distribution of the relative intensity level (log₁₀ LFQ) of all detected proteins (n = 3). The bar that includes the abundance of DoxA is highlighted. (B) LC-MS analysis of crude extracts of MAG304 pMS82, MAG306 (doxA-1), MAG307 (doxA-2) and MAG308 (doxA-3) cultivated in E1 medium. Extracted ion chromatograms showing the mass peaks [M + H]⁺ of compounds 7–12. For all strains, the main peak corresponded to N,N-dimethyl-13-deoxydaunorubicin (9), and N,N-dimethyldoxorubicin (12) could not be detected. (C) HPLC analysis of DoxA enzymatic assays. UV-Vis chromatogram traces were recorded at 490 nm. A reaction mixture without the addition of NADP⁺ was used as the negative control. The activity of DoxA with the natural substrates 13-deoxydaunorubicin (3) and daunorubicin (5). (D) The activity of DoxA with N,N-dimethyl substrates N,N-dimethyl-13-deoxydaunorubicin (9) and N,N-dimethyldaunorubicin (11).

FIGURE 6

Toxicity of N,N-dimethyldoxorubicin. (A) G001, MAG301, MAG302, MAG303 and MAG304 were streaked on SFM agar plates (supplemented with 20 μg mL⁻¹ thiostrepton for strains harbouring pRDS). The development is blocked by the production of anthracyclines. MAG304 exhibited the most pronounced inhibition of development. In contrast, deletion of the glycosyltransferase gene dnrS in MAG302 stimulated development. (B) MAG305 pWHM3-oriT and MAG309 (drrAB) were spotted on SFM agar plates supplemented with increasing concentrations of doxorubicin (6) or N,N-dimethyldoxorubicin (12). For each strain, 5 µL of spore or mycelium stock was spotted at a concentration of 1.0.10⁴ CFU per spot and the plates were incubated at 30 °C for 4 days. Dashed circles indicate the highest concentration that does not inhibit growth. For MAG305, the inhibitory concentration of N,N-dimethyldoxorubicin (12) is 16-fold lower than that of doxorubicin (6). Overexpression of drrAB increased resistance to both compounds eight-fold. (C) LC-MS analysis of crude extracts of MAG305 pWHM3-oriT and MAG309 (drrAB) cultivated in E1 medium. Extracted ion chromatograms showing the mass peaks [M + H]⁺ of compounds 7–12. Overexpression of drrAB resulted in a 3.7-fold increased production of N,N-dimethyldaunorubicin (11).