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.