Overexpression Self-Repressing Regulator Dnro Enhances Production of Daunorubicin in Blda Deficient Streptomyces Peucetius ATCC 27952

In daunorubicin (DNR) biosynthesis pathway of Streptomyces peucetius ATCC 27952, dnrO is the first regulatory gene to be activated. DnrO is self-repressing gene, which activates rest of the DNR biosynthesis pathway through cascades of regulatory events. S. peucetius was found to contain no functional copy of bldA -tRNA while closer look at dnrO codons reveals the presence of TTA codon, which is rarely encoded by bldA. The dnrO gene contains 341 codons, where 86^th codon was found to be the rare TTA codon. Therefore, for evaluating the role of dnrO in DNR production, we generated multiple engineered strains of S. peucetius by heterologously expressing bldA, dnrO and combination of bldA and dnrO. Using these strains, we evaluated the effects of heterologously expressed bldA and overexpressed dnrO on pathway specific regulators, cell densities and production of DNR. The increase in transcription level of dnrO and dnrI along with enhanced DRN production in strain with bldA as compared to wild type strain was found.The DNR biosynthesis was found to be ~1.7 mg/l higher than that of dnrO overexpression strain. S. peucetius and its variants had optimum mycelial density in NDYE medium from 48 h to 60 h and maximum anthracycline compounds production at 60 h. S. peucetius ATCC 27952 produces 3.24 mg/l of DRN while the strains with bldA and dnrO produces 4.72 mg/l and 4.15 mg/l of DRN, respectively. Combining the results of transcriptional level of pathway specific regulators and biosynthesis of DNR from strain with bldA (heterologous expression), dnrO (overexpression), and combination of both, we found the translation of UUA codon on dnrO mRNA is partially bldA dependent.