MilR2, a novel TetR family regulator involved in 5-oxomilbemycin A3/A4 biosynthesis in Streptomyces hygroscopicus

Milbemycins produced by several Streptomyces species are a group of 16-membered macrolides with potent insecticidal and anthelminthic activity. Milbemycin A3/A4, the main components of the milbemycins biosynthetic pathway, and 5-oxomilbemycin A3/A4, the analogs of milbemycin A3/A4 without the reduction of the C-5 keto group, have been developed as acaricides, insecticides, and anthelmintics. However, so far, little is known about the regulation of milbemycins biosynthesis, which has greatly hampered the generation of high producing strains by metabolic engineering. Herein, a TetR family regulator MilR2 (encoded by sbi_00792) was identified being involved in activation of 5-oxomilbemycin A3/A4 biosynthesis in a high 5-oxomilbemycins-producing strain Streptomyceshygroscopicus SIPI-KF. ThemilR2 mutant with an in-frame deletion of theMilR2 DNA-binding domain resulted in significantly reduced 5-oxomilbemycin A3/A4 production (approximately 36.9 and 39.7%) at tested two time points, and accordingly introduction of an extra copy of milR2 into SIPI-KF led to enhanced production by 12.6 and 34.4%. We further showed that MilR2 could directly repress the transcription of the gene sbi_00791 encoding a putative hydrolase, which is located divergently from milR2. The precise MilR2-binding site consisting of a 7-nt perfect inverted repeat separated by 10-nt (5-ACCAACCAGCTGGTAAGGGTTGGT-3) was defined. In situ mutagenesis of the MilR2-binding site resulted in 19.7 and 13.5% decreases in 5-oxomilbemycin A3/A4 production, which is much lower than the decreased rates of milR2. Collectively, the results demonstrated that MilR2 serves as an activator for 5-oxomilbemycin A3/A4 production and the function of MilR2 is only partially mediated through its repression on the transcription of sbi_00791.