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

Abstract

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 Streptomyces hygroscopicus SIPI-KF. The ΔmilR2 mutant with an in-frame deletion of the MilR2 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 ΔmilR2. 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.

Enhancement of ascomycin production via a combination of atmospheric and room temperature plasma mutagenesis in Streptomyces hygroscopicus and medium optimization

“…The genomes of Streptomyces sp. are characterized by extensive polycistronic organizations (Wei et al 2018; Zhang et al 2016b), thus, the whole ascomycin biosynthetic gene cluster was divided into seven co-transcription units, and the transcription of these units was analyzed by RT-PCR. The transcriptional levels of several genes, including fkbW / U / B / C / L / K / J / I / H / O / P / A / D / M , were significantly upregulated.…”

Low-Toxicity and High-Efficiency Streptomyces Genome Editing Tool Based on the Miniature Type V–F CRISPR/Cas Nuclease AsCas12f1

“…SIPI-054 is an industrial high-yield milbemycin-producing strain, which is a genetically intractable microorganism. 11 Here, four different sizes of DNA fragments were selected as deletion targets (Figure 6A), including milR2 (390 bp, encoding a TetR family regulator involved in regulation of milbemycin biosynthesis), 35 milF (603 bp, encoding a C5-ketoreductase responsible for transforming 5-oxomilbemycins to milbemycins), 36 kelBCD (3.3 kb) from the kel BGC responsible for the biosynthesis of the yellow compound KEL, 37 and a 62-kb functionally unknown DNA region, which divides the milbemycin BGC into two parts, including the large part (from milA2 to milR) and the small part (milA1and milD). 38 First, milR2 and milF were tested for AsCas12f1-based editing.…”

Modular polyketide synthase-derived insecticidal agents: from biosynthesis and metabolic engineering to combinatorial biosynthesis for their production

“…92 The introduction of a second copy of milR2 , another regulator of milbemycin BGC, resulted in a 1.3-fold increase in the production of 5-oxomilbemycin A3 ( 25 ) and 5-oxomilbemycin A4 ( 26 ) (3600 mg L −1 ). 93…”

“…92 The introduction of a second copy of milR2, another regulator of milbemycin BGC, resulted in a 1.3-fold increase in the production of 5-oxomilbemycin A3 (25) and 5-oxomilbemycin A4 (26) (3600 mg L −1 ). 93 4.4.2 Ribosome recycling and sigma factor. Ribosome recycling factors are responsible for the degradation and recycling of the post-termination ribosome complex and can be utilized to improve the expression of PKS genes.…”