ABSTRACT Mildiomycin is a representative peptidyl nucleoside antibiotic and was first isolated from Streptoverticillium rimofaciens , which has been used as an important biological agent to control powdery mildew in plants. Despite its importance, the biosynthetic pathways and regulatory mechanisms remain to be fully elucidated. In this study, we identified MilO as a positive pathway-specific regulator of mildiomycin biosynthesis in the heterologous host Streptomyces avermitilis . Gene disruption of milO resulted in almost loss of mildiomycin production, and it was restored to the level comparable to that in the wild-type strain in complemented strain. Overexpression of milO using host native promoter rpsJ p, engineered promotor SP44, and kasO p* led to a 50%, 6.5-fold, and 9.2-fold increase in mildiomycin production compared with the wild-type strain, respectively. Quantitative real-time PCR and electrophoretic mobility shift assay (EMSA) experiments revealed that MilO directly enhances the transcription of the milA gene by 20 folds after 48 h fermentation and indirectly regulates the transcription levels of other genes from milB to milM . Using DNase I footprinting assays, milO was revealed to bind to a 44 bp DNA sequence of the milA promoter region. The binding region consists of three imperfect direct repeats of TGTC(N) 3 CGGT separated by two-nucleotide spacers and each repeat is important to efficient binding to MilO. In addition, we identified two related compounds by overexpressing milO in a structural gene milN -deficient mutant. Taken together, this study indicates that pathway-specific regulator MilO is essential for mildiomycin biosynthesis and provides an effective strategy to improve the production of mildiomycin and its intermediates. IMPORTANCE As an important biological agent to control powdery mildew on plants, mildiomycin has been commercialized and used in various plants. However, its regulatory mechanisms and biosynthetic pathways remain unknown. This study provides new insights into the regulation of mildiomycin biosynthesis through MilO, a LuxR family protein that modulates mildiomycin production by directly enhancing the transcription of milA . The yield of mildiomycin was significantly improved by overexpressing milO in a heterologous host. In addition, the positive regulatory effect of milO helped to discover two related compounds, which provide important clues for the timing of uploading of two amino acid side chains during mildiomycin biosynthesis for the first time. In brief, our findings on transcriptional regulation of mildiomycin biosynthesis by milO will be valuable to further increase the yield of mildiomycin and explore its biosynthetic pathways.

Zhiyu Li

Yuli Wang

Chen Lin

Yu Wen

Zixin Deng

Ming Jiang

Xinyi He

Applied and Environmental Microbiology

应用和环境微生物学 (AEM) 发表的论文对 (A) 应用微生物学，包括生物技术、蛋白质工程、生物修复和食品微生物学，(B) 微生物生态学，包括环境、有机体和基因组微生物学，以及 (C) 做出了重大贡献。 ) 跨学科微生物学，包括无脊椎动物微生物学、植物微生物学、水生微生物学和地球微生物学。

The journal 'Applied and Environmental Microbiology' publishes papers in the following areas: (A) Applied Microbiology, including biotechnology, protein engineering, bioremediation, and food microbiology; (B) Microbial Ecology, covering environmental microbiology, organismal microbiology, and genomic microbiology; (C) Interdisciplinary Microbiology, which includes invertebrate microbiology, plant microbiology, aquatic microbiology, and earth microbiology. These studies have made significant contributions to their respective fields.

《Applied and Environmental Microbiology》期刊发表的论文涵盖以下领域：(A) 应用微生物学，包括生物技术、蛋白质工程、生物修复和食品微生物学；(B) 微生物生态学，涉及环境微生物学、有机体微生物学和基因组微生物学；(C) 跨学科微生物学，包括无脊椎动物微生物学、植物微生物学、水生微生物学和地球微生物学。这些研究对相关领域做出了重要贡献。

Category	Quartile
生物学	2
生物学, 生物工程与应用微生物	2
生物学, 微生物学	3

Positive regulation of a LuxR family protein, MilO, in mildiomycin biosynthesis

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