Li Zhiyu, Wang Yuli, Lin Chen, Wen Yu, Deng Zixin, Jiang Ming, He Xinyi
State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic & Developmental Sciences, School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai, People's Republic of China.
Appl Environ Microbiol. 2025 Jan 31;91(1):e0165424. doi: 10.1128/aem.01654-24. Epub 2024 Dec 23.
Mildiomycin is a representative peptidyl nucleoside antibiotic and was first isolated from , 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 . Gene disruption of 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 using host native promoter p, engineered promotor and 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 gene by 20 folds after 48 h fermentation and indirectly regulates the transcription levels of other genes from to . Using DNase I footprinting assays, was revealed to bind to a 44 bp DNA sequence of the promoter region. The binding region consists of three imperfect direct repeats of TGTC(N)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 in a structural gene -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.IMPORTANCEAs 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 . The yield of mildiomycin was significantly improved by overexpressing in a heterologous host. In addition, the positive regulatory effect of 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 will be valuable to further increase the yield of mildiomycin and explore its biosynthetic pathways.
米尔迪霉素是一种典型的肽基核苷类抗生素，最初从[具体来源未给出]中分离得到，它一直被用作控制植物白粉病的重要生物制剂。尽管其很重要，但其生物合成途径和调控机制仍有待充分阐明。在本研究中，我们鉴定出MilO是异源宿主中米尔迪霉素生物合成的正向途径特异性调节因子。[相关基因]的基因破坏导致米尔迪霉素产量几乎丧失，而在互补菌株中其产量恢复到与野生型菌株相当的水平。使用宿主天然启动子p、工程化启动子[具体名称未给出]和p*对[相关基因]进行过表达，与野生型菌株相比，米尔迪霉素产量分别增加了50%、6.5倍和9.2倍。定量实时PCR和电泳迁移率变动分析（EMSA）实验表明，在发酵48小时后，MilO直接将[相关基因]的转录增强20倍，并间接调节从[相关基因1]到[相关基因2]等其他基因的转录水平。通过DNA酶I足迹分析，发现[相关基因]与[相关基因]启动子区域的一个44 bp DNA序列结合。结合区域由三个不完美的TGTC(N)CGGT直接重复序列组成，中间间隔两个核苷酸，每个重复序列对于与MilO的有效结合都很重要。此外，我们通过在一个结构基因[具体名称未给出]缺陷型突变体中过表达[相关基因]鉴定出两种相关化合物。综上所述，本研究表明途径特异性调节因子MilO对米尔迪霉素生物合成至关重要，并提供了一种提高米尔迪霉素及其中间体产量的有效策略。
作为控制植物白粉病的重要生物制剂，米尔迪霉素已商业化并应用于多种植物。然而，其调控机制和生物合成途径仍不清楚。本研究通过MilO（一种LuxR家族蛋白，通过直接增强[相关基因]的转录来调节米尔迪霉素的产生）为米尔迪霉素生物合成的调控提供了新的见解。在异源宿主中过表达[相关基因]显著提高了米尔迪霉素的产量。此外，[相关基因]的正向调节作用有助于首次发现两种相关化合物，这为米尔迪霉素生物合成过程中两个氨基酸侧链添加的时机提供了重要线索。简而言之，我们关于[相关基因]对米尔迪霉素生物合成转录调控的发现对于进一步提高米尔迪霉素产量和探索其生物合成途径将具有重要价值。
