State Key Laboratory of Rice Biology and Breeding, Ministry of Agriculture Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Zhejiang Key Laboratory of Biology and Ecological Regulation of Crop Pathogens and Insects, Institute of Biotechnology, Zhejiang University, Hangzhou, 310058, China.
Institute of Eco-Environmental Protection, Shanghai Academy of Agricultural Sciences, Shanghai, 201403, China.
Microb Cell Fact. 2024 Sep 28;23(1):255. doi: 10.1186/s12934-024-02527-6.
Cyclo (Phe-Pro) (cFP), a cyclic dipeptide with notable antifungal, antibacterial, and antiviral properties, shows great promise for biological control of plant diseases. Produced as a byproduct by non-ribosomal peptide synthetases (NRPS), the regulatory mechanism of cFP biosynthesis remains unclear. In a screening test of 997 Tn5 mutants of Burkholderia seminalis strain R456, we identified eight mutants with enhanced antagonistic effects against Fusarium graminearum (Fg). Among these, mutant 88's culture filtrate contained cFP, confirmed through HPLC and LC-MS, which actively inhibited Fg. The gene disrupted in mutant 88 is part of the Dct transport system (Dct-A, -B, -D), responsible for C4-dicarboxylate transport. Knockout mutants of Dct genes exhibited higher cFP levels than the wild type, whereas complementary strains showed no significant difference. Additionally, the presence of exogenous C4-dicarboxylates reduced cFP production in wild type R456, indicating that these substrates negatively regulate cFP synthesis. Given that cFP synthesis is related to NRPS, we previously identified an NRPS cluster in R456, horizontally transferred from algae. Specifically, knocking out gene 2061 within this NRPS cluster significantly reduced cFP production. A Fur box binding site was predicted upstream of gene 2061, and yeast one-hybrid assays confirmed Fur protein binding, which increased with additional C4-dicarboxylates. Knockout of the Fur gene led to up-regulation of gene 2061 and increased cFP production, suggesting that C4-dicarboxylates suppress cFP synthesis by enhancing Fur-mediated repression of gene 2061.
环（苯丙脯氨酸）（cFP）是一种具有显著抗真菌、抗菌和抗病毒特性的环状二肽，有望成为防治植物病害的生物防治手段。cFP 作为非核糖体肽合成酶（NRPS）的副产物产生，其生物合成的调控机制尚不清楚。在对 997 个拜氏芽胞杆菌 R456 的 Tn5 突变体进行筛选测试中，我们鉴定出了 8 个对禾谷镰刀菌（Fg）具有增强拮抗作用的突变体。其中，突变体 88 的培养液滤液中含有 cFP，通过 HPLC 和 LC-MS 确认，该物质可积极抑制 Fg。突变体 88 中被破坏的基因是 Dct 转运系统（Dct-A、-B、-D）的一部分，负责 C4-二羧酸的转运。Dct 基因的敲除突变体比野生型具有更高的 cFP 水平，而互补菌株则没有显著差异。此外，外源性 C4-二羧酸的存在降低了野生型 R456 中的 cFP 产量，表明这些底物负调控 cFP 合成。鉴于 cFP 合成与 NRPS 有关，我们之前在 R456 中鉴定出一个来自藻类的 NRPS 簇，它是水平转移的。具体来说，敲除该 NRPS 簇内的基因 2061 显著降低了 cFP 的产量。在基因 2061 上游预测到了一个 Fur 盒结合位点，酵母单杂交实验证实了 Fur 蛋白的结合，并且随着 C4-二羧酸的增加，这种结合也增加。敲除 Fur 基因导致基因 2061 的上调和 cFP 产量的增加，表明 C4-二羧酸通过增强 Fur 介导的基因 2061 抑制来抑制 cFP 合成。
