Technische Universität Berlin, Chair of Bioprocess Engineering, Berlin, Germany.
Technische Universität Berlin, Chair of Bioanalytics, Berlin, Germany.
J Biol Chem. 2024 Aug;300(8):107523. doi: 10.1016/j.jbc.2024.107523. Epub 2024 Jul 4.
Despite the ever-growing research interest in polyhydroxyalkanoates (PHAs) as green plastic alternatives, our understanding of the regulatory mechanisms governing PHA synthesis, storage, and degradation in the model organism Ralstonia eutropha remains limited. Given its importance for central carbon metabolism, PHA homeostasis is probably controlled by a complex network of transcriptional regulators. Understanding this fine-tuning is the key for developing improved PHA production strains thereby boosting the application of PHAs. We conducted promoter pull-down assays with crude protein extracts from R. eutropha Re2058/pCB113, followed by liquid chromatography with tandem mass spectrometry, to identify putative transcriptional regulators involved in the expression control of PHA metabolism, specifically targeting phasin phaP1 and depolymerase phaZ3 and phaZ5 genes. The impact on promoter activity was studied in vivo using β-galactosidase assays and the most promising candidates were heterologously produced in Escherichia coli, and their interaction with the promoters investigated in vitro by electrophoretic mobility shift assays. We could show that R. eutropha DNA-binding xenobiotic response element-family-like protein H16_B1672, specifically binds the phaP1 promoter in vitro with a K of 175 nM and represses gene expression from this promoter in vivo. Protein H16_B1672 also showed interaction with both depolymerase promoters in vivo and in vitro suggesting a broader role in the regulation of PHA metabolism. Furthermore, in vivo assays revealed that the H-NS-like DNA-binding protein H16_B0227 and the peptidyl-prolyl cis-trans isomerase PpiB, strongly repress gene expression from PphaP1 and PphaZ3, respectively. In summary, this study provides new insights into the regulation of PHA metabolism in R. eutropha, uncovering specific interactions of novel transcriptional regulators.
尽管人们对聚羟基烷酸酯 (PHA) 作为绿色塑料替代品的研究兴趣与日俱增，但我们对模式生物 Ralstonia eutropha 中控制 PHA 合成、储存和降解的调控机制的理解仍然有限。鉴于其对中心碳代谢的重要性，PHA 动态平衡可能受到转录调控因子的复杂网络控制。了解这种微调是开发改良 PHA 生产菌株的关键，从而提高 PHA 的应用。我们用 R. eutropha Re2058/pCB113 的粗蛋白提取物进行启动子下拉测定，然后用液相色谱串联质谱进行分析，以鉴定参与 PHA 代谢表达调控的潜在转录调控因子，特别是针对phaP1 相蛋白和解聚酶phaZ3 和 phaZ5 基因。我们在体内用β-半乳糖苷酶测定法研究了启动子活性，并用最有前途的候选物在大肠杆菌中异源表达，并在体外通过电泳迁移率变动分析研究它们与启动子的相互作用。我们可以证明 R. eutropha DNA 结合 Xenobiotic 反应元件家族样蛋白 H16_B1672 特异性地在体外与 phaP1 启动子结合，K 值为 175 nM，并在体内抑制该启动子的基因表达。蛋白 H16_B1672 还在体内和体外与两个解聚酶启动子相互作用，表明其在 PHA 代谢调控中具有更广泛的作用。此外，体内测定表明 H-NS 样 DNA 结合蛋白 H16_B0227 和肽基脯氨酰顺反异构酶 PpiB 分别强烈抑制 PphaP1 和 PphaZ3 的基因表达。总之，本研究为 R. eutropha 中 PHA 代谢的调控提供了新的见解，揭示了新型转录调控因子的特定相互作用。
