Abstract：
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 KD 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.
摘要：
尽管人们对聚羟基链烷酸酯（PHA）作为绿色塑料替代品的研究兴趣不断增长，我们对PHA合成调控机制的理解，storage,模型生物Ralstoniaeutropha的降解仍然有限。鉴于其对中心碳代谢的重要性，PHA稳态可能由复杂的转录调节因子网络控制。了解这种微调是开发改进的PHA生产菌株从而促进PHA应用的关键。我们使用来自R.eutrophaRe2058/pCB113的粗蛋白提取物进行启动子下拉测定，然后进行LC-MS/MS，鉴定参与PHA代谢表达控制的推定转录调节因子，特异性靶向phasinphaP1和解聚酶phaZ3和phaZ5基因。使用β-半乳糖苷酶测定法在体内研究了对启动子活性的影响，最有希望的候选物是在大肠杆菌中异源产生的，并且通过电泳迁移率转移测定法在体外研究了它们与启动子的相互作用。我们可以证明，与红藻DNA结合的XRE家族样蛋白H16_B1672在体外以175nM的KD特异性结合phaP1启动子，并在体内抑制该启动子的基因表达。蛋白H16_B1672在体内和体外也显示出与两种解聚酶启动子的相互作用,表明在PHA代谢的调节中具有更广泛的作用。此外，体内实验表明，H-NS样DNA结合蛋白H16_B0227和肽基-脯氨酸顺反异构酶PpiB，分别强烈抑制PphaP1和PphaZ3的基因表达。总之,这项研究提供了新的见解，PHA代谢在R.eutropha，揭示新型转录调节因子的特定相互作用。
