大气压冷等离子体对酿酒酵母胞内钙离子浓度调控机制及对其代谢过程强化研究

The free calcium ion in a special concentration range plays essential roles in microbial growth and product formation. It has been discovered that the intracellular free-Ca2+ concentration ([Ca2+]i) is increased, promoting the cell growth and improving ethanol yield in Saccharomyces cerevisiae, by cold plasma at atmospheric pressure under the specific discharge parameters in our previous study. The calcium channel may be stimulated opening，which induces the formation of calcium signaling, when cellular membrane is depolarized by the active species in plasma. Correspondingly, the transmembrane proton motive force is enhanced by calcium signaling. This drives the dephosphorylation of ATP to ADP. The lowered ATP/ADP ratio alters the cofactors metabolism of NADH/NAD+ and NADPH/NADP+. These changes have impacts on the activities of the cofactor-dependent enzymes, leading to the redistribution of the carbon flow and the metabolism flux in the central metabolism. As a result, cell growth and ethanol yield of Saccharomyces cerevisiae are improved. To verify the hypothesis, the methods of mRNA expression, protein expression, gene disruption and metabolic flux analysis are intended to use in this project. The mechanism will be explored that the metabolism processing in Saccharomyces cerevisiae is enhanced due to perturbation of cofactor metabolism under the mediation of the calcium signaling induced by cold atmospheric plasma. This result will provide a theoretical groundwork for a novel approach of metabolic regulation, control of cold plasma at atmospheric pressure, and also lay a foundation for an equipment of in-situ enhanced microbial fermentation by cold plasma.

在微生物转化中，一定浓度的钙离子有助于菌体生长和代谢产物形成。我们前期研究发现：大气压冷等离子体在特定放电条件下，可以提高酿酒酵母胞内钙离子浓度，促进菌体生长和提高乙醇转化率。其可能机制是特定放电性质等离子体活性因子使细胞膜去极化，激活钙通道开放形成钙信号；钙信号通过增强质子泵跨膜动力，促进ATP去磷酸化形成ADP；ATP/ADP比率降低会改变其它辅因子NAD+和NADP+的代谢，从而影响辅因子依赖代谢关键酶活性，导致中心代谢途径碳流向和代谢通量重新分布，促进菌体生长和乙醇转化率提高。对这一推断的证实，有利于将大气压冷等离子体发展为一种新型代谢调控方法。因此，本项目拟运用mRNA表达、蛋白质表达、功能基因缺失、代谢通量分析等方法，揭示大气压冷等离子体调控的钙信号，通过扰动辅因子代谢，强化细胞代谢过程的机理。该研究最终为建立大气压冷等离子体在线强化微生物发酵装置奠定基础。

在微生物转化过程中，钙离子是重要第二信使，参与细胞多种生理活动，因此调控钙通道开放对提高微生物转化能力具有重要意义。本项目首先建立了荧光探针Fluo-3 AM结合多功能酶标仪精确表征等离子体处理后的酿酒酵母细胞内绝对钙离子浓度的新方法；明确了等离子体诱导后细胞内钙离子来源问题和细胞膜通透性提高的机制，建立大气压冷等离子体调控细胞膜通透性的新方法。从代谢水平揭示大气压冷等离子体间接调控钙依赖ATPase表达，扰动辅因子（ATP和NADH）代谢，强化酿酒酵母代谢的机制。通过多尺度微环境中活性因子研究，明确大气压冷等离子体活性因子在微生物强化过程中的级联反应机制和长效机制。设计和创制了具有四对平行上下电极的等离子体强化新装置，解决了处理样品的平行性和重复性问题。本课题的研究成果有利于推动大气压冷等离子体生物过程强化技术的创建和发展。