大气压冷等离子体对酿酒酵母细胞膜通透性的调控

Cellular membrane permeability can have a dramatic influence on microbial productivity in whole-cell biotransformation. Activation of ion channel is one of the reasons that the membrane permeability is enhanced. Calcium ion, as an essential messenger, mediates a wide variety of cellular processes, such as cell growth. Therefore, the significance to regulate the activation of calcium channel is well established. It has been discovered that permeability of cellular membrane in microbes is increased, promoting microbial growth, by cold plasma at atmospheric pressure under the specific discharge parameters in our previous study. The calcium channel may be stimulated opening when cellular membrane is depolarized by the active species in plasma. Correspondingly, the specific activity and protein expression of calcium ion-dependent ATPase pump are enhanced with an immediate and transient elevation of the calcium concentration in the cytosol, in order to drive the secondary transport of metabolic products and nutrients, alleviating the inhibition of products and promoting cell growth as well as formation of target products. To verify the hypothesis, two methods of membrane protein expression and gene disruption are intended to use in this project. The mechanism will be explored that increased cellular membrane permeability of Saccharomyces cerevisiae at the two different levels of calcium channel activated directly and calcium ion-dependent ATPase pump induced indirectly by cold plasma at atmospheric pressure. These results will provide a theoretical basis 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.

在微生物转化过程中，细胞膜通透性直接影响菌体生产能力。细胞膜通透性提高机制之一是离子通道开放。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具有重要意义。我们前期研究发现：大气压冷等离子体在特定放电条件下，可以提高微生物细胞膜通透性，促进菌体生长。其提高细胞膜通透性的可能机制是等离子体活性因子使细胞膜去极化，激活钙通道开放，胞内钙离子流瞬间变化，提高钙依赖ATPase离子泵跨膜动力，引起代谢产物和营养物质二次运输，解除代谢产物抑制，促进细胞生长和目标产物形成。对这一推断的证实有利于将大气压冷等离子体发展成为一种新型代谢调控方法。因此，本项目拟运用膜蛋白表达和其功能基因缺失两种方法，揭示大气压冷等离子体直接调控酿酒酵母钙通道和间接调控其钙依赖离子泵，从而提高细胞膜通透性的机理。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。

本研究将大气压介质阻挡放电等离子体作为一种新型代谢调控方法，用于提高工业微生物细胞膜通透性，从而强化菌体生产能力。大气压介质阻挡放电等离子体放电过程中，可以在气相环境、气液环境和液相环境中产生大量活性粒子，如活性氧和活性氮；经等离子体诱导的克雷伯氏菌（Klebsiella pneumoniae）细胞内活性氧呈现先升高再降低的趋势，经等离子体诱导的酿酒酵母细胞内可以产生大量ROS和RNS。等离子体诱导的K. pneumoniae接种体在6%甘油发酵培养基中进行间歇发酵，在发酵过程中细胞膜通透性始终高于对照组。从酵母浸粉中分离筛选出一株高产乙醇菌株DL5168，经形态学观察、生理生化学及分子生物学鉴定，确定该菌株为酵母属的酿酒酵母（Saccharomyces cerevisiae）。将该菌株用于等离子体强化葡萄糖发酵生产乙醇中。建立响应面方法优化实验条件，并将该方法应用到优化与等离子体放电相关的实验参数中。优化条件处理酿酒酵母接种体，发酵最终菌体生物量、乙醇浓度和乙醇转化率分别比对照提高24%，42%和33%。等离子体不同处理时间S. cerevisiae 接种体在发酵不同时期，细胞膜电位、膜通透性和细胞质内钙离子浓度均出现不同程度增加或减少，进一步我们鉴定了等离子体激活酿酒酵母钙通道类型。大气压冷等离子体诱导酿酒酵母细胞膜氢质子泵表达量均高于对照。综上所述，我们得到初步结论，大气压介质阻挡放电等离子体可以通过调控细胞膜钙通道和H+-ATPase提高细胞膜通透性，从而强化工业微生物生产能力。