光滑球拟酵母能量代谢调控其环境适应性的生理机制

ATP is the global current in the industrial strain,and play vital role on the physiological function. Among them, it was found that a higher intracellular ATP content could enhance the tolerance of industrial strain to environmental stresses. However, the exploitation of microorganisms in the industrial biotechnology requires a comprehensive understanding of manipulation mechanism of energy metabolism on fitness. The availability of genome sequences and development of cell fluorescence allows gaining understanding of microbial physiology at the global level. In this project, a mutant Torulopsis glabrata INH1, which intracellular ATP concentration was efficient tunable control by copper ion inducible promoter MT-1, was used as research model. The effect of different ATP contents on T. glabrata INH1 intracellular micro-environment, the profiles of gene and protein expression were investigated by the combination of chemostatic culture and -omics technology. The up- and down-regulated transcriptome and proteome data was use to annotate of physiological function by GO annotation and the regulatory proteins which manipulation by ATP were identified. Based on the above results, the targeted genes were searched through ChIP-seq protocol and the physiological function was annotated. In order to elucidate the physiological role of the regulatory proteins and its targeted genes on the fitness of industrial stain, a serial of mutants which delete/over-expressed the corresponding genes were re-constructed. Furthermore, the physiological function of those corresponding genes was demonstrated by RT-PCR and physiological parameters. From the above results, the relationship of ATP content- regulatory protein- regulatory model - target gene and its function- microbial fitness was described. The mechanism presented here not only gain improved insight into yeast physiology, but may provide novel strategy to acquire the desired phenotype.

能量代谢是影响微生物生理功能的重要因素，提高胞内ATP含量能显著增强微生物对恶劣环境的适应能力。然而，能量代谢如何通过胞内微环境、转录调节因子调控环境适应性的生理机制尚缺乏全局性和机理性的认识。光滑球拟酵母是一种具有重要工业应用价值的假丝酵母，ATP含量显著影响其环境适应性。本项目以能精确调控胞内ATP水平的T. glabrata INH1突变菌株为研究模型，借助恒化培养、生理生化分析、组学技术、靶基因插入失活与过量表达等研究策略，从胞内微环境、转录调控蛋白、靶基因功能等三方面系统地研究胁迫环境下能量代谢对环境适应性的影响，从能量代谢-转录调控蛋白-调控模式-靶向功能模块-环境适应性的关联机制角度解析光滑球拟酵母能量代谢调控其环境适应性的生理机制。研究结果有助于增加对假丝类酵母发酵生理的理解，并为改进微生物环境适应性提高生物技术过程经济性提供理论基础，具有重要的理论和实践意义。

ATP是微生物细胞内重要的能源物质和重要辅因子，通过影响代谢网络、信号转导和物质转运，进而影响微生物细胞的生理功能。本项目在完成基因组规模辅因子代谢网络模型构建的基础上，发现ATP通过ATP-NAD、ATP-乙酰CoA、ATP-NADP(H)等交互作用而影响微生物生理代谢。将细胞内ATP水平降低65.5%、62.0%、48.4%，则使菌体干重分别降低了46.9%、44.2%和59.8%。结合转录组数据分析，鉴定已确定了受能量代谢调控的8 个中介体蛋白(Med 2、Med 3、Med5、Med 7、Med 8、Med 10、Med15、Med 33)和11个转录调控蛋白(如Asg1p、Hal9p、Crz1p等)。进一步通过转录组学数据发现，与出发菌株wt比较，菌株Cgmed3ABΔ脂肪酸延伸与合成基因发生下调，甘油磷脂合成过程中关键基因发生下调。导致 Cgmed3ABΔ 细胞内角鲨烯含量上升30 倍；羊毛甾醇、酵母甾醇、粪甾醇和麦角固醇分别降低了88%、88%、92%和82%；主要脂肪酸C16:0、C16:1、C18:0、C18:1 和C18:2 含量降低了55%、54%、29%、39%和41%。磷脂酰胆碱(PC)和磷脂酰乙醇胺(PE)分别降低60%和73%。这一组分变化导致突变菌株Cgmed3ABΔ 的细胞膜刚性降低12%、细胞膜质子泵H+-ATPase 活性降低了75%。在pH2.0 时突变菌株Cgcrz1Δ 细胞壁多糖β-葡聚糖、单不饱和脂肪酸C16:1 和C18:1 含量和UFA/SFA 比例下降23%、10%和30%和4.47 个单位。从而导致膜甾醇成分—角鲨烯、粪甾醇和麦角甾醇含量分别下降了70%、84%和30%，突变菌株Cgcrz1Δ 的总磷脂含量下降，磷脂酸 (PA)和磷脂酰肌醇 (PI) 含量分别下降了25%和23%，磷脂的不饱和度和碳链长度降低，在pH2.0 时，与出发菌株wt 相比，突变菌株Cgcrz1Δ 的细胞膜完整性、流动性和质子泵H+-ATPase 活力分别下降了45%、9%和52%。上述研究表明，能量通过中介体和转录因子调控膜细胞组分合成，从而影响膜刚性的生理机制。从而阐明了能量代谢-中介体(转录因子)-脂质代谢-细胞膜组分-细胞膜刚性-环境胁迫的生理机制。