Analysis and optimization of lysine fermentation based on proteome analysis

本研究的目的是开发一种基于二维电泳（2-DE）蛋白质组分析获得的细胞内信息来分析和优化生物过程的方法。利用黄短杆菌进行苏氨酸限制赖氨酸发酵，进行蛋白质组学分析。在不同的生理状态下，包括指数生长阶段、初始赖氨酸生产阶段和最终赖氨酸生产阶段，从赖氨酸发酵的批量培养中提取样品。用玻璃珠均质获得无细胞提取物。苯基甲基磺酰氟（蛋白酶抑制剂）和丙酮沉淀的使用大大提高了从完整细胞中提取细胞内蛋白质的效率。在指数生长期和赖氨酸初始生产期样品中，2-DE分别检测到大约480和450个蛋白点。然而，在赖氨酸最终生产阶段的样品中，蛋白质斑点的数量减少到大约320个，这表明在发酵结束时微生物活性下降。利用肽质量指纹图谱分析谷氨酸粘连杆菌蛋白数据库，对2-DE图谱进行蛋白点识别。在指数生长期，鉴定出与糖酵解和TCA循环有关的蛋白质，而与赖氨酸生物合成有关的蛋白质未观察到。然而，在赖氨酸初始和最终生产阶段的样品中，明确鉴定了与赖氨酸生物合成有关的2个蛋白（DapA和Doh）以及与糖酵解和TCA循环有关的蛋白。这表明，利用2-DE图可以在蛋白质水平上识别赖氨酸发酵生理状态的差异。因此，利用2-DE进行蛋白质组分析可以有效地获取细胞内信息，从而更好地了解生物过程。

The aim of this study is to develop a methodology to analyze and optimize bioprocesses based on intracellular information obtained by proteome analysis using a two-dimensional electrophoresis (2-DE). Threonine-limited lysine fermentation using Brevibacterium flavum was employed for the proteome analysis. Samples were taken from batch cultures of lysine fermentation at different physiological states including an exponential growth phase, an initial lysine production phase and a final lysine production phase. The cell-free extracts were obtained by homogenization using glass beads. The use of phenylmethylsulfonyl fluoride (protease inhibitor) and acetone precipitation greatly improved the extraction efficiency of intracellular proteins from intact cells. Approximately 480 and 450 protein spots were detected by 2-DE in the samples of the exponential growth phase and the initial lysine production phase, respectively. However, the number of protein spots decreased to approximately 320 in the sample of the final lysine production phase, which suggested the decline of microbial activity at the end of fermentation. The image analysis was then performed to identify protein spots of 2-DE maps by use of the database of Conrynebacterium glutamicum proteins analyzed by a peptide mass fingerprinting. At the exponential growth phase, proteins concerning glycolysis and TCA cycle were identified, while the proteins related to lysine biosynthesis was not observed. However, in the sample of the initial and final lysine production phases, 2 proteins (DapA and Doh) concerning lysine biosynthesis was clearly identified as well as proteins concerning glycolysis and TCA cycle. This shows that the difference in physiological states of lysine fermentation could be recognized at a protein level using 2-DE maps. Thus, the proteome analysis using 2-DE was found to be effective to obtain the intracellular information for the better understanding of bioprocesses.