Analysis and optimization of lysine fermentation based on proteome analysis

这项研究的目的是开发一种方法学来分析和优化生物过程，该方法基于使用双向电泳(2-DE)进行蛋白质组分析所获得的细胞内信息。采用黄短杆菌苏氨酸限制性赖氨酸发酵进行蛋白质组学分析。样品取自不同生理状态下的赖氨酸发酵分批培养物，包括指数生长期、初始赖氨酸生产阶段和最终赖氨酸生产阶段。用玻璃微珠均质，得到无细胞提取物。使用苯甲基磺酰氟(酶抑制剂)和丙酮沉淀法大大提高了从完整细胞中提取胞内蛋白的效率。在指数生长期和赖氨酸产生初期的样品中，2-DE分别检测到约480个和450个蛋白质斑点。然而，在最终赖氨酸生产阶段的样品中，蛋白质斑点数量减少到约320个，这表明发酵结束时微生物活性下降。然后利用多肽质量指纹图谱分析谷氨酸康氏杆菌蛋白质的数据库，进行图像分析，以确定2-DE图谱上的蛋白质斑点。在指数生长期，发现了与糖酵解和TCA循环有关的蛋白质，而没有观察到与赖氨酸生物合成有关的蛋白质。然而，在赖氨酸产生的起始和结束阶段的样本中，明确鉴定了与赖氨酸生物合成有关的两个蛋白质(DAPA和DOH)以及与糖酵解和TCA循环有关的蛋白质。这表明利用2-DE图谱可以在蛋白质水平上识别赖氨酸发酵过程中生理状态的差异。因此，利用2-DE进行蛋白质组分析可以有效地获取细胞内的信息，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.