Analysis and optimization of lysine fermentation based on proteome analysis

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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.