Determination Of Amino Acids And Inorganic Ions In Cultures And Its Application In Bacitracin Fermentation Process

Metabolic engineering is the intentional directed improvement of cellular properties through the modification of specific biochemical reactions or the introduction of new one by means of recombinant DNA and genetics technology. As an important branch of metabolic engineering research, metabolic flux focuses on how to describe metabolic intermediates, including amino acids, organic acids and inorganic ions, especially carbohydrates, pass through their ways in metabolite networks by mathematics formulas.The determination of in vivo metabolic fluxes is a demanding task. It requires extensive instruments, expensive media, and sophisticated analytical and computational techniques. In this research, an emphasis was made on analysis of amino acids and inorganic ions in cultures to disclose the exact metabolic pathway of Bacillus licheniformis which produces bacitracin in its fermentation process by capillary electrophoresis (CE) method.In the first part, CE method is applied to analyze the accumulated amino acids in broth during bacitracin fermentation process. Followed by studying the relationship between the amount of amino acids and bacitracin titer, it is indicated that in lower concentrations of nitrogen sources, EMP pathway plays the leading role in its metabolite network, meanwhile the transformation from 3-PG to PEP is strictly blocked. When the concentration of nitrogen sources is raised, the block is relieved, accompanied with TCA cycle strengthens. In this process, the bacitracin titer increases.In the second part of this research, CE methods are built and optimized to separate ions in organic nitrogen sources. The results of phosphorus, calcium and magnesium determination are compared with that of traditional chemical methods. It shows that CE method has advantages in linearity range, automation, detection speed and sample amount.