Study on Metabolic Flux of Recombinant Escherichia coli BL21 at Different Specific Growth Rates

• Main Authors: Hsien-Chih Pi, 皮先智
• Other Authors: Kow-Jen Duan
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/02121815390251869349

碩士 === 大同大學 === 生物工程研究所 === 91 === Employing genetic engineering E. coli for mass production of protein is very efficient. Metabolic flux calculation is a method to understand digest of nutrient in the E. coli. The present study performed continuous fermentation in analyzing difference of metabolic products between genetic engineering and wild E. coli. We hope results of this study can be applied in enhancing production of protein from genetic engineering E. coli. Continuous process was performed to achieve steady state fermentation of this study. Metabolic products including organic acids and amino acids inside cell and excretion to broth were analyzed. The dilution rate of the continuous fermentation were 0.13, 0.23, 0.4, 0.57 h-1. The dry cell weight of E. coli BL21 (DE3) in steady state fermentation were 2.73, 2.66, 2.37, 1.5 g/L for dilution rate of 0.13, 0.23, 0.4, 0.57 respectively. On the other hand, the dry cell weight of E. coli BL21 (DE3) w/PET20-LacZ were 2.5, 2.43, 2.5, 1.81 g/L for dilution rate of 0.13, 0.23, 0.4, 0.57 respectively. Glucose-6-phosphate and D-3-phosphoglyceric acid were accumulated in glycolysis. The results indicate rate determining steps regarding these two compounds. a-Ketoglutarate and oxaloacetate accumulated in the cell both for E. coli BL21 (DE3) and E. coli BL21 (DE3) w/PET 20-lacZ. These metabolites in the TCA cycle will be further digested to amino acids. Lactic acid, acetic acid, glycerol accumulated in the cell at high dilution rate. Part of energy from nutrients was stored in these metabolites when carbon source supply was sufficient. Uptake of amino acids from the medium increased with dilution rate. The accumulation of amino acids, particularly for the aspartate family, increased with dilution rate. Synthesis of D-3-phosphoglyceric acid remained constant for all dilution rates. There is no accumulation of serine in the cell. It seems that serine was excreted to the medium to avoid synthesis inhibition of D-3-phosphoglyceric acid.