Approaches to Production of L-Phenylalanine by Genetic Engineering

• Main Authors: Lai, Zhang-Jian, 賴建彰
• Other Authors: Chao Yun-Peng
• Format: Others
• Language: zh-TW
• Published: 1998
• Online Access: http://ndltd.ncl.edu.tw/handle/65762586648838470540

碩士 === 逢甲大學 === 化學工程研究所 === 86 === In Escherichia coli, aspartate aminotransferase (encoded by aspC) and aromatic amino acid aminotransferase (encoded by tyrB) share overlapping substrate specificity in the synthesis of aromatic amino acids. Through the transamination reactions catalyzed by AspC or TyrB, L-phenylalanine can be produced from phenylpyruvate with aspartic acid as the amino donor. Consequently, oxaloacetate is produced as the by-product. To modulate and enhance the production levels of proteins, both aspC and tyrB were subcloned into a runaway vector. Employing resting cells of AspC- or TryB-overproducting E. coli K-12 strains for L-phenylalanine productions resulted in molar yields of 70% and 50%, respectively. Further introduction of phosphoenolpyruvatecarboxykinase (encoded by pck) into the bioconversion reactions, the conversion yields were improved to 93% from 70% and 70% from 50% in relatively short time. These results correspond to 1.68 mg and 1.26 mg of L-phenylalanine produced per mg dry cell weight per hour, which account for 30- and 40-fold increase compared to the previous report [13].Pck is the enzyme capable of converting oxaloacetate to phosphoenolpyruvate .Accordingly, itsuggests that the increased yields by additional involvement of Pck may be due to the relief of the inhibitory effect of oxaloacetate on aminotransferases.A transcriptional fusion was constructed by placing lacZ gene downstream of the tyrB promoter. By altering environmental conditions, the responding activities of LacZ were measured and used to determine the optimal condit ions for production of TyrB protein on a runaway vector. It was found that the highest yield of TyrB protein was obtained when the culture was continued for 7 hours in Luria-Bertani medium or 11 hours in M9 minimal medium supplemented with 0.2% of glucose after shifting the temperature from 30℃ to 40℃at 0.5 OD550 mm unit.