Control of L-phenylalanine production by dual feeding of glucose and L-tyrosine

Abstract

Control of L-phenylalanine production by a recombinant of Escherichia coli AT2471 by means of the dual feeding of glucose and L-tyrosine was investigated. A novel method was developed for on-line monitoring of the maximum glucose uptake rate (MGUR), in which the length of time required for the consumption of added glucose was measured. Accumulation of acetic acid was successfully prevented throughout the whole period of the culture when the glucose concentration was kept below 0.1 g/L by controlling the glucose feeding on the basis of on-line monitoring of the MGUR and the cell concentration with a laser sensor.In a batch culture with glucose feeding, after L-tyrosine was depleted cell growth and the L-phenylalanine production rate decreased along with decreases in the specific enzyme activities of chorismate mutase-p-prephenate dehydratase (CMP) and 3-deoxy-D-arabinoheputulosonate 7-phosphate synthase (DAHP), which are the key enzymes in the L-phenylalanine synthesis pathway. Increasing the L-tyrosine feed rate by an appropriate amount, but not so far as to cause L-tyrosine accumulation in the culture, increased the activities of the enzymes and the specific rates of growth and production while the product yield based on glucose consumption decreased.The average specific rates of growth, production, and MGUR could be expressed as functions of the specific L-tyrosine consumption rate during both the earlier and later periods of L-tyrosine feeding. Estimations of the amount of L-phenylalanine produced, the product yield, and the cost factor by using these functions with several different combinations of two specific L-tyrosine consumption rates for two 10-h periods resulted in a suggested optimum L-tyrosine feeding strategy giving a lower specific L-tyrosine consumption rate in the later period, to suppress cell growth, in comparison to that in the earlier period. During L-tyrosine feeding, the three specific rates (growth, production, and MGUR) could be successfully controlled by adjusting the specific L-tyrosine consumption rate to the predicted value. The cost factor was lowest in this controlled culture, demonstrating experimentally the effectiveness of the strategy. (c) 1996 John Wiley & Sons, Inc.