Takagi M, Nishio Y, Oh G, Yoshida T
International Center for Biotechnology, Osaka University, 2-1, Yamada-oka, Suita, Osaka 565, Japan.
Biotechnol Bioeng. 1996 Dec 20;52(6):653-60. doi: 10.1002/(SICI)1097-0290(19961220)52:6<653::AID-BIT3>3.0.CO;2-N.
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.
研究了通过葡萄糖和L-酪氨酸双补料对重组大肠杆菌AT2471生产L-苯丙氨酸的控制。开发了一种在线监测最大葡萄糖摄取速率（MGUR）的新方法，该方法通过测量消耗添加葡萄糖所需的时间来实现。通过基于MGUR的在线监测和激光传感器测量的细胞浓度来控制葡萄糖补料，使葡萄糖浓度保持在0.1 g/L以下，从而在整个培养期间成功防止了乙酸的积累。在补加葡萄糖的分批培养中，L-酪氨酸耗尽后，细胞生长和L-苯丙氨酸生产率随着分支酸变位酶-p-预苯酸脱水酶（CMP）和3-脱氧-D-阿拉伯庚酮糖酸7-磷酸合酶（DAHP）的比酶活性降低而下降，这两种酶是L-苯丙氨酸合成途径中的关键酶。适当增加L-酪氨酸补料速率，但不至于使L-酪氨酸在培养物中积累，可提高酶的活性以及生长和生产的比速率，而基于葡萄糖消耗的产物得率会降低。在L-酪氨酸补料的前期和后期，生长、生产和MGUR的平均比速率均可表示为特定L-酪氨酸消耗速率的函数。使用这些函数，结合两个10小时时间段内两种特定L-酪氨酸消耗速率的几种不同组合，对L-苯丙氨酸产量、产物得率和成本因子进行估算，结果表明建议采用一种最佳的L-酪氨酸补料策略，与前期相比，后期采用较低的特定L-酪氨酸消耗速率以抑制细胞生长。在L-酪氨酸补料期间，通过将特定L-酪氨酸消耗速率调整到预测值，可以成功控制生长、生产和MGUR这三个比速率。在这种受控培养中成本因子最低，通过实验证明了该策略的有效性。（c）1996约翰威立父子公司
