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首页> 外文期刊>Biotechnology Progress >Serial 13C-based flux analysis of an L-phenylalanine-producing E. coli strain using the sensor reactor.
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Serial 13C-based flux analysis of an L-phenylalanine-producing E. coli strain using the sensor reactor.

机译：使用传感器反应器对生产L-苯丙氨酸的大肠杆菌进行基于序列13C的通量分析。

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摘要

With the aid of the recently developed Sensor reactor system, 13C-labelling experiments were performed in order to determine, and optimize, C flux during the L-phenylalanine (L-Phe) production phase of an industrial-scale (300 l) recombinant Escherichia coli (4pF81, based on strain k 12 LJ110) culture. On the basis of the data from NMR labelling analysis, 3 subsequent flux patterns were derived for monitoring L-Phe formation during an observation window from 14 to 23.3 h process time (14-16.8, 17.2-20 and 20.5-23.3 h). Linear programming was performed to identify optimum flux patterns for L-Phe formation. Additionally, flux sensitivity analysis was used to identify the most promising metabolic engineering target. High rates of phosphoenolpyruvate conversion to pyruvate was identified as the most important reason for the deterioration of the L-Phe yield from glucose; it decreased from 20 to, finally, 11 mol%. Considering the characteristics of the enzyme kinetics involved, the working hypothesis was formulated that phosphoenolpyruvate synthase activity was increasingly hampered by increasing oxaloacetate and 2-oxoglutarate concn. Simultaneously, pyruvate kinase activity increased due to activation by fructose 1,6-diphosphate. It is suggested that overexpression of pps, the gene encoding phosphoenolpyruvate synthase, should be performed to optimize L-Phe in the recombinant strain.
机译：借助最新开发的传感器反应器系统，进行了13C标记实验，以确定和优化工业规模（300 l）重组大肠杆菌的L-苯丙氨酸（L-Phe）生产阶段的C通量大肠杆菌（4pF81，基于菌株k 12 LJ110）培养。根据NMR标记分析的数据，推导了3个后续的通量模式，以在14到23.3 h的处理时间（14-16.8、17.2-20和20.5-23.3 h）的观察窗内监视L-Phe的形成。进行线性编程以鉴定用于L-Phe形成的最佳通量模式。此外，通量灵敏度分析用于确定最有前途的代谢工程目标。磷酸烯醇丙酮酸高转化为丙酮酸被认为是葡萄糖导致L-Phe产量下降的最重要原因。它从20降低到11摩尔％。考虑到所涉及的酶动力学特征，提出了工作假设：草酰乙酸和2-氧戊二酸的浓度增加，磷酸烯醇丙酮酸合酶的活性越来越受到阻碍。同时，丙酮酸激酶活性由于果糖1,6-二磷酸的激活而增加。提示应进行过表达pps（编码磷酸烯醇丙酮酸合酶的基因）以优化重组菌株中的L-Phe。

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