Utility of an Escherichia coli strain engineered in the substrate uptake system for improved culture performance at high glucose and cell concentrations:: An fed-batch cultures
Utility of an Escherichia coli strain engineered in the substrate uptake system for improved culture performance at high glucose and cell concentrations:: An fed-batch cultures
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DOI：
10.1002/bit.21664
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发表时间：
2008-03-01
影响因子：
3.8
通讯作者：
Ramirez, Octavio T.
中科院分区：
文献类型：
--
作者：
Lara, Alvaro R.;Caspeta, Luis;Ramirez, Octavio T.
溢流代谢是大肠杆菌需氧培养的一个不良特征。这是由于葡萄糖消耗率升高导致底物转化为醋酸酯的高水平，严重影响了细胞生理和生物过程性能。这种现象通常发生在高葡萄糖浓度下的分批培养中。补料分批培养是防止溢出代谢的经典生物处理替代方法，其中葡萄糖摄取速率由外部添加的葡萄糖控制。尽管补料分批培养模式被广泛使用，但只有在有效防止溢出代谢的情况下，补料分批培养模式存在的缺点才能在较高的初始葡萄糖浓度下被更简单的分批培养克服。在本研究中，利用改良的葡萄糖转运系统，对一株缺乏磷酸烯醇式丙酮酸：碳水化合物磷酸转移酶系统的大肠杆菌(VH32)在葡萄糖浓度高达100g/L的条件下进行了分批培养，同时表达了重组绿色荧光蛋白。在所测的最高葡萄糖浓度下，亲本菌株(W31-10)的乙酸乙酯积累量最高达13.6g/L，而VH32的最高积累量仅为2g/L。因此，在葡萄糖浓度为100g/L的VH32培养中，细胞浓度和绿色荧光蛋白浓度分别达到52g/L和8.2g/L。相比之下，W31-10菌株的最大生物量和GFP分别只有工程菌的65%和48%。并与W3110的传统补料分批培养进行了比较。这项研究表明，通过代谢工程绕过溢出代谢，可以在简单的分批培养中获得较高的细胞和重组蛋白浓度。这代表了一种新的、有价值的替代经典生物处理方法的方法。
Overflow metabolism is an undesirable characteristic of aerobic cultures of Escherichia coli. It results from elevated glucose consumption rates that cause a high substrate conversion to acetate, severely affecting cell physiology and bioprocess performance. Such phenomenon typically occurs in batch cultures under high glucose concentration. Fed-batch culture, where glucose uptake rate is controlled by external addition of glucose, is the classical bioprocessing alternative to prevent overflow metabolism. Despite its wide-spread use, fed-batch mode presents drawbacks that could be overcome by simpler batch cultures at high initial glucose concentration, only if overflow metabolism is effectively prevented. In this study, an E. coli strain (VH32) lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) with a modified glucose transport system was cultured at glucose concentrations of up to 100 g/L in batch mode, while expressing the recombinant green fluorescence protein (GFP). At the highest glucose concentration tested, acetate accumulated to a maximum of 13.6 g/L for the parental strain (W31 10), whereas a maximum concentration of only 2 g/L was observed for VH32. Consequently, high cell and GFP concentrations of 52 and 8.2 g/L, respectively, were achieved in VH32 cultures at 100 g/L of glucose. In contrast, maximum biomass and GFP in W31 10 cultures only reached 65 and 48%, respectively, of the values attained by the engineered strain. A comparison of this culture strategy against traditional fed-batch culture of W3110 is presented. This study shows that high cell and recombinant protein concentrations are attainable in simple batch cultures by circumventing overflow metabolism through metabolic engineering. This represents a novel and valuable alternative to classical bioprocessing approaches.