BACKGROUND Escherichia coli is an important strain for L-threonine production. Genetic switch is a ubiquitous regulatory tool for gene expression in prokaryotic cells. To sense and regulate intracellular or extracellular chemicals, bacteria evolve a variety of transcription factors. The key enzymes required for L-threonine biosynthesis in E. coli are encoded by the thr operon. The thr operon could coordinate expression of these genes when L-threonine is in short supply in the cell. RESULTS The thrL leader regulatory elements were applied to regulate the expression of genes iclR, arcA, cpxR, gadE, fadR and pykF, while the threonine-activating promoters PcysH, PcysJ and PcysD were applied to regulate the expression of gene aspC, resulting in the increase of L-threonine production in an L-threonine producing E. coli strain TWF001. Firstly, different parts of the regulator thrL were inserted in the iclR regulator region in TWF001, and the best resulting strain TWF063 produced 16.34 g L-threonine from 40 g glucose after 30 h cultivation. Secondly, the gene aspC following different threonine-activating promoters was inserted into the chromosome of TWF063, and the best resulting strain TWF066 produced 17.56 g L-threonine from 40 g glucose after 30 h cultivation. Thirdly, the effect of expression regulation of arcA, cpxR, gadE, pykF and fadR was individually investigated on L-threonine production in TWF001. Finally, using TWF066 as the starting strain, the expression of genes arcA, cpxR, gadE, pykF and fadR was regulated individually or in combination to obtain the best strain for L-threonine production. The resulting strain TWF083, in which the expression of seven genes (iclR, aspC, arcA, cpxR, gadE, pykF, fadR and aspC) was regulated, produced 18.76 g L-threonine from 30 g glucose, 26.50 g L-threonine from 40 g glucose, or 26.93 g L-threonine from 50 g glucose after 30 h cultivation. In 48 h fed-batch fermentation, TWF083 could produce 116.62 g/L L-threonine with a yield of 0.486 g/g glucose and productivity of 2.43 g/L/h. CONCLUSION The genetic engineering through the expression regulation of key genes is a better strategy than simple deletion of these genes to improve L-threonine production in E. coli. This strategy has little effect on the intracellular metabolism in the early stage of the growth but could increase L-threonine biosynthesis in the late stage.

中文翻译：

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多个关键基因的表达调控，以改善大肠杆菌中的L-苏氨酸。

背景技术大肠杆菌是生产L-苏氨酸的重要菌株。遗传开关是用于原核细胞基因表达的普遍调节工具。为了感应和调节细胞内或细胞外化学物质，细菌会进化出多种转录因子。大肠杆菌中L-苏氨酸生物合成所需的关键酶由thr操纵子编码。当L-苏氨酸在细胞中短缺时，thr操纵子可以协调这些基因的表达。结果thrL前导调控元件被用于调控iclR，arcA，cpxR，gadE，fadR和pykF的表达，而苏氨酸激活启动子PcysH，PcysJ和PcysD被用于调控aspC的表达，从而产生L-苏氨酸的大肠杆菌菌株TWF001中L-苏氨酸产量的增加。首先，将调节子thrL的不同部分插入TWF001的iclR调节子区域，培养30小时后，得到的最佳菌株TWF063从40 g葡萄糖中产生了16.34 g L-苏氨酸。其次，将跟随不同苏氨酸激活启动子的基因aspC插入TWF063的染色体中，培养30小时后得到的最佳菌株TWF066从40 g葡萄糖中产生了17.56 g L-苏氨酸。第三，分别研究了arcA，cpxR，gadE，pykF和fadR的表达调节对TWF001中L-苏氨酸生产的影响。最后，使用TWF066作为起始菌株，单独或组合调节arCA，cpxR，gadE，pykF和fadR基因的表达，以获得生产L-苏氨酸的最佳菌株。产生的菌株TWF083，其中七个基因（iclR，aspC，培养30小时后，调节arcA，cpxR，gadE，pykF，fadR和aspC），从30 g葡萄糖中产生18.76 g L-苏氨酸，从40 g葡萄糖中产生26.50 g L-苏氨酸，或从50 g葡萄糖中产生26.93 g L-苏氨酸。在48小时的分批补料发酵中，TWF083可以生产116.62 g / L的L-苏氨酸，葡萄糖的产量为0.486 g / g，生产率为2.43 g / L / h。结论通过关键基因表达调控的基因工程是比简单删除这些基因来提高大肠杆菌中L-苏氨酸产量更好的策略。该策略在生长的早期对细胞内代谢几乎没有影响，但是在晚期可增加L-苏氨酸的生物合成。培养30 h后，从50 g葡萄糖中提取93 g L-苏氨酸。在48小时的分批补料发酵中，TWF083可以生产116.62 g / L的L-苏氨酸，葡萄糖的产量为0.486 g / g，生产率为2.43 g / L / h。结论通过关键基因表达调控的遗传工程是比简单删除这些基因来提高大肠杆菌中L-苏氨酸产量更好的策略。该策略在生长的早期对细胞内代谢几乎没有影响，但是在晚期可增加L-苏氨酸的生物合成。培养30 h后，从50 g葡萄糖中提取93 g L-苏氨酸。在48小时的分批补料发酵中，TWF083可以生产116.62 g / L的L-苏氨酸，葡萄糖的产量为0.486 g / g，生产率为2.43 g / L / h。结论通过关键基因表达调控的遗传工程是比简单删除这些基因来提高大肠杆菌中L-苏氨酸产量更好的策略。该策略在生长的早期对细胞内代谢几乎没有影响，但是在晚期可以增加L-苏氨酸的生物合成。结论通过关键基因表达调控的遗传工程是比简单删除这些基因来提高大肠杆菌中L-苏氨酸产量更好的策略。该策略在生长的早期对细胞内代谢几乎没有影响，但是在晚期可增加L-苏氨酸的生物合成。结论通过关键基因表达调控的遗传工程是比简单删除这些基因来提高大肠杆菌中L-苏氨酸产量更好的策略。该策略在生长的早期对细胞内代谢几乎没有影响，但是在晚期可增加L-苏氨酸的生物合成。