Identification of riboflavin: revealing different metabolic characteristics between Escherichia coli BL21(DE3) and MG1655.
Identification of riboflavin: revealing different metabolic characteristics between Escherichia coli BL21(DE3) and MG1655.
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DOI：
10.1093/femsle/fnv071
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发表时间：
2015-06
影响因子：
2.1
通讯作者：
Xinran Wang;Qian Wang;Q. Qi
中科院分区：
文献类型：
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作者：
Xinran Wang;Qian Wang;Q. Qi
由于来源不同，大肠杆菌B和K-12菌株之间存在着许多生理上的差异。更深入地了解这些菌株的代谢和调节机制将有助于改善这些工业主力的使用。在本研究中，我们观察到BL21发酵液在培养过程中逐渐变黄。通过光谱分析和液-质联用鉴定，首次确定发酵液中积累的黄色物质为核黄素。通过对BL21和MG1655核黄素代谢相关酶序列的比较，发现BL21核黄素双功能蛋白激酶/FMN腺苷转移酶(RIBF)115位点突变。His115Leu突变使酶活性降低到MG1655的55%，这可能是核黄素在BL21中积累的原因之一。定量聚合酶链式反应分析表明，BL21核黄素和FAD生物合成途径的整个分支的基因表达上调。BL21和MG1655的几个生理代谢特性不同，可能与核黄素的积累有关。
There are many physiological differences between Escherichia coli B and K-12 strains, owing to their different origins. Deeper insight into the metabolic and regulative mechanisms of these strains will inform improved usage of these industrial workhorses. In the present study, we observed that BL21 fermentation broth gradually turned yellow during cultivation. By spectral analysis and liquid chromatography-mass spectrometry identification, we confirmed for the first time that the yellow substance accumulated in the fermentation broth is riboflavin. Comparing the enzyme sequences involved in riboflavin metabolism between BL21 and MG1655, we identified a site mutation on the 115 residue of bifunctional riboflavin kinase/FMN adenylyltransferase (RibF) in BL21. This His115Leu mutation was found to reduce enzyme activity to 55% of that of MG1655, which is probably one reason for riboflavin accumulation in BL21. Quantitative PCR analysis showed that genes of the entire branch of the riboflavin and FAD biosynthesis pathways in BL21 were up-regulated. Several physiological and metabolic characteristics of BL21 and MG1655 were found to be different, and may also be related to the riboflavin accumulation.