BIOSYNTHETICALLY DIRECTED FRACTIONAL C-13-LABELING OF PROTEINOGENIC AMINO-ACIDS - AN EFFICIENT ANALYTICAL TOOL TO INVESTIGATE INTERMEDIARY METABOLISM
BIOSYNTHETICALLY DIRECTED FRACTIONAL C-13-LABELING OF PROTEINOGENIC AMINO-ACIDS - AN EFFICIENT ANALYTICAL TOOL TO INVESTIGATE INTERMEDIARY METABOLISM
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DOI：
10.1111/j.1432-1033.1995.tb20829.x
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发表时间：
1995-09-01
期刊：
影响因子：
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通讯作者：
SZYPERSKI, T
中科院分区：
文献类型：
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作者：
SZYPERSKI, T
通过在含有[C-13（6）]葡萄糖和具有天然同位素丰度的葡萄糖的混合物作为唯一碳源的基本培养基上表达蛋白质来实现蛋白质原氨基酸的生物合成导向的分级C-13标记。随后蛋白质的水解产生游离氨基酸。在灵敏的二维异质结[C-13，H-1]-相关光谱（2D [C-13，H-1]-COSY）中观察C-13-C-13自旋-自旋标量耦合精细结构，允许人们识别由来自葡萄糖的单个源分子的完整的二碳和三碳片段并入氨基酸中产生的非随机C-13标记模式。由于2D [C-13，H-1]-COSY足以分辨所有相关共振，因此可以分析氨基酸混合物而无需进一步分离其组分。概率方程与观察到的多重峰强度的C-13精细结构的完整的碳片段的相对丰度。它们能够定量分析生物合成途径网络中的碳通量，从而使用蛋白质氨基酸作为探针来研究中间代谢。本文表明，生物合成导向的分数C-13标记的氨基酸提供了一个有效的分析工具，定量研究糖酵解，丙酮酸代谢，戊糖磷酸途径，三羧酸循环和C-1代谢。该方法的可能应用包括探索未知的生物合成途径和快速阐明已知生物合成反应网络对生长条件或遗传操作变化的响应。结合同位素、人力和NMR仪器时间的相对低的成本，这使得蛋白质氨基酸的生物合成部分C-13标记对于支持生物技术中的工艺设计和代谢工程特别有吸引力，由于筛选程序变得可行，其能够系统地表征细胞的代谢状态作为生物技术优化中涉及的参数的函数，流程.
Biosynthetically directed fractional C-13 labeling of proteinogenic amino acids is achieved by expression of proteins on a minimal medium which contains a mixture of [C-13(6)]glucose and glucose with natural isotope abundance as the sole carbon source. Subsequent hydrolysis of the proteins yields the free amino acids. The observation of C-13-C-13 spin-spin scalar coupling fine structures in sensitive two-dimensional heteronuclear [C-13,H-1]-correlation spectroscopy (2D [C-13,H-1]-COSY) allows one to identify non-random C-13-labeling patterns arising from the incorporation of intact two-carbon and three-carbon fragments from a single source molecule of glucose into the amino acids. Since 2D [C-13,H-1]-COSY suffices to resolve all relevant resonances, the mixture of amino acids can be analyzed without further separation of its components. Probabilistic equations relate the observed multiplet intensities of the C-13 fine structures to the relative abundance of the intact carbon fragments. They enable a quantitative analysis of the carbon flux in the network of biosynthetic pathways, thus using the proteinogenic amino acids as probes to study intermediary metabolism. This paper shows that biosynthetically directed fractional C-13 labeling of amino acids provides an efficient analytical tool to quantitatively investigate glycolysis, pyruvate metabolism, pentose phosphate pathway, tricarboxylic acid cycle and C-1 metabolism. Possible applications of the method include both the exploration of unknown biosynthetic pathways and the rapid elucidation of the response of a known biosynthetic reaction network to changes in growth conditions or genetic manipulations. In conjunction with the relatively low costs for isotopes, manpower and NMR instrument time, this makes biosynthetic fractional C-13 labeling of proteinogenic amino acids particularly attractive to support process design and metabolic engineering in biotechnology, since screening procedures become feasible which enable a systematic characterization of the cell's metabolic state as a function of parameters that are involved in the optimization of biotechnological processes.