低丰度~(13)C标记的酿酒酵母代谢通量分析探讨研究
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摘要
通过对酿酒酵母发酵液进行低浓度的[1-13C]葡萄糖标记,并采用GC-C-IRMS测定发酵菌体中蛋白氨基酸的δ13C值,最终显示在0.5%~2%的标记浓度范围内,15种菌体蛋白氨基酸δ13C值与标记浓度呈现出很好的线性关系。证明了0.5%或者更低的浓度标记都可以用于13CMFA中,大大降低了碳同位素标记代谢流成本,为13CMFA应用于传统白酒和黄酒大生产发酵过程中的复杂代谢机理研究提供一种手段。
Stable carbon isotope compositions of proteinogenic amino acids in S.cerevisiae fermenting liquid at low labeled tracer substrate were determined by gas chromatography-combustion-isotope ratio mass spectrometry(GC-C-IRMS) in the experiments. The results suggested that, within 0.5 %~2 % labeled range, δ13C values of 15 kinds of amino acids in protein had good linear relationships with the labeled concentration. The experiments demonstrated that 0.5 % or even lower labeled concentration was feasible in13CMFA, which could reduce greatly the metabolic flux cost and provide a new approach to13CMFA application in the research on the complex metabolism of the fermentation of traditional liquor and yellow rice wine.
Stable carbon isotope compositions of proteinogenic amino acids in S.cerevisiae fermenting liquid at low labeled tracer substrate were determined by gas chromatography-combustion-isotope ratio mass spectrometry(GC-C-IRMS) in the experiments. The results suggested that, within 0.5 %~2 % labeled range, δ13C values of 15 kinds of amino acids in protein had good linear relationships with the labeled concentration. The experiments demonstrated that 0.5 % or even lower labeled concentration was feasible in13CMFA, which could reduce greatly the metabolic flux cost and provide a new approach to13CMFA application in the research on the complex metabolism of the fermentation of traditional liquor and yellow rice wine.
引文
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[10]钟其顶,李国辉,王道兵,等.气相色谱-燃烧-同位素质谱仪(GC-C-IRMS)测定游离氨基酸的δ13C值[J].酿酒科技,2013(9):7-10.
[2]Marx A,de Graaf A A,Wiechert W,et al.Determination of the fluxes in the central metabolism of Corynebacterium glutamicum by nuclear magnetic resonance spectroscopy combined with metabolite balancing[J].Biotechnology and Bioengineering,1996,49(2):111-129.
[3]Zamboni N,Fendt S,Rühl M,et al.13C-based metabolic flux analysis[J].Nature Protocols,2009,4(6):878-892.
[4]庞伟.13C标记技术测定Escherichia coli TUQ2厌氧中心碳代谢途径通量分布[D].天津:天津大学,2008.
[5]Wittmann C,Kim H M,Heinzle E.Metabolic network analysis of lysine producing Corynebacterium glutamicum at a miniaturized scale[J].Biotechnology and Bioengineering,2004,87(1):1-6.
[6]Sauer U,Hatzimanikatis V,Bailey J E,et al.Metabolic fluxes in riboflavin-producing Bacillus subtilis[J].Nature Biotechnology,1997,15(5):448-452.
[7]Dauner M,Sauer U.GC-MS analysis of amino acids rapidly provides rich information for isotopomer balancing[J].Biotechnology Progress,2000,16(4):642-649.
[8]Wittmann C,Heinzle E.Application of MALDI-TOF MS to lysine‐producing Corynebacterium glutamicum[J].European Journal of Biochemistry,2001,268(8):2441-2455.
[9]Velagapudi V R,Wittmann C,Schneider K,et al.Metabolic flux screening of Saccharomyces cerevisiae single knockout strains on glucose and galactose supports elucidation of gene function[J].Journal of Biotechnology,2007,132(4):395-404.
[10]钟其顶,李国辉,王道兵,等.气相色谱-燃烧-同位素质谱仪(GC-C-IRMS)测定游离氨基酸的δ13C值[J].酿酒科技,2013(9):7-10.