Yang Tae Hoon, Wittmann Christoph, Heinzle Elmar
Biochemical Engineering Institute, Saarland University, Bldg. A 1.5, Postbox 151150, D-66041 Saarbrücken, Germany.
Metab Eng. 2006 Sep;8(5):417-31. doi: 10.1016/j.ymben.2006.03.001. Epub 2006 Jul 17.
A novel method for (13)C flux analysis based on on-line CO(2) labeling measurements is presented. This so-called respirometric (13)C flux analysis requires multiple parallel (13)C labeling experiments using differently labeled tracer substrates. In Part I of the work, a membrane-inlet mass spectrometry-based measurement system with 6 parallel reactors with each 12 ml liquid volume and associated experimental and computational methods for the respirometric (13)C data acquisition and evaluation are described. Signal dynamics after switching between membrane probes follow exactly first-order allowing extrapolation to steady state. Each measurement cycle involving 3 reactors takes about 2 min. After development of a dynamic calibration method, the suitability and reliability of the analysis was examined with a lysine-producing mutant of Corynebacterium glutamicum using [1-(13)C(1)], [6-(13)C(1)], [1,6-(13)C(2)] glucose. Specific rates of oxygen uptake and CO(2) production were estimated with an error less than +/-0.3 mmol g(-1) h(-1) and had +/-3% to +/-10% deviations between parallel reactors which is primarily caused by inaccuracies in initial biomass concentration. The respiratory quotient could be determined with an uncertainty less than +/-0.02 and varied only +/-3% between reactors. Fractional labeling of CO(2) was estimated with much higher precision of about +/-0.001 to +/-0.005. The detailed statistical analysis suggested that these data should be of sufficient quality to allow physiological interpretation and metabolic flux estimation. The obtained data were applied for the respirometric (13)C metabolic flux analysis in Part II.
本文提出了一种基于在线二氧化碳标记测量的新型（13）C通量分析方法。这种所谓的呼吸测量（13）C通量分析需要使用不同标记的示踪底物进行多个平行的（13）C标记实验。在工作的第一部分，描述了一种基于膜进样质谱的测量系统，该系统有6个平行反应器，每个反应器的液体体积为12毫升，以及用于呼吸测量（13）C数据采集和评估的相关实验和计算方法。膜探针切换后的信号动力学恰好遵循一级反应，允许外推至稳态。每个涉及3个反应器的测量周期约需2分钟。在开发了一种动态校准方法后，使用[1-（13）C（1）]、[6-（13）C（1）]、[1,6-（13）C（2）]葡萄糖对谷氨酸棒杆菌的赖氨酸生产突变体进行了分析，检验了该分析方法的适用性和可靠性。估计的氧气摄取和二氧化碳产生的比速率误差小于±0.3 mmol g（-1）h（-1），平行反应器之间的偏差为±3%至±10%，这主要是由初始生物量浓度的不准确造成的。呼吸商的测定不确定度小于±0.02，反应器之间的变化仅为±3%。二氧化碳的标记分数估计精度更高，约为±0.001至±0.005。详细的统计分析表明，这些数据应具有足够的质量，以允许进行生理学解释和代谢通量估计。在第二部分中，将获得的数据应用于呼吸测量（13）C代谢通量分析。
