LUNAM Université, Université de Nantes, CNRS, GEPEA, UMR 6144, Bât. CRTT, 37 boulevard de l'Université, BP 406, F-44602 Saint-Nazaire Cedex, France.
Biotechnol Bioeng. 2012 Dec;109(12):3030-40. doi: 10.1002/bit.24575. Epub 2012 Jun 27.
Adaptive metabolic behavior of photoautotrophic microorganisms toward genetic and environmental perturbations can be interpreted in a quantitative depiction of carbon flow through a biochemical reaction network using isotopic non-stationary (13) C-metabolic flux analysis (INST (13) C-MFA). To evaluate (13) C-metabolic flux maps for Chlamydomonas reinhardtii, an original experimental framework was designed allowing rapid, reliable collection of high-quality isotopomer data against time. It involved (i) a short-time (13) C labeling injection device based on mixing control in a torus-shaped photobioreactor with plug-flow hydrodynamics allowing a sudden step-change in the (13) C proportion in the substrate feed and (ii) a rapid sampling procedure using an automatic fast filtration method coupled to a manual rapid liquid nitrogen quenching step. (13) C-substrate labeling enrichment was controlled through the total dissolved inorganic carbon concentration in the pulsed solution. First results were obtained from steady-state continuous culture measurements allowing the characterization of the kinetics of label incorporation into light-limited growing cells cultivated in a photobioreactor operating at the maximal biomass productivity for an incident photon flux density of 200 µmol m(-2) s(-1). (13)C label incorporation was measured for 21 intracellular metabolites using IC-MS/MS in 58 samples collected across a labeling experiment duration of 7 min. The fastest labeling rate was observed for 2/3-phosphoglycerate with an apparent isotopic stationary state reached after 300 s. The labeling rate was consistent with the optimized mixing time of about 4.9 s inside the reactor and the shortest reliable sampling period assessed at 5 s.
采用同位素非稳态（13）C-代谢通量分析（INST（13）C-MFA），可通过生化反应网络中碳流的定量描述，来阐释光自养微生物对遗传和环境扰动的适应性代谢行为。为了评估莱茵衣藻的（13）C-代谢通量图谱，我们设计了一个原始的实验框架，可快速、可靠地收集随时间变化的高质量同位素分馏数据。该框架涉及：（i）一种基于混合控制的短时间（13）C 标记注入装置，该装置在具有塞流流动动力学的环形光生物反应器中运行，可使基质进料中的（13）C 比例突然发生阶跃变化；（ii）采用自动快速过滤法和手动快速液氮淬灭步骤的快速采样程序。（13）C-底物标记丰度通过脉冲溶液中的总溶解无机碳浓度进行控制。稳态连续培养测量得到了初步结果，可对在光生物反应器中培养的、在 200 µmol m(-2) s(-1) 的入射光子通量密度下以最大生物质生产率运行的光限制生长细胞的标记掺入动力学进行表征。在 7 分钟的标记实验持续时间内，采用 IC-MS/MS 对 58 个样品中的 21 种细胞内代谢物进行了（13）C 标记掺入测量。在 300 s 后，二磷酸甘油酸达到了明显的同位素稳态，此时的标记速率最快。标记速率与反应器内约 4.9 s 的优化混合时间以及评估的最短可靠采样时间 5 s 一致。
