Wittemeier Luisa, Rajarathinam Yogeswari, Erban Alexander, Hagemann Martin, Kopka Joachim
Max-Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476, Potsdam, Germany.
Environmental and Biochemical Sciences, The James Hutton Institute, Invergowrie, Dundee, DD2 5DA, UK.
New Phytol. 2025 Oct;248(1):401-414. doi: 10.1111/nph.70412. Epub 2025 Jul 24.
Photoautotrophic organisms fix inorganic carbon (Ci) by RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE/OXYGENASE (RUBISCO) and PHOSPHOENOLPYRUVATE CARBOXYLASE (PEPC). Monitoring Ci assimilation rates in vivo is a major challenge in analyzing photoautotrophic metabolism and engineering improved photosynthesis, as conventional methods do not distinguish between these two fluxes. We explored widely applied gas chromatography mass spectrometry (GC-MS) metabolite profiling for C-positional fractional C enrichment (EC) analyses of aspartate to differentiate within one molecule between PEPC, RUBISCO, and CBB cycle activities by C pulse-labeling. We validated this method using two GC-MS instruments and two prevailing chemical derivatization methods. We selectively determined EC at each carbon position of aspartate with accuracy < 1% and precision < 2.5%. In combination with dynamic CO labeling of Synechocystis cultures, we determined PEPC activity in vivo alongside assessments of RUBISCO and CBB cycle activities. We demonstrate that RUBISCO is inactive in the dark, whereas PEPC remains active but at a lower rate than during the day. Accurate quantifications of aspartate concentrations and positional ECs provide molar Ci assimilation rates of photoautotrophic Synechocystis cultures. This technology can be combined with C-positional analyses of other metabolites, for example 3-phosphoglycerate, and may be adapted to characterize natural and biosynthetically engineered Ci-assimilation.
光合自养生物通过1,5-二磷酸核酮糖羧化酶/加氧酶（RUBISCO）和磷酸烯醇式丙酮酸羧化酶（PEPC）固定无机碳（Ci）。在分析光合自养代谢和设计改进光合作用时，监测体内Ci同化率是一项重大挑战，因为传统方法无法区分这两种通量。我们探索了广泛应用的气相色谱-质谱联用（GC-MS）代谢物谱分析方法，用于对天冬氨酸进行C位置分数C富集（EC）分析，以通过C脉冲标记在一个分子内区分PEPC、RUBISCO和卡尔文-本森-巴斯德循环（CBB循环）的活性。我们使用两台GC-MS仪器和两种常用的化学衍生化方法验证了该方法。我们以<1%的准确度和<2.5%的精密度选择性地测定了天冬氨酸每个碳位置的EC。结合集胞藻培养物的动态CO标记，我们在评估RUBISCO和CBB循环活性的同时测定了体内PEPC活性。我们证明RUBISCO在黑暗中无活性，而PEPC仍有活性，但速率低于白天。准确量化天冬氨酸浓度和位置EC可提供光合自养集胞藻培养物的摩尔Ci同化率。该技术可与其他代谢物（如3-磷酸甘油酸）的C位置分析相结合，并可用于表征天然和生物合成工程改造的Ci同化。
