Isotopically nonstationary metabolic flux analysis (INST-MFA) provides a versatile platform to quantitatively assess in vivo metabolic activities of autotrophic systems. By applying INST-MFA to recombinant aldehyde-producing cyanobacteria, we identified metabolic alterations that correlated with increased strain performance in order to guide rational metabolic engineering. We identified four reactions adjacent to the pyruvate node that varied significantly with increasing aldehyde production: pyruvate kinase (PK) and acetolactate synthase (ALS) fluxes were directly correlated with product formation, while pyruvate dehydrogenase (PDH) and phosphoenolpyruvate carboxylase (PPC) fluxes were inversely correlated. Overexpression of enzymes for PK or ALS did not result in further improvements to the previous best-performing strain, while downregulation of PDH expression (through antisense RNA expression) or PPC flux (through expression of the reverse reaction, phosphoenolpyruvate carboxykinase) provided significant improvements. These results illustrate the potential of INST-MFA to enable a systematic approach for iterative identification and removal of pathway bottlenecks in autotrophic host cells.

同位素非平稳代谢流分析 (INST-MFA) 提供了一个通用平台来定量评估自养系统的体内代谢活动。通过将 INST-MFA 应用于重组产醛蓝藻，我们确定了与菌株性能增加相关的代谢改变，以指导合理的代谢工程。我们确定了与丙酮酸节点相邻的四个反应，这些反应随着醛产量的增加而显着变化：丙酮酸激酶（PK）和乙酰乳酸合酶（ALS）通量与产物形成直接相关，而丙酮酸脱氢酶（PDH）和磷酸烯醇丙酮酸羧化酶（PPC）通量与产物形成直接相关。呈负相关。PK 或 ALS 酶的过度表达并没有导致对先前表现最佳菌株的进一步改善，而 PDH 表达（通过反义 RNA 表达）或 PPC 通量（通过逆反应磷酸烯醇丙酮酸羧激酶的表达）的下调提供了显着的改善。这些结果说明了 INST-MFA 能够采用系统方法迭代识别和消除自养宿主细胞中的途径瓶颈的潜力。