Department of Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, TN, USA.
Department of Biological Sciences, Vanderbilt University, Nashville, TN, USA.
Metab Eng. 2020 Jul;60:56-65. doi: 10.1016/j.ymben.2020.03.007. Epub 2020 Mar 25.
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.
同位素非稳态代谢通量分析（INST-MFA）为定量评估自养系统的体内代谢活性提供了一个通用的平台。通过将 INST-MFA 应用于重组醛产生蓝细菌，我们确定了与提高菌株性能相关的代谢变化，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变化显著的反应：与产物形成直接相关的丙酮酸激酶（PK）和乙酰乳酸合酶（ALS）通量，而丙酮酸脱氢酶（PDH）和磷酸烯醇丙酮酸羧激酶（PPC）通量则呈负相关。PK 或 ALS 酶的过表达并没有导致在前最佳表现菌株上进一步改进，而 PDH 表达的下调（通过反义 RNA 表达）或 PPC 通量的下调（通过表达相反的反应，磷酸烯醇丙酮酸羧激酶）提供了显著的改进。这些结果说明了 INST-MFA 能够系统地识别和消除自养宿主细胞中途径瓶颈的潜力。
