Kutscha Regina, Tomin Tamara, Birner-Gruenberger Ruth, Bekiaris Pavlos Stephanos, Klamt Steffen, Pflügl Stefan
Institute of Chemical, Environmental and Bioscience Engineering, Technische Universität Wien, Gumpendorfer Straße 1a, 1060, Vienna, Austria.
Institute of Chemical Technologies and Analytics, Technische Universität Wien, Vienna, Austria.
Biotechnol Biofuels Bioprod. 2024 Aug 5;17(1):110. doi: 10.1186/s13068-024-02534-0.
Due to increasing ecological concerns, microbial production of biochemicals from sustainable carbon sources like acetate is rapidly gaining importance. However, to successfully establish large-scale production scenarios, a solid understanding of metabolic driving forces is required to inform bioprocess design. To generate such knowledge, we constructed isopropanol-producing Escherichia coli W strains.
Based on strain screening and metabolic considerations, a 2-stage process was designed, incorporating a growth phase followed by a nitrogen-starvation phase. This process design yielded the highest isopropanol titers on acetate to date (13.3 g L). Additionally, we performed shotgun and acetylated proteomics, and identified several stress conditions in the bioreactor scenarios, such as acid stress and impaired sulfur uptake. Metabolic modeling allowed for an in-depth characterization of intracellular flux distributions, uncovering cellular demand for ATP and acetyl-CoA as limiting factors for routing carbon toward the isopropanol pathway. Moreover, we asserted the importance of a balance between fluxes of the NADPH-providing isocitrate dehydrogenase (ICDH) and the product pathway.
Using the newly gained system-level understanding for isopropanol production from acetate, we assessed possible engineering approaches and propose process designs to maximize production. Collectively, our work contributes to the establishment and optimization of acetate-based bioproduction systems.
由于生态问题日益受到关注，利用醋酸盐等可持续碳源进行微生物生产生物化学品正迅速变得重要起来。然而，要成功建立大规模生产方案，需要深入了解代谢驱动力以指导生物工艺设计。为了获得此类知识，我们构建了产异丙醇的大肠杆菌W菌株。
基于菌株筛选和代谢考量，设计了一个两阶段工艺，包括一个生长阶段，随后是氮饥饿阶段。该工艺设计产生了迄今为止以醋酸盐为底物的最高异丙醇滴度（13.3 g/L）。此外，我们进行了鸟枪法和乙酰化蛋白质组学分析，并确定了生物反应器场景中的几种应激条件，如酸应激和硫摄取受损。代谢建模能够深入表征细胞内通量分布，揭示细胞对ATP和乙酰辅酶A的需求是将碳导向异丙醇途径的限制因素。此外，我们强调了提供NADPH的异柠檬酸脱氢酶（ICDH）通量与产物途径通量之间平衡的重要性。
利用新获得的关于从醋酸盐生产异丙醇的系统层面理解，我们评估了可能的工程方法，并提出了使产量最大化的工艺设计。总的来说，我们的工作有助于基于醋酸盐的生物生产系统的建立和优化。
