背景:由于越来越多的生态问题,从可持续的碳源,如乙酸盐,微生物生产的生物化学品正在迅速获得重要性。然而,成功建立大规模生产场景,需要对代谢驱动力有深入的了解才能为生物过程设计提供信息。为了产生这样的知识,我们构建了产异丙醇大肠杆菌W菌株。
结果:基于菌株筛选和代谢考虑,设计了一个两阶段的过程,包含生长阶段,然后是氮饥饿阶段。该方法设计在乙酸盐上产生了迄今为止最高的异丙醇滴度(13.3gL-1)。此外,我们进行了鸟枪和乙酰化蛋白质组学,并确定了生物反应器场景中的几种应力条件,如酸胁迫和硫吸收受损。代谢模型可以对细胞内通量分布进行深入表征,揭示细胞对ATP和乙酰辅酶A的需求是将碳引向异丙醇途径的限制因素。此外,我们断言提供NADPH的异柠檬酸脱氢酶(ICDH)的通量与产物途径之间平衡的重要性。
结论:使用新获得的从乙酸盐生产异丙醇的系统级理解,我们评估了可能的工程方法,并提出了工艺设计,以最大限度地提高产量。总的来说,我们的工作有助于建立和优化基于乙酸盐的生物生产系统。
BACKGROUND: 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.
RESULTS: 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-1). 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.
CONCLUSIONS: 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.