Bafna-Rührer Jonas, Bhutada Yashomangalam D, Orth Jean V, Øzmerih Süleyman, Yang Lei, Zielinski Daniel, Sudarsan Suresh
The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2800 Kongens Lyngby, Denmark.
Department of Bioengineering, University of California, San Diego, CA 92093-0412, USA.
PNAS Nexus. 2024 Sep 2;3(9):pgae376. doi: 10.1093/pnasnexus/pgae376. eCollection 2024 Sep.
Engineering microbial cells for the commercial production of biomolecules and biochemicals requires understanding how cells respond to dynamically changing substrate (feast-famine) conditions in industrial-scale bioreactors. Scale-down methods that oscillate substrate are commonly applied to predict the industrial-scale behavior of microbes. We followed a compartment modeling approach to design a scale-down method based on the simulation of an industrial-scale bioreactor. This study uses high cell-density scale-down experiments to investigate knockout strains of five major glucose-sensitive transcription factors (Cra, Crp, FliA, PrpR, and RpoS) to study their regulatory role during glucose oscillations. RNA-sequencing analysis revealed that the glucose oscillations caused the down-regulation of several stress-related functions in . An in-depth analysis of strain physiology and transcriptome revealed a distinct phenotype of the strains tested under glucose oscillations. Specifically, the knockout strains of Cra, Crp, and RpoS resulted in a more sensitive transcriptional response than the control strain, while the knockouts of FliA and PrpR responded less severely. These findings imply that the regulation orchestrated by Cra, Crp, and RpoS may be essential for robust production strains. In contrast, the regulation by FliA and PrpR may be undesirable for temporal oscillations in glucose availability.
对微生物细胞进行工程改造以实现生物分子和生化物质的商业生产，需要了解细胞在工业规模生物反应器中如何应对动态变化的底物（饱食-饥饿）条件。通常采用振荡底物的缩小规模方法来预测微生物在工业规模下的行为。我们采用隔室建模方法，基于对工业规模生物反应器的模拟来设计一种缩小规模方法。本研究利用高细胞密度缩小规模实验，研究了五种主要的葡萄糖敏感转录因子（Cra、Crp、FliA、PrpR和RpoS）的敲除菌株，以研究它们在葡萄糖振荡过程中的调控作用。RNA测序分析表明，葡萄糖振荡导致了几种与应激相关功能的下调。对菌株生理学和转录组的深入分析揭示了在葡萄糖振荡条件下测试菌株的独特表型。具体而言，Cra、Crp和RpoS的敲除菌株比对照菌株产生了更敏感的转录反应，而FliA和PrpR的敲除菌株反应则不那么强烈。这些发现表明，由Cra、Crp和RpoS协调的调控可能对稳健的生产菌株至关重要。相比之下，FliA和PrpR的调控可能不利于葡萄糖可用性的时间振荡。
