Efficiency of acetate-based isopropanol synthesis in Escherichia coli W is controlled by ATP availability

ABSTRACT: Due to increasing ecological concerns, recombinant microbial production of biochemicals from sustainable carbon sources like acetate is rapidly gaining importance. However, for establishing successful 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-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 modelling allowed for an in-depth characterization of intracellular flux distributions, uncovering ATP availability as a determining factor for routing carbon towards 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.

INSTRUMENT(S):

ORGANISM(S): Escherichia Coli

SUBMITTER: Tamara Tomin  

LAB HEAD: Ruth Birner-Gruenberger

PROVIDER: PXD047939 | Pride | 2024-10-17

REPOSITORIES: Pride