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

Biotechnol Biofuels Bioprod

Institute of Chemical, Environmental and Bioscience Engineering, Technische Universität Wien, Gumpendorfer Straße 1a, 1060, Vienna, Austria.

Published: August 2024

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). 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.

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Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC11302359PMC
http://dx.doi.org/10.1186/s13068-024-02534-0DOI Listing

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