ABSTRACT. This talk will present a forward-looking perspective on how my laboratory, in collaboration with and supported by others, has applied quantitative metabolomics, isotope-based metabolic flux analysis, and computational modeling to uncover the unique metabolic constraints of Clostridium thermocellum. I will highlight evidence that its central metabolism, particularly glycolysis and ethanol fermentation as well as other pathways, operates near thermodynamic equilibrium and imposes unusually high enzyme and protein allocation costs. Using quantitative flux, thermodynamic, and resource balance analyses, we identify phosphofructokinase and pyrophosphate-dependent reactions as key bottlenecks shaping metabolic flux, yield, and product titers. Finally, I will discuss how thermodynamics-guided metabolic engineering, including redesign of glycolysis and cofactor usage, can overcome these constraints and improve ethanol production, providing general principles for engineering cellulolytic Clostridium species.

ABSTRACT. Climate change and poor recycling of waste is threatening global biosustainability. Humankind is thus facing a pressing need for sustainable production of chemicals, fuels, and food and improved waste recycling. Acetogen bacteria have become attractive biocatalysts for converting inexpensive and abundant solid and gaseous waste feedstocks into high-value products using gas fermentation. However, our knowledge of limits of acetogen metabolism is scarce but this is required for both fundamental understanding of cellular behaviour and rational metabolic engineering. We thus challenged the model-acetogen Clostridium autoethanogenum to grow faster or at lower pH in autotrophic continuous cultures and used adaptive laboratory evolution (ALE) and reverse genetic engineering to improve growth limits. We were able to obtain steady-states at up to specific growth rates of ~2.8 day-1 (~0.12 h-1) with faster growth supporting both higher yields and productivities of reduced by-products ethanol and 2,3-butanediol. Lowering pH led to significant changes in product and transcriptional profiles. Autotrophic ALE yielded superior strains that can grow faster, without complex nutrients, and are robust for operating continuous cultures. Reverse genetic engineering of mutations in genes potentially involved in regulatory networks recovered all three superior features of our ALE strains through triggering significant proteomic rearrangements. These results advance our understanding of limits of acetogen metabolism and offer engineering targets in an industrially relevant cell factory.