(621b) Assessing the Impact of Allosteric Enzyme Regulations Limiting Ethanol Titer in Clostridium Thermocellum Using a Core Kinetic Model

Clostridium thermocellum is a promising candidate for consolidated bioprocessing due to its ability to consume and ferment cellobiose to ethanol. Despite significant efforts, achieved yields and titers fall below industrially relevant targets. This implies that there still exist enzymatic, regulatory and possibly thermodynamic bottlenecks that throttle back metabolic flow. This work aims to reveal some of these factors by elucidating internal metabolic fluxes in wild-type C. thermocellum grown on cellobiose via ¹³C-metabolic flux analysis (¹³C-MFA). Flux elucidation results confirmed that that both pyruvate, phosphate dikinase and the malate shunt are pyruvate contributors. ¹³C labeling data interpreted by ¹³C-MFA alluded to serine generation via the mercaptopyruvate pathway, with candidate genes Clo1313_0176 and Clo1313_0149 identified for encoding this activity in C. thermocellum. Using the resultant flux distribution in conjunction with batch fermentation process yield data for various mutant strains, we constructed an updated kinetic model of C. thermocellum core metabolism (i.e. k-ctherm101). During kinetic parameterization, we introduced a systematic method for identifying a minimal set of putative regulatory mechanisms required for agreement with the experimental data using the K-FIT kinetic parameterization algorithm. Subsequently, we use k-ctherm101 to explore the effect of removing allosteric regulations on ethanol yield and titer. By exploring all possible simultaneous (up to four) regulation removals we identified combinations that lead to 76% model predicted improvements in ethanol titers. Top strategies include the simultaneous removal of acetyl-CoA inhibition of acetaldehyde dehydrogenase, ethanol inhibition of pyruvate ferredoxin oxidoreductase, and ethanol inhibition of alcohol dehydrogenase combined with nadh inhibition of either alcohol dehydrogenase or acetaldehyde dehydrogenase.