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 enzymatic, regulatory, and possibly thermodynamic bottlenecks that throttle back metabolic flow still exist. 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) using three distinct datasets. Subsequently, 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 that helps to achieving good model agreement with experimental data using the K-FIT kinetic parameterization algorithm. 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. The pathway was shown to be thermodynamically feasible and candidate gene Clo1313_0176 was identified for encoding this activity in C. thermocellum. Statistical analysis on k-ctherm101 confirmed that the C. thermocellum-specific regulatory mechanisms on lactate dehydrogenase (activated by fructose 1,6-bisphosphate), alcohol dehydrogenase (inhibited by ethanol and NAD+), and pyruvate, phosphate dikinase (activated by ammonia) were essential to accurate training data recapitulation.

13C Metabolic flux analysis and kinetic modeling of Clostridium thermocellum grown on cellobiose

ACS Fall 2020 Virtual Meeting

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