Engineering Clostridium thermocellum for production of 2,3-butanediol from cellulose

Jilani, S. Bilal; Ashok, Nandhini; Bomble, Yannick J.; Guss, Adam M.; Olson, Daniel G.

doi:10.1016/j.mec.2025.e00269

Engineering Clostridium thermocellum for production of 2,3-butanediol from cellulose

Journal Article · Wed Dec 24 00:00:00 EST 2025 · Metabolic Engineering Communications

DOI:https://doi.org/10.1016/j.mec.2025.e00269· OSTI ID:3012491

Jilani, S. Bilal ^[1]; ^[2]; Bomble, Yannick J. ^[3]; ^[2]; ^[1]

Dartmouth College, Hanover, NH (United States)
Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)
National Renewable Energy Laboratory (NREL), Golden, CO (United States)

Clostridium thermocellum is a promising host for consolidated bioprocessing due to its ability to directly ferment cellulose into fuels and chemicals. However, natural product formation in this organism is limited. Here, we report engineering C. thermocellum for the production of 2,3-butanediol (23BD), a valuable industrial chemical. We functionally expressed a thermophilic 23BD pathway in this organism resulting in a 23BD titer of 19.7 mM from cellulose, representing a metabolic yield of 24%. We used a cell-free systems biology approach to identify limiting steps in the 23BD pathway, revealing that exogenous 23BD dehydrogenase (BDH) activity was essential for production, while native acetolactate synthase (ALS) and acetolactate decarboxylase (ALDC) activities were present but limiting in the parent strain. This approach also revealed redox balance limitations. We demonstrated that this improved understanding of redox balance limitations could be used to increase 23BD titer in vivo, showing that adding acetate could be used to increase 23BD yield. This work establishes a foundation for developing C. thermocellum into a robust platform for 23BD production directly from cellulose and highlights the utility of cell-free systems for guiding metabolic engineering in non-model organisms.

View Accepted Manuscript (DOE)

Research Organization:: Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)

Sponsoring Organization:: National Institutes of Health (NIH); USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: AC05-00OR22725; SC0022175

OSTI ID:: 3012491

Journal Information:: Metabolic Engineering Communications, Journal Name: Metabolic Engineering Communications Vol. 22; ISSN 2214-0301

Publisher:: ElsevierCopyright Statement

Country of Publication:: United States

Language:: English

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