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

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

doi:10.1016/j.mec.2025.e00269

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

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

Synthetic Biology

Research output: Contribution to journal › Article › peer-review

Abstract

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.

Original language	English
Article number	e00269
Journal	Metabolic Engineering Communications
Volume	22
DOIs	https://doi.org/10.1016/j.mec.2025.e00269
State	Published - Jun 2026

Funding

Funding for the contributions of NA and AMG to this work was also provided by the Center for Bioenergy Innovation ( CBI ), U.S. Department of Energy , Office of Science , Biological and Environmental Research Program under Award Number ERKP 886 . Funding for the contributions of SBJ, YJB, and DGO to this work was provided by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, Genomic Science Program under Award Number DE-SC0022175. Funding for the contributions of NA and AMG to this work was also provided by the Center for Bioenergy Innovation (CBI), U.S. Department of Energy, Office of Science, Biological and Environmental Research Program under Award Number ERKP886. This work was authored in part by Oak Ridge National Laboratory, which is managed by UT-Battelle, LLC, for the U.S. Department of Energy under contract DE-AC05-00OR22725. Protein expression and purification work was supported in part by the bioMT facility at Dartmouth College through NIHNIGMS grant P20-GM113132. This work was authored in part by Oak Ridge National Laboratory, which is managed by UT-Battelle, LLC, for the U.S. Department of Energy under contract DE-AC05-00OR22725. Funding for the contributions of SBJ, YJB, and DGO to this work was provided by the U.S. Department of Energy , Office of Science , Office of Biological and Environmental Research , Genomic Science Program under Award Number DE-SC 0022175 . Protein expression and purification work was supported in part by the bioMT facility at Dartmouth College through NIH NIGMS grant P 20-GM113132 .

Keywords

Acetivibrio thermocellus
Clostridium thermocellum
Hungateiclostridium thermocellum
Ruminiclostridium thermocellum

Access to Document

10.1016/j.mec.2025.e00269

Cite this

@article{838359d9c14c477cae375ba5e2017ee1,

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

abstract = "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.",

keywords = "Acetivibrio thermocellus, Clostridium thermocellum, Hungateiclostridium thermocellum, Ruminiclostridium thermocellum",

author = "Jilani, \{S. Bilal\} and Nandhini Ashok and Bomble, \{Yannick J.\} and Guss, \{Adam M.\} and Olson, \{Daniel G.\}",

note = "Publisher Copyright: {\textcopyright} 2026 The Authors.",

year = "2026",

month = jun,

doi = "10.1016/j.mec.2025.e00269",

language = "English",

volume = "22",

journal = "Metabolic Engineering Communications",

issn = "2214-0301",

}

TY - JOUR

T1 - Engineering Clostridium thermocellum for production of 2,3-butanediol from cellulose

AU - Jilani, S. Bilal

AU - Ashok, Nandhini

AU - Bomble, Yannick J.

AU - Guss, Adam M.

AU - Olson, Daniel G.

PY - 2026/6

Y1 - 2026/6

N2 - 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.

AB - 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.

KW - Acetivibrio thermocellus

KW - Clostridium thermocellum

KW - Hungateiclostridium thermocellum

KW - Ruminiclostridium thermocellum

UR - https://www.scopus.com/pages/publications/105027406159

U2 - 10.1016/j.mec.2025.e00269

DO - 10.1016/j.mec.2025.e00269

M3 - Article

AN - SCOPUS:105027406159

SN - 2214-0301

VL - 22

JO - Metabolic Engineering Communications

JF - Metabolic Engineering Communications

M1 - e00269

ER -

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

Abstract

Funding

Keywords

Access to Document

Other files and links

Fingerprint

Cite this