Mutant selection and phenotypic and genetic characterization of ethanol-tolerant strains of Clostridium thermocellum

Xiongjun Shao; Babu Raman; Mingjun Zhu; Jonathan R. Mielenz; Steven D. Brown; Adam M. Guss; Lee R. Lynd

doi:10.1007/s00253-011-3492-z

Mutant selection and phenotypic and genetic characterization of ethanol-tolerant strains of Clostridium thermocellum

Xiongjun Shao, Babu Raman, Mingjun Zhu, Jonathan R. Mielenz, Steven D. Brown, Adam M. Guss, Lee R. Lynd

Synthetic Biology

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70 Scopus citations

Abstract

Clostridium thermocellum is a model microorganism for converting cellulosic biomass into fuels and chemicals via consolidated bioprocessing. One of the challenges for industrial application of this organism is its low ethanol tolerance, typically 1-2% (w/v) in wild-type strains. In this study, we report the development and characterization of mutant C. thermocellum strains that can grow in the presence of high ethanol concentrations. Starting from a single colony, wild-type C. thermocellum ATCC 27405 was sub-cultured and adapted for growth in up to 50 g/L ethanol using either cellobiose or crystalline cellulose as the growth substrate. Both the adapted strains retained their ability to grow on either substrate and displayed a higher growth rate and biomass yield than the wild-type strain in the absence of ethanol. With added ethanol in the media, the mutant strains displayed an inverse correlation between ethanol concentration and growth rate or biomass yield. Genome sequencing revealed six common mutations in the two ethanol-tolerant strains including an alcohol dehydrogenase gene and genes involved in arginine/pyrimidine biosynthetic pathway. The potential role of these mutations in ethanol tolerance phenotype is discussed.

Original language	English
Pages (from-to)	641-652
Number of pages	12
Journal	Applied Microbiology and Biotechnology
Volume	92
Issue number	3
DOIs	https://doi.org/10.1007/s00253-011-3492-z
State	Published - Nov 2011

Funding

Acknowledgements The authors are grateful for the support provided by funding grants from the BioEnergy Science Center (BESC), a US Department of Energy (DOE) Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science and Mascoma Corporation. The authors are also grateful for the genome sequencing support provided by the DOE Joint Genome Institute (JGI). Oak Ridge National Laboratory is managed by University of Tennessee UT-Battelle LLC for the Department of Energy under contract no. DE-AC05-00OR22725.

Funders	Funder number
BioEnergy Science Center
DOE Office of Science and Mascoma Corporation
University of Tennessee UT-Battelle LLC
U.S. Department of Energy	DE-AC05-00OR22725
Biological and Environmental Research

Keywords

Clostridium thermocellum
Ethanol tolerance
Genome sequencing
Mutations
Strain adaptation

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abstract = "Clostridium thermocellum is a model microorganism for converting cellulosic biomass into fuels and chemicals via consolidated bioprocessing. One of the challenges for industrial application of this organism is its low ethanol tolerance, typically 1-2% (w/v) in wild-type strains. In this study, we report the development and characterization of mutant C. thermocellum strains that can grow in the presence of high ethanol concentrations. Starting from a single colony, wild-type C. thermocellum ATCC 27405 was sub-cultured and adapted for growth in up to 50 g/L ethanol using either cellobiose or crystalline cellulose as the growth substrate. Both the adapted strains retained their ability to grow on either substrate and displayed a higher growth rate and biomass yield than the wild-type strain in the absence of ethanol. With added ethanol in the media, the mutant strains displayed an inverse correlation between ethanol concentration and growth rate or biomass yield. Genome sequencing revealed six common mutations in the two ethanol-tolerant strains including an alcohol dehydrogenase gene and genes involved in arginine/pyrimidine biosynthetic pathway. The potential role of these mutations in ethanol tolerance phenotype is discussed.",

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Mutant selection and phenotypic and genetic characterization of ethanol-tolerant strains of Clostridium thermocellum

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