Ethanol-Tolerant Gene Identification in Clostridium thermocellum Using Pyro-Resequencing for Metabolic Engineering

  • Shihui Yang
  • Dawn M. Klingeman
  • Steven D. BrownEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 834)


Classic strain development that combines random mutagenesis and selection has a long history of success in generation of biocatalysts with industrially designed traits. However, the genetic loci contributing to the phenotypic strain changes are difficult to identify prior to genome sequencing technology advancement. In this chapter, we present the approach using Roche 454 next-generation pyro-resequencing to identify the genotypic changes such as single nucleotide polymorphisms (SNP) associated with an ethanol-tolerant strain of Clostridium thermocellum. The parameters used to filter the pyro-resequencing output for SNP identification are also discussed. These can help researchers to identify the genotypic change of other biocatalysts for strain improvement through metabolic engineering.

Key words

454 pyrosequencing Next-generation sequencing Single nucleotide polymorphism Genotyping Clostridium thermocellum Biofuel Consolidated bioprocessing 



The authors would like to thank Meghan M. Drake for her careful review and suggestions. The BioEnergy Science Center is a US Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science. This manuscript has been authored by UT-Battelle, LLC, under Contract No. DE-AC05-00OR22725 with the U.S. Department of Energy.


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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Shihui Yang
    • 1
    • 2
  • Dawn M. Klingeman
    • 1
  • Steven D. Brown
    • 1
    Email author
  1. 1.Biosciences Division and BioEnergy Science CenterOak Ridge National LaboratoryOak RidgeUSA
  2. 2.National BioEnergy CenterNational Renewable Energy LaboratoryGoldenUSA

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